T0028 – Effect of water on the molecular mobility of elastin
Purified and hydrated elastin is studied by both thermal and dielectric techniques to have insight into the chain dynamics of this protein. By differential scanning calorimetry, the glassy behavior of elastin is highlighted; the glass transition temperature (Tg) of elastin is found to be widely dependent on hydration, falling from 200°C in the dehydrated state to 30°C for 30% hydration. A limit of Tg at around 0°C is found when crystallizable water is present in the system, that is, when the formation of ice prevents motions of some 10 nm along the polypeptidic chains. The technique of thermally stimulated currents, carried out in the -180 to 0°C temperature range, is useful to detect localized motions. In this case, too, the localized motions vary considerably according to hydration: a first relaxation mode is observed at -145°C and it is associated with the reorientation of crystallizable water in ice I; a second relaxation mode, more complex and cooperative, occurs at around -80°C and could be attributed to the complex constituted by the dipolar groups of the polypeptidic chain and noncrystallizable water, behaving as a glassy system
T0015 – Comparison of chemical treatments on the chain dynamics and thermal stability of bovine pericardium collagen
A new approach for the replacement of heart
valves consists of obtaining an acellular matrix from animal
aortic valves that performs mechanically, is nonantigenic,
and is free from calcification and fibroblast proliferation.
Novel biochemical treatments must be developed for this
purpose. In this work, we focus on the characterization of
collagen in acellular bovine cardiovascular tissues, fresh or
glutaraldehyde treated, and stored in different solutions
[phosphate-buffered saline (PBS), ethanol, octanol, and glutaraldehyde],
to determine whether the resulting fibrous
material is structurally preserved. The preservation of the
triple helical structure of collagen is checked by differential
scanning calorimetry (DSC), which is a well suited technique
to analyze thermal transitions in proteins, such as denaturation.
To get insight into the molecular dynamics of collagen
in the nanometric range, we used thermally stimulated currents,
a dielectric technique running at low frequency, that
measure the dipolar reorientations in proteins submitted to
a static electrical field. The combined use of these two techniques
allowed us to evaluate the physical structure and
conformation of collagen after the different chemical treatments.
We have found that the glutaraldehyde treatment
followed by octanol storage preserves the triple helical conformation
of the polypeptidic chains of collagen, contrary to
the ethanol and PBS storage that induce drastic changes in
the thermal and dielectric behavior of the protein. Moreover,
this particular chemical treatment stabilizes the collagen
structure (shift toward high temperature of the collagen denaturation
and stiffening of the chains by a cross-linking
action) when compared to the control sample, and so could
provide interesting fibrous material for the conception of
bioprosthetic heart valve.
T0014 – Molecular stability of elastin : effect of molecular architecture
The thermal and dielectric properties of elastin and two soluble derivatives ( k-elastin and derived elastin
peptides from enzymatic elastolysis) were investigated in the freeze-dried state in a wide temperature range
(from -180 to +220°C). The glass transition of these amorphous proteins was studied by differential
scanning calorimetry (DSC). The dielectric relaxations of both proteins were followed by thermally stimulated
currents (TSC), an isochronal dielectric spectrometry running at variable temperature, analogous to a lowfrequency
spectroscopy (10^(-3) - 10^(-2) Hz) and by dynamic dielectric spectroscopy (DDS), performed
isothermally with the frequency varying from 10^(-2) to 3 x 10^6 Hz. The combination of TSC and DDS
experiments and the determination of the activation parameters of the relaxation times inform about the
molecular mobility of the proteins, both in the glassy state and in the liquid state. Major differences between
the relaxation behavior of elastin and its soluble derivatives have been discussed and correlated with the
molecular architecture of the proteins.
T0009 – The beta-alpha branching in D-sorbitol as studied by thermally stimulated depolarization currents (TSDC)
The molecular motions in D-sorbitol (D-glucitol) have been studied by thermally stimulated depolarization currents (TSDC) in the temperature region between -160 and 10°C. The relaxation appears as a broad global peak between -160 and -50°C and its features were compared with those of the relaxation of maltitol (a D-glucitol derivative). A study of the relaxation of sorbitol, which shows a maximum intensity at approximately -1°C, is also presented, and from the obtained data, the fragility index of this glass former is calculated. The and relaxations are observed to merge in the frequency window of the TSDC technique, and it is underlined that this merging is a consequence of the overlap of the tails of these distributions in this frequency window. In this context, the merging observed by TSDC images the branching of the most probable times of the two distributions predicted by dielectric relaxation spectroscopy at the branching temperature T.
E0087 – Etude par analyse thermique de trois matrices lipidiques
B3279 – Microcalorimetric study on conformational change of denatured RNase A adsorbed onto a moderately hydrophobic surface
The microcalorimetric method was used to measure the displacement adsorption heat of denatured (by 1.8 mol L?1 guanidine hydrochloride (GuHCl)) Ribonuclease A (RNase A) adsorbed onto a moderately hydrophobic surface (PEG-600) from various ammonium sulfate ((NH4)2SO4) concentration solutions at 298 K. According to the thermodynamics of the stoichiometric displacement theory for adsorption (SDT-A) and the measured adsorption isotherms, the adsorption thermodynamic functions, ?G, ?S, ?H, and their fractions were obtained. In combination with FTIR analysis, the regulation of conformational change of adsorbed denatured RNase A was found. The results showed that the moderately hydrophobic surface can provide energy to denatured protein and make it gain more ordered conformation with (NH4)2SO4 concentration increment. The analysis of thermodynamic fractions showed that the contribution of four subprocesses associated with the displacement adsorption of RNase A was different in various (NH4)2SO4 concentrations.
B3272 – DNA interaction with flavone and hydroxyflavones
In recent years flavonoids have attracted a great interest as potential therapeutic drugs against a wide range of diseases. While the antioxidant activity of these natural polyphenolic compounds is well known, their binding to DNA characteristics is not fully understood despite the fact that many of them exert their biological effects by reversibly binding to nucleic acids. The present study aims to investigate the interaction of flavone and four hydroxyflavone isomers with double stranded DNA, occurring in bulk solution. A combination of micro-DSC and UV spectroscopy has been used to study the effect of these compounds and of their structure on the structure and stability of the DNA molecule. The characteristics of DNA thermal denaturation have been used as a measure of the effect of the compounds on the stability of the double helix. Micro-DSC has been used to determine the temperature dependence of the heat capacity of the process of thermal denaturation of DNA in solutions containing flavone and the following hydroxyflavones: 3-hydroxyflavone, 5-hydroxyflavone, 6-hydroxyflavone and 7-hydroxyflavone. The observed enthalpy of transition and the transition temperature of DNA thermal denaturation were determined for each compound. UV thermal denaturation curves were also recorded, under the same experimental conditions as the DSC measurements. The transition temperature values and the thermodynamic parameters of thermal denaturation were determined.
Differential scanning calorimetry and ultraviolet spectroscopy measurements have evidenced an interaction between the studied flavones and DNA, showing a stabilization of DNA structure, due mainly to intercalation of the flavones. The results also show a noteworthy effect of the structure of the hydroxyflavone isomers on this stabilization.
B3271 – The effect of compatible solute ectoines on the structural organization of lipid monolayer and bilayer membranes
Compatible solutes are small organic osmolytes responsible for osmotic balance and at the same time compatible with the cellular metabolism. Here, we have investigated the effect of the compatible solutes, ectoine and hydroxyectoine, on the fluid–rigid domain structure of lipid monolayer and bilayer membranes. Mainly saturated dipalmitoyl-phosphatidylcholine membranes exhibiting a clear le/lc phase transition were used. Fluorescence microscopy showed that ectoines added to the aqueous subphase expand and fluidize the lipid monolayers especially at surface pressures below 30 mN/m. The domain structure at the le/lc phase transition is sensitively modified leading to smaller but more numerous domains in the presence of ectoines. Hydroxyectoine was more efficient than ectoine. These results are explained by the replacement theory assuming that the ectoines are likely to be expelled from the membrane surface thus favoring the hydration of the lipid membrane. This effect reduces the line tension, which is the interfacial energy at the domain edges leading to reduced domain sizes and increased number of rigid domains. Isotherms of negatively charged phosphatidylglycerol membranes show a similar expansion, while unsaturated lipids are less affected. Mixed phosphatidylcholine/phosphatidylglycerol membranes exhibit the same effect on the line tension increasing the tendency for a phase separation. This could be shown also in bilayer vesicles, where the compatible solutes have only a minor effect on the lipid main phase transition in pure DPPC membranes but reduce the extent of the pretransition. In mixed DPPC/DPPG bilayer membranes ectoines cause a phase separation leading to the enrichment of expanded DPPC domains. In conclusion, our study gives for the first time evidence that ectoines have an effect on lipid membranes increasing the hydration of the surface and thus increasing the mobility of the lipid head groups and fluidizing the lipid layer accordingly. This increased fluidity may be of advantage for cell membranes to withstand extreme conditions like temperature or osmotic pressure and might also accelerate cellular repair mechanisms.
B3270 – Thermodynamic and structural characterization of Liposomal-Locked in-Dendrimers as drug carriers
A new Liposomal-Locked in-Dendrimer (LLD) formed by DPPC-DPPG and PAMAM 3.5 incorporating the anticancer drug DOX was studied by means of spectroscopic and DSC investigations. Multilamellar Lipid Bilayers were also considered for the sake of comparison. The results were in line with a picture of phase separation between DPPC-DPPG lipids and dendrimer that promotes the stability of the liposome membrane and the cooperativity of the relevant gel-to-liquid-crystal transition, which is enhanced in the presence of the dendrimer and the drug. As a result, the inner core of the liposome contained large amounts of dendrimer-DOX complex and was protected by a very stable membrane. This view was given a more general validation through investigations performed with other types of dendrimers, namely PG1 and PG2. The thermodynamic interpretation of the DSC data allowed a better understanding of the physico-chemical factors that justify this behaviour that makes these LLDs very promising as a new class of Modulatory Liposomal Controlled Release System (MLCRS) that could lead to drug formulations with higher safety and efficacy profiles.
B3267 – Studies on the conformational change of adsorbed BSA onto a moderately hydrophobic surface at different denaturant concentrations and surface coverages
This paper is aimed at investigating the conformational change of denatured bovine serum albumin (BSA) in combination with thermodynamic functions and their fractions, adsorption isotherms, Fourier transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC). Microcalorimetric measurements of displacement adsorption enthalpies DeltaH of denatured BSA (by guanidine hydrochloride (GuHCl)) adsorbed onto a moderately hydrophobic surface (PEG-600) from solutions were carried out. The contents of secondary structure elements of BSA in solutions and in the adsorbed state were determined by FTIR and the thermal stability of adsorbed BSA was measured by DSC. The adsorption thermodynamic functions DeltaH, DeltaS, DeltaG, and their fractions were calculated based on the thermodynamics of the stoichiometric displacement theory for adsorption (SDT-A) and adsorption isotherms. The results showed that the surface can provide energy to denatured BSA and make it gain a more ordered conformation with GuHCl concentration increment. At a given GuHCl concentration, although the ordered secondary structure of adsorbed BSA molecules decreased, their tertiary structure may be more perfect with surface coverage increment. The thermodynamic analysis of four subprocesses associated with adsorption also confirmed the increment of conformational gain.
B3240 – Freeze-drying of nanostructure lipid carriers by different carbohydrate polymers used as cryoprotectants
Freeze-drying technique preserves the stability of nanoparticles. The objective of this study was optimization of freeze-drying condition of nano lipid carriers (NLCs). NLCs were prepared by emulsion-solvent evaporation followed by ultra-sonication method. Different carbohydrate and polymeric cryoprotectants including Microcelac® (mixture of lactose and Avicel), Avicel PH102 (microcrystalline cellulose), mannitol, sucrose, Avicel RC591 (mixture of microcrystalline cellulose and sodium carboxymethyl cellulose), maltodextrine, Aerosil and PEG4000 were tested initially. The NLCs showing lower particle size growth and greater absolute zeta potential after freeze drying were chosen for further investigation using Taguchi optimization method. Studied factors included cryoprotectant type and concentration, freezing temperatures applied at different time periods and sublimation time. Sucrose, Avicel RC591 and Aerosil were selected as cryoprotectants from initial screening tests. Increasing their concentration increased the particle size. 1% of Avicel RC591, 24 h of freezing at ?70 °C and 48 h sublimation time showed lower growth in particle size.
B3234 – Production of yeast chitin–glucan complex from biodiesel industry byproduct
Crude glycerol from the biodiesel industry was used as carbon source for high cell density fed-batch cultivation of Pichia pastoris aiming at producing a chitin–glucan complex (CGC). More than 100 g L?1 biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g g?1 during the batch phase and 0.63 g g?1 during the fed-batch phase. The chitin–glucan complex was recovered from the yeast cell wall by hot alkaline extraction. CGC content in the cell wall was found to be relatively constant throughout the cultivation (18–26%) with a volumetric productivity of 1.28 g L?1 h?1 at the end of the fed-batch phase. The molar ratio of chitin:?-glucan in the extracted biopolymer was 16:84, close to other CGC extracted from Aspergillus biomass. The extracted polymer was characterized by Differential Scanning Calorimetry (DCS) and solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and compared with commercial biopolymers, namely, crab shell chitin and/or chitosan, algal ?-glucan (laminarin) and fungal chitin–glucan complex (kiOsmetine).
B3229 – Characteristics, phase behavior and control release for copolymer–liposome with both pH and temperature sensitivities
A pH- and thermo-sensitive berberine hydrochloride liposome modified by the poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate) was synthesized. The morphology and size were determined by atomic force microscope (AFM) and dynamic light scattering (DLS). The phase transition temperature (Tpm) and phase transition pH (pH*) of the copolymer–liposome were obtained by differential scanning calorimetry (DSC). The results indicate that the size of copolymer–liposome depends on the mass ratios of the copolymer to soya bean lecithin (SPC) and the solution pH value, and the size can reach a maximum value at phase transition pH (pH*). The Tpm and pH* were affected by the mass ratios of the copolymer to SPC and properties of the copolymer. The release of berberine hydrochloride (BH) from the copolymer–liposome was investigated and the maximum release of BH was caused at Tpm and pH* of the copolymer–liposome. The results illustrate that BH released from the liposome is due to the phase transition of the copolymer–liposome, where both Tpm and pH* were acted as switches of the phase transition, and both spacial and temporal releases can be carried out by adjusting the temperature, pH value and release time
B3225 – Cell behavior on a CCN1 functionalized elastin-mimetic protein polymer
We report the design of an elastin-mimetic triblock copolymer with the ability to guide endothelial cell adhesion, spreading, and migration while maintaining the elastomeric properties of the protein polymer. The V2 ligand sequence from matricellular protein CCN1 (cysteine-rich 61, CYR61) was multimerized and cloned into elastin polymer LysB10, creating LysB10.V2. Cell adhesion studies demonstrated that a LysB10.V2 surface density of at least 40 pmol/cm2 was required to elicit cell attachment. Peptide blocking studies confirmed V2 specific engagement with integrin receptor ?v?3 (P < 0.05) and we observed the formation of actin stress fiber networks and vinculin clustering, characteristic of focal adhesion assembly. Haptotatic migration assays demonstrated the ability of LysB10.V2 surfaces to stimulate migration of endothelial cells (P < 0.05). Significantly, we illustrated the ability of LysB10.V2 to support a quiescent endothelium. The CCN1 molecule functions to support many key biological processes necessary for tissue repair and thus presents a promising target for bioengineering applications. Collectively, our results demonstrate the potential to harness CCN1 specific function in the design of new scaffold materials for applications in regenerative medicine.
B3223 – Food protein aggregates as vitamin-matrix carriers: Impact of processing conditions
We studied the ability of protein aggregates for loading and protection of ?-tocopherol, a model of heat-and light-sensitive bioactive compounds. Aqueous dispersions of whey proteins (4.5 wt.%, pH 6.7) in the absence and presence of ?-tocopherol (4 wt.%) were prepared using an ultradisperser (10,000 rpm for 10 min and 65 °C), and then submitted to further high-pressure homogenisation (HPH) at 300 or 1200 bar for 12 cycles. Relative to free-vitamin dispersions, increasing HPH conditions in the presence of vitamin led to higher protein denaturation, more tryptophan quenching and wavelength blue-shift (by 10 nm), in parallel with increased zeta potential values (by -10 mV), particle sizes (by 50%), and newly formed protein dimers, trimers and high molecular weight aggregates. As a result, the degree of vitamin degradation under increasing HPH and long-term storage was shown to decrease from 66% (ultradisper) to 50%, or to 30% (subject to further treatments at 300 or 1200 bar, respectively).
B3222 – Structural perturbation of diphtheria toxoid upon adsorption to aluminium hydroxide adjuvant
Aluminium-containing adjuvants are often used to enhance the potency of vaccines. In the present work we studied whether adsorption of diphtheria toxoid to colloidal aluminium hydroxide induces conformational changes of the antigen. Diphtheria toxoid has a high affinity for the aluminium hydroxide particles based on a high adsorption degree, adsorption rate and adsorptive capacity. The conformation and stability of diphtheria toxoid in solution and adsorbed to aluminium hydroxide adjuvant were characterized using five physicochemical techniques: intrinsic and extrinsic fluorescence spectroscopy, circular dichroism, infrared spectroscopy and differential scanning calorimetry. Diphtheria toxoid adsorbed to aluminium hydroxide resulted in a minimal shift of the tryptophan fluorescence spectrum, whereas a large increase in the emission of the Bis-ANS probe was observed, indicating that hydrophobic sites of the protein became accessible due to adsorption. In addition, circular dichroism and infrared spectroscopy revealed that adsorption to aluminium hydroxide caused an increase of ?-sheet content and a decrease of ?-helix content in diphtheria toxoid. Differential scanning calorimetry demonstrated a major decrease in the enthalpy of denaturation upon adsorption. In conclusion, the adsorption of diphtheria toxoid to aluminium hydroxide adjuvant leads to substantial conformational changes in the antigen. Since physicochemical methods can be used to monitor these conformational changes, these analytical methods might offer a tool in regulatory required vaccine quality control by demonstrating consistency in production.
B3211 – Hydration of Proteins: Excess Partial Enthalpies of Water and Proteins
Isothermal batch calorimetry was applied to study the hydration of proteins. The hydration process was analyzed by the simultaneous monitoring of the excess partial enthalpies of water and the proteins in the entire range of water content. Four unrelated proteins (lysozyme, chymotrypsinogen A, human serum albumin, and ?-lactoglobulin) were used as models. The excess partial quantities are very sensitive to the changes in the state of water and proteins. At the lowest water weight fractions (w1), the changes of the excess thermochemical functions can mainly be attributed to water addition. A transition from the glassy to the flexible state of the proteins is accompanied by significant changes in the excess partial quantities of water and the proteins. This transition appears at a water weight fraction of 0.06 when charged groups of proteins are covered. Excess partial quantities reach their fully hydrated values at w1 > 0.5 when coverage of both polar and weakly interacting surface elements is complete. At the highest water contents, water addition has no significant effect on the excess thermochemical quantities. At w1 > 0.5, changes in the excess functions can solely be attributed to changes in the state of the proteins.
B3204 – Effect of shear on vesicle and lamellar phases of DDAB/lecithin ternary systems
The influence of shear flow on bilayer structures (vesicle and planar lamellar phases, L?), formed in DDAB/lecithin ternary systems, is studied by means of conventional rheology, Rheo NMR, and optical microscopy. The vesicles in the diluted (Lam,) phase are polydisperse multilamellae which turn into smaller monodisperse vesicles under shear. The concentrated (Lam2) phase is formed by non-oriented lamellae that do not surprisingly exhibit any pronounced shear-induced alignment prior to the transition into giant multilamellar vesicles. The biphasic region (Lam, + Lam2) shows a mosaic texture with a powder pattern indicating the prevalence of lamellae that transform into onions under shear.
B3194 – C- and N-truncated antimicrobial peptides from LFampin 265 – 284: Biophysical versus microbiology results
Lactoferrin is a glycoprotein with two globular lobes, each having two domains. Since the discovery of its antimicrobial properties, efforts have been made to find peptides derived from this protein showing antimicrobial properties. Most peptides initially studied were derived from Lactoferricin B, obtained from the protein by digestion with pepsin. More recently, a new family of antimicrobial peptides (AMPs) derived from Lactoferrin was discovered by Bolcher et al, and named Lactoferrampin (LFampin). The original sequence of LFampin contained residues 268 - 284 from the N1 domain of Lactoferrin. From this peptide, the Bolscher's group synthesized a collection of peptides obtained by extension and / or truncation at the C or N-terminal sides, in order to unravel the main structural features responsible for antimicrobial action. Here, we present results for three of these peptides, namely LFampin 265 - 284, LFampin 265 - 280, and LFampin 270 - 284. The peptides were tested against bacteria (E. coli and S. sanguinis), fungi (C. albicans), and model membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG), and their mixtures at a ratio of 3 : 1 (DMPC : DMPG (3 : 1)). The ability to adopt a helical conformation was followed by a circular dichroism (CD), and the perturbation of the gel to the liquid-crystalline phase transition of the membrane was characterized by differential scanning calorimetry (DSC). Distinct behavior was observed in the three peptides, both from the microbiology and model membrane studies, with the biophysical results showing excellent correlation with the microbiology activity studies. LFampin 265 - 284 was the most active peptide toward the tested microorganisms, and in the biophysical studies it showed the highest ability to form an ?-helix and the strongest interaction with model membranes, followed by LFampin 265 - 280. LFampin 270 - 284 was inactive, showing marginal secondary structure and no interaction with the pathogen model membranes.
B3098 – E, Z and positional-monoenoic phyto-fatty acids influencing membrane fluidity: DSC and NMR experiments
The membrane fluidity of biological tissues is highly influenced by the ?-bond position and isomeric configuration in the long chain of phyto-fatty acids (FAs). Z, E and positional isomeric monoenoic lipids, i.e. the phytomolecules oleic (OA), elaidic (EA), vaccenic acid (TV) and its Z-isomer (CV), have been evaluated for their effects on the fluidity of cellular membranes. To this purpose the Differential Scanning Calorimetry (DSC) and Deuterium Nuclear Magnetic Resonance (2H-NMR), are suitable techniques to understand the supramolecular lamellar structure during the order (gel)-disorder (fluid) transition. It was found that the presence of CV concentration, induces the biomimetic system to reach the first step to fluid phase earlier than the membrane containing OA. DSC showed that the endothermic peak onset of the membrane containing CV occurs at a lower temperature than that of a membrane containing an equal amount of OA. 2H-NMR investigation confirmed the last statement. In fact the study of the main phase transition of the two different systems, revealed that model membrane containing a 3% (w/w) of CV goes in ripple phase, i.e. the first step to the fluid state, at a lower temperature as compared to the membrane of an identical system with OA.
B3075 – Microstructure study of liposomes decorated by hydrophobic magnetic nanoparticles
Magnetoliposomes, consisting of liposomes and magnetic nanoparticles (MNPs), have been tailored as very promising delivery vehicles in biotechnology and biomedicine applications. In this paper, liposomes with hydrophobic MNPs were prepared. The hydrophobic MNPs were successfully embedded in the lipid bilayer, which was proved by the results obtained from transmission electron microscope, atomic force microscope, differential scanning calorimetry and steady state fluorescence measurements. Moreover, systematic researches were carried out to investigate the effects of hydrophobic MNPs concentration on the morphology and microstructure of liposomes. The results show that the lipid bilayer was saturated with the hydrophobic MNPs when the mass ratio of MNPs to lipid reached 0.002.
B3074 – A study on the nature of intermolecular links in the cryotropic weak gels of hyaluronan
In this study, the influence of acidification and salting effect on the properties of hyaluronan (HA) aqueous solutions and cryotropic weak gels were investigated by dynamic rheometry, polarizing and optical microscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and FTIR spectroscopy. The forming mechanism of HA cryotropic weak gels was also discussed. Experimental results indicated that the HA weak gel showing a thermoreversible property was constructed by entangled bundle-like structures that could be melted at elevated temperature above 70 °C, and that the junction knots of three-dimensional polymeric network were not the ordinary microcrystalline zones that are generally of detectable crystallinity and thermal effect. The intermolecular hydrogen bonding induced from –COOH and –NHCOCH3 in HA chains played a predominant role in respect to the network formation and stabilization of HA weak gel.
B3071 – Yeast (1-3),(1-6)-d-glucan films: Preparation and characterization of some structural and physical properties
Cell wall polysaccharide suspensions (mainly ?-glucan) was isolated from baker's yeasts (Saccharomyces cerevisiae) and used for the preparation of films. Glycerol was added as a plasticizer. Purity and composition of the films were tested by elemental analysis, enzymatic assay of ?- and ?-glucans, monosaccharide composition analysis (total hydrolysis, HPAEC) and vibration spectroscopy (FTIR, FT Raman). Surface properties and the degree and type of crystallinity, together with ageing effects, were estimated by scanning electron microscopy (SEM), atomic force microscopy (AFM) and X-ray diffraction (XRD). Mechanical and thermal properties were characterized by tensile tests and difference scanning calorimetry (DSC), respectively. The prepared films were water insoluble, compact, non-porous, exhibit no pronounced crystallinity and consist of granular-like and fibre microstructures, which could be assigned as cell wall residues and released polysaccharide macromolecules. Certain structural changes in the film surface during one-year shelf storage can be related to reorientation and decomposition of surface macromolecules due to reaction with the ambient atmosphere, rather than to crystallization phenomena.
B3068 – Temperature-dependent stability and DPPH scavenging activity of liposomal curcumin at pH 7.0
This paper investigated the influences of temperature on the stability and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of curcumin encapsulated in liposome at pH 7.0. Liposomal curcumin showed higher stability and DPPH scavenging activity than free curcumin at 25 °C. When temperature increased from 25 to 80 °C, liposomal curcumin degraded more pronouncedly above the phase transition temperature (Tm = 45.7 °C) of liposome than lower temperatures, suggesting a weaker curcumin protection from the liquid crystalline phase of phospholipid bilayer than that from the gel phase. Moreover, the presence of remarkable "jump" increases around Tm in the values of observed pseudo-first-order rate constant and the percent of DPPH scavenging activity of liposomal curcumin indicated that the liquid crystalline phase of phospholipid bilayer is more beneficial for curcumin to reduce DPPH. This study reveals that changing the microstructure of encapsulation carrier may effectively control the properties of phytochemicals like curcumin.
B3066 – Synthesis and characterization of a novel fish scale-immobilized chitosan adsorbent—Preliminary features of dichlorophenol sorption by solution calorimetry
Brazilian Corvina fish scales were cross linked with polyglutaraldehyde and chemically modified with chitosan gel. Characterization has pointed that chitosan has good and stable adhesion on the fish scales. The sorption of dichlorophenol-2,6-indophenol (DPI) on the novel material was studied by isothermal solution calorimetry. The non-symmetric shapes of the calorimetric plots indicate that the DPI sorption sites of the adsorbent are not energetically uniform. The enthalpies of the DPI sorption processes were highly exothermic (from -536.7 to -50.9 kJ mol?1). The analysis of both the characterization of the materials and the calorimetric results has suggested that the interactions at the fish scales/DPI interface are due to surface reactions. The present work underlines the excellent features of the new fish scale-based adsorbent for use in phenol sorption applications at solid/solution interfaces.
B3037 – Effects of temperature and relative humidity on fibrillar collagen in parchment: A micro differential scanning calorimetry (micro DSC) study
Micro DSC measurements were used to investigate the synergistic effects of temperature and relative humidity on deterioration of parchment. Samples were obtained by exposing new parchments to various temperature and relative humidity atmospheres for increasing times in controlled test chambers. The impact of this accelerated ageing was assessed by measuring the thermodynamic parameters associated with the thermal denaturation of fibrillar collagen: variation of denaturation temperature, Tmax, indicated changes in collagen thermal stability, increases of DSC peak half-width, DT1/2, revealed greater thermal heterogeneity and structural disorder, whereas parallel decrease of denaturation enthalpy, DH, and DSC peak maximum height, Cp max, measured the loss of fibrillar structure. Deconvolution of DSC denaturation peaks provided a valuable illustration of both the dynamics and the pattern of the deterioration
of fibrillar collagen. Impact of temperature and relative humidity on parchment integrity and stability is discussed in terms of their implications for conservation science.
B2980 – Thermal and dynamic behaviour of the actin monomer in case of different cations
Actin is one of the important elements of the striated muscle that transmits force from the myosin filaments and as a part of the cytoskeleton plays an important role in shape determination of cells. It is a known experience that removal of the divalent cation affects the dynamic behaviour of actin in both forms. Paramagnetic probes and electron paramagnetic resonance (EPR) spectroscopy provide direct technique by which the rotation and the orientation of specifically labelled proteins can be followed during biochemical manipulations. The spectroscopic measurements could be combined with DSC measurements that report domain stability and interactions and allow the calculation of the thermodynamic parameters during the melting process. Actin was spin-labelled with maleimide or fluoro-dinitro proxyl probe molecules which are bound to the Cys-374 or Lys-61 residues of the smaller domain. EPR spectroscopy spectra were recorded in monomer form in Ca- and EGTA-state as a function of temperature up to the melting point. Similarly, DSC measurements were performed and analyzed using the kinetic
theory. The measurements showed that removal of the divalent cation from the globular actin induced significant local and global structural change both in the thermodynamic properties and the rotational mobility of actin detected by DSC and EPR. On the basis of the results derived by deconvolution of the DSC pattern we can suggest a non-interactive two-domain melting for the
monomer actin after removing the divalent cations.
B2972 – Examination of a coffee product enriched with calcium by the isoperibolic method
The main reason of osteoporosis, one of the most frequently occurring civilization disease nowadays, is the calcium-defficient and phosphorus-surplus nutrition, respectively. One of the possible preventions is to complete the foods with predominance of phosphorus with Ca at least till Ca:P ratio of 1:1. To complete foods with Ca today primarilly organic Casalts (e.g. Ca-malate, -lactate, -citrate) are used, their absoption has clinically been approved. These Ca-enrichers are food additives and have E-numbers. The raw material of a Ca-enricher has been developed by us is the Ca-rich whey of fermented fresh cheese being indigenous in the Carpathian basin, and which is further enriched in Ca (first of all in Ca-malate) during the technology. This product being under EU-patentization, contains the main adjuvants (milk protein, lactose, oligosaccharide, micro elements) promoting the Ca-absorption and incorporation in bones. It is an additive-food, its bioavailability has clinically been proved. Its further advantage is that owing to its colloidchemical stucture it can be instantized. In our experiments separately 20–20 mg from calcium-malate (CaM), fresh cheese whey powder enriched in Ca (QC), its instant version (QCi), instant coffee (DE), mixture of 50–50% instant coffee and instant fresh cheese whey
powder enriched in Ca (DEQCi) were measured into the bottom of a mixing batch vessel and 180 mg distilled water into the upper part of it (in the reference one 20 + 180 mg distilled water).
B2970 – AMP.PNP affects the dynamical properties of monomer and polymerized actin
Actin is the component of several biological systems and it plays important role in different biological processes, especially in cell motility. The actin-based motility is accompanied with ATP-consume, and the irreversible ATP hydrolysis is coupled with the polymerization of monomer actin into filamentous form. When an actin monomer is incorporated into a filament, the ATPase is activated, and thereby the polymer formation is promoted. The polymer formation and the ATP hydrolysis is associated with internal motions and significant changes of the conformation in reaction partners. In this article, the ATP nucleotide in monomer actin was exchanged by its non-hydrolyzable analogue adenylyl-imidodiphosphate (AMP.PNP), and using two biophysical methods, electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC), we studied the local and global changes in globular and fibrous actin following the nucleotide exchange. The paramagnetic probe molecule—a maleimide spin label—was attached to Cys-374 site of monomer actin, and its rotational mobility was derived at different temperature. In DSC measurements the transition temperatures of samples with different bound nucleotides were compared. From the measurements we could conclude, that the nucleotide exchange induces changes in the internal rigidity of the actin systems, AMP.PNP-actins showed longer rotational correlation time and increased thermal transition temperature.
B2961 – Exploring antibiotic resistant mechanism by microcalorimetry II. Determination of thermokinetic parameters of imipenem hydrolysis with metallo-?-lactamase ImiS
In an effort to understand the reaction of antibiotic hydrolysis with B2 metallo-?-lactamases (MbLs), the thermodynamic parameters of imipenem hydrolysis catalyzed by metallo-?-lactamase ImiS from Aeromonas veronii bv. sobria were determined by microcalorimetric method. The values of activation free energy ?G?‡ are 86.400 ± 0.043, 87.543 ± 0.034, 88.772 ± 0.024, and 89.845 ± 0.035 kJ mol-1 at 293.15, 298.15, 303.15, and 308.15 K, respectively, activation enthalpy ?H?‡ is 18.586 ± 0.009 kJ mol-1, activation entropy ?S?‡ is -231.34 ± 0.12 J mol-1 K-1, apparent activation energy E is 21.084 kJ mol-1, and the reaction order is 1.5. The thermodynamic parameters reveal that the imipenem hydrolysis catalyzed by metallo-?-lactammase ImiS is an exothermic and spontaneous reaction.
B2960 – Differential scanning calorimetry (DSC) analysis of human plasma in melanoma patients with or without regional lymph node metastases
Melanoma malignum (MM) is a common type of skin cancer, and its incidence is increasing in the general population. We aimed to detect blood plasma components with differential scanning calorimetry (DSC) in 15 white adult MM patients, who had histopathologically diagnosed, operable cutaneous MM without any distant metastases. We observed that thermal changes (second Tm, calorimetric enthalpy) in blood plasma showed correlation with tumor thickness and the extent of regional invasion. Further studies are needed to elucidate these relationships, but our preliminary work has provided DSC should be a new tool for the early diagnosis and monitoring of MM patients.
B2959 – Differential scanning calorimetric examination of the interfacial membrane in failed hip joint replacements
Aseptic and septic periprosthetic osteolysis following total hip arthroplasty has become increasingly recognized as a major clinical problem. An aggressive granulomatous tissue, the interfacial membrane, develops at the interface between the bone and the prostheses or the bone and the cement. Our hypothesis was that during the septic and aseptic loosening of the total hip arthroplasty, there is a clear pathological abnormality in the tissue elements building up the interfacial membrane, which is responsible for the different aetiologies of the disease and
could be monitored besides the classical methods by differential scanning calorimetry. In our study, the interfacial membrane pieces removed during operations of revision hip arthroplasties in the cases of aseptic loosening and during prosthesis removals in the cases of septic implant loosening. We investigated stem parts of cemented hip arthroplasties only. Our measurements were carried out on eight septic and 12 aseptic samples. With our investigations, we could demonstrate that DSC is a useful and well-applicable method for the investigation of the interfacial
membrane that develops in septic and aseptic loosening of hip arthroplasty. DSC scans clearly demonstrated significant differences between the different types and conditions of samples (aseptic membrane: Tm = 62.2 °C and ?Hcal = 2.13 J/g, septic membrane: Tm = 60.2 °C and ?Hcal = 3.22 J/g,). These investigations can help us make a correct diagnosis in the problematic cases of loosened total hip arthroplasty. To decide the possible septic feature of a given sample, calorimetry could serve as one of the quickest procedures available.
B2957 – Differential scanning calorimetric examination of the human hyaline cartilage of the femoral head after femoral neck fracture
The femoral neck fracture continues to be unsolved fractures and the guidelines for management are still evolving. The primary complications arising from femoral neck fractures are non-union and avascular necrosis. The various methods currently available for predicting the vascularity of the head at the time of fracture are not sufficiently quantitative to be used on a routine clinical basis. The hypothesis was that after the femoral neck fracture there are clear pathological abnormalities in the cartilage of the femoral head, which could be monitored besides the classical methods by differential scanning calorimetry. The thermal denaturation of human samples was monitored by a SETARAM Micro DSC-II calorimeter. All the experiments were performed between 0 and 100 °C. The heating rate was 0.3 K/min. DSC scans clearly demonstrated significant differences between the control and different stages avascular samples (control, fresh fractures: Tm = 68.2 °C, ?Hcal = 2.87 J/g, avascular necrosis: Tm = 70.7 °C, ?Hcal = 3.61 J/g,). These observations could be explained with the structural alterations caused by the
biochemical processes during the degeneration of the cartilage due to avascular femoral head necrosis. With the investigations the authors could demonstrate that DSC is a useful and well-applicable method for the investigation of hyaline cartilage of the fractured human femoral head. It was confirmed significances between the changes of calorimetric results and the elapsed time from the primary femoral neck fracture.
B2925 – Thermal unfolding of monomeric and dimeric ?-lactoglobulins
The thermal stabilities of dimeric bovine ?-lactoglobulin and monomeric equine ?-lactoglobulin were investigated at neutral pH by means of differential scanning calorimetry, circular dichroism, tryptophan fluorescence, and by binding of an hydrophobic probe. Differential scanning calorimetry showed the presence of two structural domains with different thermal stabilities in both proteins. Thermodynamic analysis of the calorimetric signal revealed that the two domains unfold independently according to a mechanism where an equilibrium step is followed by an irreversible transition. The spectroscopic data supported this model and allowed recognition of the structural regions corresponding to the more thermally stable domain. The differences in
thermal stability between the two proteins can be primarily ascribed to the properties of the less stable domain
B2924 – Glassy Behavior of Denatured DNA Films Studied by Differential Scanning Calorimetry
We use differential scanning calorimetry (DSC) to study the properties of DNA films, made of oriented fibers, heated above the thermal denaturation temperature of the double helical form. The films show glassy properties that we investigate in two series of experiments, a slow cooling at different rates followed by a DSC scan upon heating and aging at a temperature below the glass transition. Introducing the fictive temperature to characterize the glass allows us to derive quantitative information on the relaxations of the DNA films, in particular to evaluate their enthalpy barrier. A comparison with similar aging studies on PVAc highlights some specificities of the DNA samples
B2910 – Structural and biomechanical characterizations of porcine myocardial extracellular matrix
Extracellular matrix (ECM) of myocardium plays an important role to maintain a multilayered helical architecture of cardiomyocytes. In this study, we have characterized the structural and biomechanical properties of porcine myocardial ECM. Fresh myocardium were decellularized in a rotating bioreactor using 0.1 % sodium dodecyl sulfate solution. Masson’s trichrome staining and SEM demonstrated the removal of cells and preservation of the interconnected 3D cardiomyocyte lacunae. Movat’s pentachrome staining showed the preservation of cardiac elastin ultrastructure and vascular elastin distribution/alignment. DNA assay result confirmed a 98.59 % reduction in DNA content; the acellular myocardial scaffolds were found completely lack of staining for the porcine a-Gal antigen; and the accelerating enzymatic degradation assessment showed a constant degradation rate. Tensile and shear properties of the acellular myocardial scaffolds were also evaluated. Our observations showed that the acellular myocardial ECM possessed important traits of biodegradable scaffolds, indicating the potentials in cardiac regen-
eration and whole heart tissue engineering.
B2840 – Catalytic Carbon Monoxide Oxidation Using Bio-Templated Platinum Clusters
The catalytic properties of highly dispersed, bacterial surface layer supported nanoscale platinum clusters immobilized at alumina particles are studied with respect to carbon monoxide oxidation. Compared to samples prepared from platinum impregnated alumina, the templated metal clusters are catalytically active at lower temperatures. The catalytic behaviour of the samples is discussed with respect to their cluster morphology studied by TEM.
B2835 – Differential scanning calorimetry study of glycerinated rabbit psoas muscle fibres in intermediate state of ATP hydrolysis
Thermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins (actin and myosin) in their native environment in striated muscle fibres. The interaction of actin with myosin in the highly organized muscle structure is affected by internal forces; therefore their altered conformation and interaction may differ from those obtained in solution. The energetics of long functioning intermediate states of ATP hydrolysis cycle was studied in muscle fibres by differential scanning calorimetry (DSC).
B2820 – Apparent Molar Heat Capacities and Apparent Molar Volumes of Aqueous 1,1,1,3,3,3-Hexafluoroisopropanol at Different Temperatures
Specific heats and apparent molar heat capacities of aqueous 1,1,1,3,3,3-hexafluoroisoproanol (HFIP) have been determined at temperatures from 20.0 to 45.0°C using micro differential scanning calorimetry in the molality range of 0.06741 to 1.24053 mol-kg?1. Densities and apparent molar volumes have also been determined for aqueous HFIP at temperatures from 10.3 to 30.0°C using digital densimetry in the molality range of 0.04009 to 0.67427 mol-kg?1. The results of these measurements have been used to calculate the following partial molar quantities and temperature derivatives for aqueous HFIP as a function of temperature. The contribution of the ----F atom to the partial molar heat capacity and volume has been calculated. The results have been explained in terms of structural changes in water in aqueous HFIP solution. The results obtained in this work contain essential information needed for the development of an equation of state for this system, when used in combination with other thermodynamic properties of aqueous HFIP.
B2807 – Partial Molar Volumes of Amino Acids and Peptides in Aqueous Salt Solutions at 25?C and a Correlation with Stability of Proteins in the Presence of Salts
Partial molar volumes for a homologous series of amino acids and peptides have been measured in aqueous 1M sodium acetate, sodium thiocyanate, and sodium sulfate at 25°C. These data have been utilized in conjunction with the data in water to deduce partial molar volumes of transfer V 0 2,m(tr) from water to these aqueous salt solutions. The volumes of transfer for the amino acids and peptides are found to be positive. The interpretation is that this result arises from the dominant interaction of the sodium salts with the charged centers of amino acids and peptides. Thermal denaturation of the structurally homologous proteins lysozyme and ?-lactalbumin has been studied in the presence of these salts. Significant thermal stabilization of hen egg-white lysozyme has been observed in the presence of sodium acetate and sodium sulfate. However, the thermal stabilization observed for ?-lactalbumin is very small in the presence of
these salts and sodium thiocyanate leads to a lowering of its thermal denaturation temperature. The rise in the surface tension of aqueous salt solutions with salt concentration has
been correlated with the calorimetric and volumetric measurements. The results show that V 0 2,m(tr) depends less on the type of electrolyte than on the ionic strength of the solution. The V 0 2,m(tr) values correlate very well with the increase in the surface tension of aqueous salt solutions, indicating significant role of surface tension in interactions of amino acids, peptides, or protein with the salts.
B2806 – Apparent Molar Heat Capacities and Apparent Molar Volumes of Aqueous Nicotinamide at Different Temperatures
Specific heat capacities and apparent molar heat capacities of aqueous nicotinamide have been determined from 25.0 to 55.0°C using microdifferential scanning calorimetry in the molality range of 0.07433 to 1.50124 mol-kg?1. Densities and apparent molar volumes have also been determined for aqueous nicotinamide from 10.30 to 34.98°C using a digital densimeter in the molality range 0.07804–2.02435 mol-kg?1. The results of these measurements have been used to calculate the following partial molar quantities and temperature derivatives for aqueous nicotinamide as a function of temperature. The results are discussed in terms of the changes in the packing of nicotinamide molecules in the crystal, interactions in the aqueous form, and its structurepromoting ability with rise in temperature.
B2802 – Sequential flow injection analysis of complex systems using calorimetric detection
The application of the calorimetric detection seems to be an interesting possibility for the determination of single substrate components in mixtures or for the identification of substrate systems. The flow injection technique is used for a sequential initialisation of different enzyme catalysed reactions. The method is developed by use of a conventional flow-through
calorimeter and demonstrated for a ternary system containing glucose, urea and penicillin G. More effective working conditions are realised by application of our IC calorimeters. First results from investigations with a flow-through IC calorimeter are communicated
B2800 – Temperature Dependent Protease Activity and Structural Properties of Human HtrA2 Protease
Human HtrA2 belongs to a new class of oligomeric serine protease, members of which are found in most organisms. Mature HtrA2 is released from mitochondria into the cytosol in response to apoptotic stimuli. In this report, the effect of temperature on proteolytic activity of HtrA2 and related structural properties were investigated. In the range from 25 to 55°C, the proteolytic activity of HtrA2 rapidly increased with temperature, and it drastically decreased at and over 60°C. Structural analysis using far-UV CD spectroscopy and gel filtration revealed no significant change in the secondary structure of HtrA2 from 25 to 70°C, or in the oligomeric size between 25 and 55°C. However, a significant change at the tertiary level, as examined using near-UV CD, was observed for HtrA2 in the range from 25 to 60°C. Differential scanning calorimetry indicated that HtrA2 exhibits a thermal transition beginning at around 61°C. The fluorescence intensity of ANS interacting with HtrA2 decreased with increasing temperature. HtrA2 was found to be able to complement DegP function at 44°C, indicating that HtrA2 could have protective functions in mitochondria.
B2799 – Thermokinetic characterization of imipenem hydrolysis with metallo-lactamase CcrA from Bacteroides fragilis
In an effort to study on the antibiotic resistance in bacteria, we first report the thermokinetic parameters of the imipenem hydrolysis with B1 subclasses metallo-?-lactamase CcrA from Bacteroides fragilis. The values of activation free energy ?G?# is 87.54 ± 0.03, 88.78 ± 0.03, 89.90 ± 0.04 and 91.06 ± 0.02 kJ mol?1 at 293.15, 298.15, 303.15 and 308.15 K, respectively, the activation enthalpy ?G?# is 19.10 ± 0.01 kJ mol?1, the activation entropy ?S?# is ?233.56 ± 0.09 J mol?1 K?1 and the apparent activation energy E is 21.598 kJ mol?1. The results reveal that the imipenem hydrolysis catalyzed by CcrA is an exothermic and spontaneous reaction with reaction order of 1.4, and has a lower ?G?# and ?S?# than the cefazolin hydrolysis with same enzyme.
B2795 – A Study on the Stability and Enzymatic Activity of Yeast Alcohol Dehydrogenase in Presence of the Self-Assembling Block Copolymer Poloxamer 407
Yeast alcohol dehydrogenase (ADH) is an enzyme widely studied for biotechnological applications due to its involvement in fermentation industry, and various attempts to improve its catalytic properties and its thermal stability have been carried out. In this paper, the influence of a block copolymer (Poloxamer 407) on ADH enzymatic activity and thermal behaviour has been studied in order to get new insights about the use of poloxamers in formulation of sustained release systems for therapeutic proteins. Poloxamer 407 has the ability to form micelles and gel due to its self-assembling and thermoresponsive properties. The effect of the copolymer towards thermal stress and pH changes, which often reduce enzymes activity it has been investigated by means of enzymatic assays and differential scanning calorimetry. Results showed that at pH 9.1 and 7.3, the Poloxamer in the form of unimeric, micellar and gel state is able to effectively preserve the enzyme from thermoinactivation. In addition by calorimetric data Poloxamer 407 has showed an effect in preserving ADH from aggregation at pH 7.3. In conclusion, Poloxamer 407 seems to be very effective in protecting ADH from stress related events, like alkaline inactivation and aggregation.
B2794 – The effects of polyols on the thermal stability of calf thymus DNA
The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, L(?) and D(+) arabitol, D-mannitol, D-sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the
concentration of these additives a decrease in both the denaturation enthalpy (?dH) and temperature of the maximum of the denaturation peak (Tmax) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric
alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of L(?) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for L(?) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of L(?) arabitol indicates
a conformational change of polynucleotide towards a more extended conformation.
B2793 – Characterisation and evaluation of the environmental impact on historical parchments by differential scanning calorimetry
Our recent developments concerning the assessment of parchments deterioration using DSC are reported. Measurements performed on samples in excess water conditions, in static air and gas flow provided qualitative and quantitative information on parchment ageing and deterioration at microscopic and mesoscopic level, when assembly of fibres/fibrils is weakened, partially and eventually completely lost, and at molecular level, when triple helix uncoiling occurs. A damage ranking scale based on a large collection of DSCparameters obtained by investigating
artificially aged samples was set up. Deconvolution of the DSC thermal denaturation peaks in excess water enabled evaluating and discriminating stability of parchments with similar damage levels. Further experimental evidences such as softening of the crystalline fraction of collagen, thermaloxidation and collagen gelatinisation were detected by DSC measurements in gas flow and static air, and related to specific deterioration patterns. DSC measurement of wet samples provided an objective and reliable method for evaluating parchment shrinkage temperature overcoming the limitations of conventional methods.
B2792 – Role of the N-terminal region for the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius
In order to clarify the role played by the N-terminal region for the conformational stability of the thermophilic esterase 2 (EST2) from Alicyclobacillus acidocaldarius, two mutant forms have been investigated: a variant obtained by deleting the first 35 residues at the N-terminus (EST2-36del), and a variant obtained by mutating Lys102 to Gln (K102Q) to perturb the N-terminus by destroying the salt bridge E43-K102. The temperature- and denaturant-induced unfolding of EST2 and the two mutant forms have been studied by means of circular dichroism (CD), differential scanning calorimetry (DSC) and fluorescence measurements. In line with its thermophilic origin, the denaturation temperature of EST2 is high: Td=91 °C and 86 °C if detected by recording the CD signal at 222 nm and 290 nm, respectively. This difference suggests that the thermal denaturation process, even though reversible, is more complex than a two-state N?D transition. The non-two-state behaviour is more pronounced in the case of the two mutant forms. The complex DSC profiles of EST2 and both mutant forms have been analysed by means of a
deconvolution procedure. The thermodynamic parameters characterizing the two transitions obtained in the case of EST2 are: Td,1=81 °C, ?dH1=440 kJ mol?1, ?dCp,1=7 kJ K?1mol?1, Td,2=86 °C, ?dH2=710 kJ mol?1, and ?dCp,2=9 kJ K?1mol?1. The first transition occurs at lower temperatures in the two mutant forms, whereas the second transition is always centred at 86 °C. The results indicate that EST2 possesses two structural domains whose coupling is tight in the wild-type protein, but markedly weakens in the two mutant forms as a consequence of the perturbations in the N-terminal region.
B2791 – On the stability of the ripple phase in the DPPC/PLPC/water ternary system
The effect of incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine (PLPC) on the structure of the Pb% ripple mesophase in aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) has been studied by differential scanning calorimetry (DSC) and scanning dilatometry (SD). For samples containing 34 wt. % 2H2O and 0–15 wt. % PLPC, a pretransition was observed by DSC. The pretransition disappears at 15 wt. % PLPC. The behavior of thermodynamic functions at the pretransition and main transition gives new insights on the structural changes produced by PLPC on bilayers of DPPC.
B2789 – Differential scanning calorimetry of chromatin at different levels of condensation
The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 ?C, while the other two occur at 63 ?C and 74 ?C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone
complexes is nearly the same as that of DNA in solution; (b) the transition at 63 ?C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.
B2788 – Thermodynamic analysis of the contributions of the copper ion and the disulfide bridge to azurin stability: synergism among multiple depletions
The stabilizing potential of the copper ion and the disulfide bridge in azurin has been explored with the aim of inspecting the ways in which these two factors influence one another. Specifically, whether copper and disulfide contributions to protein stability are additive has been examined. To this aim, the thermal unfolding of a copper-depleted mutant lacking the disulfide bridge between Cys3 and Cys26 (apo C3A/C26A azurin) was studied by differential scanning calorimetry. A comparison of the unfolding parameters of holo and apo C3A/C26A azurin with the apo C3A/C26A protein has shown that the effects of simultaneous copper and disulfide depletion are additive only at two temperatures: T ¼ 15 °C and T ¼ 67 °C. Within this range the presence of the copper ion and the disulfide bridge has a positive synergistic effect on azurin stability. These findings might have implications for the rational use of the stabilizing potential of copper and disulfides in copper protein engineering.
B2787 – Polymersomes Containing Iron Sulfide (FeS) as Primordial Cell Model
According to Wächtershäuser’s “Iron-Sulfur-World” one major requirement for the development of life on the prebiotic Earth is compartmentalization. Vesicles spontaneously formed from amphiphilic components containing a specific set of molecules including sulfide minerals may have lead to the first autotrophic prebiotic units. The iron sulfide minerals may have been formed by geological conversions in the environment of deep-sea volcanos (black smokers), which can be observed even today. Wächtershäuser postulated the evolution of chemical pathways as fundamentals of the origin of life on earth. In contrast to the classical Miller-Urey experiment, depending on external energy sources, the “Iron-Sulfur-World” is based on the catalytic and energy reproducing redox system FeS + H2S ? FeS2 + H2. The energy release out of this redox reaction (?rG°=?38 kJ/mol, pH 0) could be the cause for the subsequent synthesis of complex organic molecules and the precondition for the development of more complex units similar to cells known today. Here we show the possibility for precipitating iron sulfide inside vesicles composed of amphiphilic block-copolymers as a model system for a first prebiotic unit. Our findings could be an indication for a chemoautotrophic FeS based origin of life.
B2695 – Thermodynamic investigation of effect of salt concentrations on denatured ?-Amylase adsorbed onto a moderately hydrophobic surface
The displacement adsorption enthalpies (?H) of denatured ?-Amylase (by 1.8 mol L-1 GuHCl) adsorbed onto a moderately hydrophobic surface (PEG-600, the endgroup of polyethylene glycol) from solutions (x mol L-1(NH4)2SO4, 0.05 mol L-1 KH2PO4, pH 7.0) at 298 K are determined by microcalorimeter. Further, entropies (?S), Gibbs free energies (?G) and the fractions of ?H, ?S, and ?G for net adsorption of protein and net desorption of water are calculated in combination with adsorption isotherms of ?-Amylase based on the stoichiometric displacement theory for adsorption (SDT-A) and its thermodynamics. It is found that the displacement adsorptions of denatured a-Amylase onto PEG-600 surface are exothermic and enthalpy driven processes, and the processes of protein adsorption are accompanied with the hydration by which hydrogen bond form between the adsorbed protein molecules favor formation of ?-sheet and ?-turn structures. The Fourier transformation infrared spectroscopy (FTIR) analysis shows that the contents of ordered secondary structures of adsorbed
?-Amylase increase with surface coverages and salt concentrations increment.
B2692 – Differential scanning calorimetric examination of ruptured lower limb tendons in human
The tendon ruptures are serious injuries of the lover limb in middle age and physically active population. While the Achilles tendon rupture is common, the patellar ligament and quadriceps ligament ruptures are an absolutely rare injury. Usually there is no correlation between the velocity of the trauma and the supervening of the rupture. The aetiology of the degenerative changes in the collagen structures of the tendons and ligaments which could be disposed for the rupture are still not clear. Our hypothesis was that before the injury there are clear pathological abnormalities in the tissues of the tendons, which are predisposed for the rupture, and could be monitored besides the classical histological methods by differential
scanning calorimetry. The thermal denaturation of human samples was monitored by a SETARAM Micro DSC-II calorimeter. All the experiments were performed between 0 and 100 C. The heating rate was 0.3 K/min. DSC scans clearly demonstrated significant differences between the control and ruptured samples (control: Tm = 59.7 C, T1/2 = 1.4 C and ?Hcal = 8.54 J/g; ruptured Achilles tendon: Tm = 62.75 C, T1/2 = 2.6 C and ?Hcal = 1.54 J/g, ruptured Quadriceps tendon: Tm = 64.8 C, T1/2 = 1.6 C and ?Hcal = 1.53 J/g, ruptured Patellar tendon: Tm = 63.9 C, T1/2 = 1.41 C and ?Hcal = 0.97 J/g). These observations could be explained with the structural alterations caused by the biochemical processes. With our
investigations we could demonstrate that DSC is a useful and well applicable method for the investigation of collagen tissue of the degenerated human tendons and ligaments.
We can prove with this method that the degenerative changes of the tissue elements increase the thermal stability of collagen tissues of the tendons which could be disposed for the rupture.
B2691 – Inter-monomer cross-linking affects the thermal transitions in F-actin
Chemical cross-links which covalently connected the Cys-374 and Glu-41 residues of adjacent monomers in the same strand of F-actin were used to follow the consequences of the modification for the motional and structural properties of the actin filaments. DSC measurements reported that the inter-monomer cross-links shifted the thermal transition temperature and affected strongly the cooperativity of the transition in comparison with uncrosslinked F-actin. Addition of HMM to F-actin induced significant decrease of the transition temperature to lower value from 69.4 to 67. 5 C.
B2616 – Calorimetric investigation of rat myometrium as a function of gestational age
The aim of the present work was the characterization of the myometrial samples of healthy control and chronic inflammation-exposed rats as a function of gestational stage by means of differential scanning calorimetry (DSC). Non-pregnant, mid-pregnant (day 14) and late-pregnant (day 21) animals were included. The spontaneous, KCl-stimulated as well as maximal motor activity was additionally recorded in vitro. Calorimetric enthalpy (?H) normalised on wet mass significantly increased as pregnancy progressed in case of non-inflamed uteri. Inflammation resulted in a significant decrease in ?H/m of samples from non-pregnant till pregnant rats. Maximal contractility increased by inflammation in all tested stages of gestation. Our present results indicate that DSC can be considered as a useful tool for the investigation of the uterine function modified by gestation or a pathological condition.
B2615 – DSC, as a new method to verify the exact warm and cold ischemic injury during small bowel surgery
The fact that small bowel is extremely sensitive to ischemia/reperfusion injury had encouraged us to compare the influences of warm and cold ischemia on the intestinal structural changes by differential scanning calorimetry (DSC) method. Warm and cold ischemia groups were established on Wistar rats with 1, 3 and 6 h ischemic times. Intestinal biopsies were collected after laparotomy and at the end of the ischemia periods. DSC measurement was performed on mucosa, on muscular layer and on the total intestinal wall. Our DSC data confirmed that longer warm ischemia period caused more severe damage in the structure of mucosa and muscular layers. According to the results of transition temperature and calorimetric enthalpy suggest that these changes reduced by cold ischemic procedure in University of Wisconsin solution. However, the thermal destruction of each layers following cold preservation injury revealed significant differences compared to normal bowel structure.
B2614 – DSC analysis of human plasma in breast cancer patients
Breast cancer is the commonest cause of cancer death in women worldwide, and its incidence has been increasing for many years in economically developed countries. Differential scanning calorimetry (DSC) is more and more often applied for oncological purposes, as it could be used as a new method for diagnose and monitoring tumours. In current research liquid plasma components was detected with DSC in breast cancer patients. The study included 19 women with different tumour diameter (0.5–75 mm) and with or without regional lymph node metastases (0–10 metastatic lymph node). Preoperatively peripheral blood samples were collected from the patients and from healthy controls, and plasma components were analysed by DSC technique. The diameter of the tumour tissue and the number of metastatic lymph nodes were evaluated on the basis of postoperative histological results. In our preliminary study we observed thermic changes (e.g. Tm, calorimetric enthalpy) in the blood plasma of breast cancer patients, and we have found correlation with tumour size and with the degree of regional lymph node invasion as well. Further studies are needed to clarify these relationships, but our application of the DSC method has provided a potential tool for the early diagnosis and monitoring of breast cancer patients.
B2613 – DSC examination of kidney tissue following warm ischemia and reperfusion injury
The fact that kidney is sensitive to ischemia/reperfusion (I/R) injury had encouraged us to compare the conventional histology and differential scanning calorimetry (DSC) methods in renal structural changes following experimental warm I/R models. Our histological findings showed that longer warm I/R period caused more severe damage in structure of kidney. The thermal parameters (transition temperature, calorimetric enthalpy) of I/R injury reflected the histological results. These processes correlated with the ischemia time and the degree of injury was more serious after reperfusion periods compared to normal kidney structure, which provides basis for further investigation in different kidney stress models.
B2612 – DSC examination of intestinal tissue following cold preservation
The fact that small bowel is extremely sensitive to cold preservation had encouraged us to compare the conventional histology and differential scanning calorimetry (DSC) methods in intestinal structural changes following experimental cold storage models. Our histological findings showed that longer cold preservation period caused more severe damage in structure of mucosa and crypts, but there were no changes in the muscular layer. According to our DSC data (transition temperature, calorimetric enthalpy) suggest that the thermal destruction of mucosa, muscular layer and total intestinal wall following preservation injury revealed significant differences compared to normal bowel structure
B2611 – Whey protein isolate–chitosan interactions: A calorimetric and spectroscopy study
Isothermal titration calorimetry (ITC) measurements were performed using solutions of whey protein isolate (WPI) and chitosan with different deacetylation degrees (DD), in acetate buffer solutions, pH 3–6. Turbidity measurements were performed in parallel in order to follow the changes in aggregation, so as to get deeper insight on the interaction mechanism. The viscosity–average molar mass of chitosan was obtained from intrinsic viscosity measurements, and the interaction enthalpies were derived at the studied pH values. Further, the denaturation process of ?-lactalbumin and ?-lactoglobulin within WPI was characterized by differential scanning calorimetry (DSC). At pH 3, where both chitosan and the proteins are positively charged, a weak carbohydrate–protein interaction is observed. When the pH is raised to 6, where the protein charge is expected to be negative, a much stronger interaction takes place. The results are discussed with special emphasis on the effect of pH on the interactions observed in this complex system.
B2610 – Improvement in physicochemical parameters of DPPC liposomes and increase in
skin permeation of aciclovir and minoxidil by the addition of cationic polymers
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes were prepared by high-pressure homogeniser and coated with two cationic polymers, chitosan (CS) and for the first time Eudragit EPO (EU), respectively. Compared to the control liposomes, the polymeric liposomes showed greater physico-chemical stability in terms of mean particle size and zeta potential at room temperature. In the present study, aciclovir and minoxidil have been used as hydrophilic and hydrophobic candidates. In the presence of the drugs, the polymeric liposomes still showed constant particle size and zeta potential. Influences of polymers and model drugs on thermotropic phase transition of DPPC liposomes were studied by micro-differential scanning calorimetry (microDSC). The influences on configuration of DPPC liposomes were investigated by Fourier transform infrared spectroscopy (FTIR). According to DSC results, cationic poly-
mers had a stabilising effect, whereas aciclovir and minoxidil changed the physical properties of the DPPC bilayers by influencing the main phase transition temperature and erasing the pre-transition. The investigation of C@O stretching bands of DPPC at 1736 cmÿ1 in FTIR spectra showed that aciclovir has strong hydrogen bonding with C@O groups of DPPC, whereas carbonyl groups were free in minoxidil presence. Moreover, the coating of liposomes with CS or EU led to higher skin diffusion for both drugs. This could be explained as an effect of positively charged liposomes to interact stronger with skin negatively charged surface and their possible interactions with structures below the stratum corneum.
Calorimetric and spectroscopic studies on the interaction of Calf thymus DNA with divalent metal ions have been carried out in order to investigate how different ionic strengths as well as different divalent cations affect the thermal stability of the DNA double helix. The thermodynamic parameters of thermal denaturation of ds-DNA have been determined from solutions containing Mn2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+ ions in different concentrations. The results obtained indicated that the nature of the interactions of the metal ions with the DNA molecule depends on metals ion concentration. At low metal concentrations there are no significant changes in the melting temperature value. However at high metal concentrations a decrease in melting temperature was observed, showing that the presence of high divalent cation concentration decreases the double helix stability. Further, it is also shown that the cations tested have significantly different interactions with DNA, even at the same concentration. This reveals clearly that not only the ionic strength is important in DNA stability, but that the changes observed in stability and thermal profile depend largely on the metal used.
B2605 – Amphiphilic and thermosensitive copolymers based on pullulan and Jeffamine®:Synthesis, characterization and physicochemical properties
The synthesis of thermosensitive copolymers based on pullulan and polyether amine was performed in water using a watersoluble carbodiimide and Nhydroxysuccinimide as activators. Jeffamine® M2005 was chosen as a polyether to impart thermosensitive character to the copolymer. Pullulan was modified into carboxymethylpullulan, to bring carboxylate groups to the polysaccharide so as to further the grafting reaction. The copolymers were characterized by FTIR, 1H NMR spectroscopy and molecular weights measurements (by SEC coupled with MALS/DRI/Viscometer lines). The thermosensitive behaviour of CMPgM2005 copolymers was studied by fluorescence spectroscopy of pyrene, by rheometry and microDSC measurements. The sol–gel transition temperature was found dependent on the solvent, the grafting degree of M2005 and the concentration of the copolymer. For example it was 35 ?C
in water, 28 ?C in acid buffer (0.1 M, pH 5.4) and 26 ?C in saline phosphate buffer (0.15 M, pH 7.4) for a grafting degree of 0.20 at a concentration of 5 wt%.
B2600 – Using heat conduction microcalorimetry to study thermal aggregation kinetics of proteins
The thermally induced irreversible aggregation of a monoclonal antibody in different pH buffers was investigated using different techniques such as micro-differential scanning calorimetry (micro-DSC), size exclusion HPLC (SEC) and isothermal microcalorimetry. The kinetics of aggregation of the protein was analyzed in terms of a Lumry–Eyring model proceeding via a non-native conformational state. The rate constants and reaction enthalpies of unfolding and consequent aggregation were obtained by fitting the isothermal microcalorimetric and SEC data based on proposed aggregation mechanisms. The consistency of rate constants obtained via isothermal microcalorimetry and SEC indicates it is possible to deconvolute the observed microcalorimetry power–time data obtained from thermally induced protein aggregation.
B2569 – A DSC study of deterioration caused by environmental chemical pollutants to parchment, a collagen-based material
A DSC study of new parchments exposed at 25 °C for 1–16 weeks to controlled atmospheres containing 50 ppm of gaseous chemical pollutants (NO2, SO2, NO2 + SO2) and 50% relative humidity (RH) was performed. Samples were exposed to chemical pollutants alone, as well as after previous heating at 100 °C for 2–16 days and/or irradiating with visible light (1.7 × 105 lx) for 4–16 h. DSC measurements were performed in both sealed crucibles in static air atmosphere at 25–200 °C and open crucibles under gas flow (nitrogen, oxygen, synthetic air) at 25–280 °C. Analysis of DSC curves provided the variation induced by ageing on the thermodynamic parameters associated with both parchment denaturation and softening of collagen crystalline fraction. All the ageing procedures decreased both temperature and enthalpy of denaturation and increased broadness of DSC peak in function of ageing time. The occurrence of thermal oxidation peaks and/or lower temperature endothermic peaks was observed. The temperature of the first softening peak always indicated a general tendency to decrease as a function of ageing time. Shrinkage temperature of collagen fibres measured by thermomicroscopy also decreased as a result of accelerated ageing treatments.
B2552 – Hydration of ?-chymotrypsin: Excess partial enthalpies of water and enzyme
A novel method has been developed for studying simultaneously the excess partial enthalpies of water and the enzyme in the entire range of water content. Bovine pancreatic ?-chymotrypsin was used as a model enzyme. The proposed method includes the measurements of the enthalpies of solution of the dried and hydrated enzyme in water at 25 °C. From these thermochemical data the excess partial enthalpies of water and ?-chymotrypsin were calculated. The partial quantities are very sensitive to the changes in the state of water and ?-chymotrypsin. A transition from the glassy to the flexible state of ?-chymotrypsin is accompanied by significant changes in the excess partial enthalpies of water and ?-chymotrypsin. This transition appears at water weight fraction (w1) of 0.06 when charged groups of ?-chymotrypsin are covered. Excess partial quantities reach their fully hydrated values at w1 > 0.4 when coverage of both polar and weakly interacting surface elements is complete.
B2543 – Thermodynamics of azurin folding. The role of copper ion
The role played by the metal ion in thermodynamics of azurin folding was addressed by studying the thermal denaturation of the apo-form by differential scanning calorimetry (DSC), and by comparing the results with data concerning the holo protein. The thermal unfolding experiments showed that at 25°C the presence of metal ion increases the thermodynamic stability of azurin by 24 kJ mol–1. A comparison between the unfolding and the copper binding free energies allow us to assert that the unfolded polypeptide chain binds copper and subsequently folds into native holo azurin, being this the thermodynamically most favourable process in driving azurin folding.
B2533 – Calorimetric and EPR studies of the thermotropic phase behavior of phospholipid membranes
Transmission electron micrographs (TEM) showed that liposome vesicles prepared from DL-a-phosphatidylcholine dimyristoyl (1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine) (DMPC) by the modified reverse-phase evaporation method (mREV) were spherical in shape and in majority of them were less than 100 nm in diameter. Differential scanning calorimetry (DSC) method was used to determine the influence of cholesterol content and pH of Tris-HCl buffer used for the preparation of liposomes on the temperature of phase transition TC of phospholipids which form the investigated liposome vesicles. The use of DSC method made it possible to determine not only the temperature of the main phase transition of phospholipids but also the temperature of the phospholipid phase transition from the tilted gel phase (L ) b’ to the ripple gel phase (P ). b’ The results were compared with those obtained with EPR study. EPR study was carried out in the temperature range from 284 to 310 K i.e. below and above the phase transition temperature TC of DMPC. On the basis of EPR spectra of spin marker 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) incorporated into the liposome, the values of parameters f were determined. Hence TEMPO can be used to observe the change in partition between aqueous and fluid
lipid regions. The change in the relative values of f determined for DMPC as a function of temperature shows that this phospholipid undergoes a transition from a ‘gel phase’ to a lamellar smectic liquid crystalline phase in the presence of excess water. The EPR study of TEMPO allowed us to determine the transition temperature TC. The results were compared with those obtained with DSC method.
B2532 – Thermal and FTIR investigation of freeze-dried protein-excipient mixtures
The effect of excipients on the secondary structure of lyophilized proteins was studied through second-derivative Fourier transform infrared (FTIR) spectroscopic analysis. The glass transition temperature (Tg), denaturation temperature (Td) and moisture content were determined by differential scanning calorimetry (DSC) and thermogravimetry (TG). Tg, Td and the preservation of protein secondary structure were found to be dependent upon the type and amount of the excipient included in the formulation. Meanwhile, the lyophilized proteins easily adsorbed amounts of moisture during storage to reduce their Tgs and stability.
B2531 – A convenient tool for studying the stability of proteins and nucleic acids. Differential scanning calorimetry
The aim of this work is to discuss the thermodynamic properties, obtained by differential scanning calorimetry (DSC), of the thermal transition of proteins and nucleic acids and to analyze these data using statistical thermodynamic relations. The denaturation of the ordered, specific structures of biological macromolecules is a cooperative process and in many cases the macromolecules undergo a two-state transition. Differential scanning calorimetry, giving direct thermodynamic information, has proved to be very useful in clarifying the energetics of macromolecule transitions and in characterizing their thermal stability. Here, various examples are discussed: i) the equilibrium thermal denaturation of ribonuclease A, a model for the use of DSC by following the temperature-unfolding of the proteins, a monomolecular transition; ii) the equilibrium thermal dissociation of a DNA double helix in two strands, an example of how DSC is used to follow a bimolecular process; iii) an example of the use of DSC for studying the melting of unimolecular and tetramolecular DNA quadruple-helices.
B2530 – Fractions of thermodynamic functions for native lysozyme adsorption onto moderately hydrophobic surface
Calorimetric measurement of adsorption enthalpies of native lysozyme(Lyz) on a moderately hydrophobic surface at 25°C, pH 7.0 and various salt concentrations was performed. Based on the thermodynamics of stoichiometric displacement theory (SDT), we calculated the fractions of thermodynamic functions involving four subprocesses during a displacement adsorption process fromthe directly determined enthalpies in combination with adsorption isotherm measurements. The thermodynamic fractions reveal the relative degree of the four subprocesses for contributions to enthalpy, entropy and free energy. The results show that native Lyz adsorption on a moderately hydrophobic surface is an entropy driven process contributed mainly by conformational loss of adsorbed Lyz
B2529 – Thermodynamic analysis of denaturated lysozyme folded on moderately hydrophobic surface at 298 K
Both calorimetric determination of displacement adsorption enthalpies DH and measurement of adsorbed amounts of lysozyme (Lyz) denatured by 1.8 mol L–1 guanidine hydrochloride (GuHCl) on a moderately hydrophobic packings at 298 K, pH 7.0 and various salt concentrations were carried out. Based on the thermodynamics of stoichiometric displacement theory (SDT)
the fractions of thermodynamic functions, which related to four subprocesses of denatured protein refolding on the surface, were calculated and thermodynamic analysis that which one of the subprocesses plays major role for contribution to the thermodynamic fractions was made in detail. The moderately hydrophobic surface can provide denatured Lyz energy and make it gain more conformation with surface coverage or salt concentration increment. The displacement adsorptions of denatured Lyz onto PEG-600 surface are exothermic, more structure-ordered and enthalpy driven processes.
B2528 – Thermal stability of DNA in DNA–induced DOTAP liposome aggregates
The influence on the melting of calf thymus DNA induced by cationic liposomes, commonly used in gene therapy, was studied by means of ultraviolet spectrophotometry and differential scanning calorimetry. Both the two methods reveal that DNA in DNA-induced liposome complexes undergoes a denaturation process at a much higher temperature than free DNA does. The extent of protection strongly depends on the charge ratio R(+/–) of liposome–DNA complexes. In the case of dioleoyl trimethyl ammonium propane (DOTAP) liposomes, the maximum of the stabilization occurs at R(+/–)=0.7, where the DNA is still native up to temperatures higher than 100°C. This protection against denaturation up to higher temperatures might be of importance for bio-technological applications, such as biomolecular separation, antigene sequencing and for drug design purpose.
B2527 – Domains in bovine seminal ribonuclease
Bovine seminal ribonuclease is the only pancreatic-type ribonuclease to possess a dimeric structure: the two identical subunits are covalently linked by two disulfide bridges. Actually, the protein exists in two different dimeric structures owing to the possibility of swapping the N-terminal -helical segments: the swapped MxM dimer, and the non-swapped M=M dimer. The thermal denaturation of the two separated forms is investigated by differential scanning calorimetry. The process is reversible and can be represented by two sequential two-state transitions, indicating the presence of two domains in BS-RNase, regardless of the swapping phenomenon. Inspection of the structural models leads to the tentative identification of an external domain and a core domain, the latter more stable
B2526 – The effect of trimethylamine N-oxide on RNase A stability . A DSC study
The thermal stability of bovine pancreatic ribonuclease (RNase A) has been investigated in the presence of trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, by means of differential scanning calorimetry (DSC) and circular dichroism (CD) measurements at neutral and acid pH conditions. It is well known that compatible osmolytes such as TMAO are effective in
stabilizing protein structure and counteracting the denaturing the effect of urea and guanidinium hydrochloride (GuHCl). Calorimetric results show that TMAO stabilizes RNase A at pH 7.0 and does not stabilize the protein at pH 4.0. RNase A thermal denaturation in the presence of TMAO is a reversible two-state N D process. We also show that TMAO counteracts the urea and
GuHCl denaturing effect at neutral pH, whereas the counteracting ability is lost at acid pH.
B2524 – DSC examination of the degenerated human palmar aponeurosis in Dupuytren disease
The Dupuytren contracture – degenerative shortening of the palmar aponeurosis – is a common disease of the hand in Europe. The aetiology of the degenerative changes in the collagen structures is still not clear. To describe the clinical manifestation of the disease we use an international classification according to Iselin. Our hypothesis was that in Dupuytren disease there is a clear pathological abnormality in the tissue elements building up the palmar aponeurosis, which is responsible for the disease, and could be monitored besides the classical histological methods by differential scanning calorimetry. The thermal denaturation of different parts of human samples was monitored by a SETARAM Micro DSC-II calorimeter. All the experiments were performed between 0 and 100°C. The heating rate was 0.3 K min–1. DSC scans clearly demonstrated significant differences between the different types and conditions of samples (control: Tm=63°C and ?Hcal=4.1 J g–1, stage I.: Tm= 63°C and ?Hcal=5.1 J g–1, stage II.: Tm=64°C and ?Hcal=5.2 J g–1, stage III.: Tm=60°C and ?Hcal=5.2 J g–1, stage IV.: Tm=60.2°C and
?Hcal=5.3 J g–1). The heat capacity change between native and denatured states of aponeurosis samples increased with the degree of structural alterations indicating significant water loosing. These observations could be explained with the structural alterations caused by the biochemical processes. With our investigations we could demonstrate that DSC is a useful and well applicable method for the investigation of collagen tissue of the human aponeurosis. Our results may be of clinical relevance in the future i.e. in the choice of the optimal time
of surgical therapy of different clinical level Dupuytren contractures.
B2523 – DSC examination of the esophagus after implantation of special stents designed for the management of acute esophagus variceal bleeding. Experimental study
Massive bleeding from esophagus varices presents a life threatening complication of portal hypertension. No effective method of treatment is available until now, that would guarantee high grade of patient wellness during the conditioning and investigation phase until the definitive treatment could be introduced. The aim of this study was to evaluate the tissue response to esophagus stents – designed for manage acute variceal bleeding – in animal experiment. Self-expandable nitinol stents were introduced into the esophagus of six porcines. Another twelve porcines were undergone the same procedure, using the new biodegradable stents made of PDO (polydioxanon). Histological investigations of the stented esophagus segments were observed after 2 and 4 weeks at the first 12 animals. To monitor the time of stent degradation, histology were performed 7 and 12 weeks after the implantation in the biodegradable group (3–3 animals). Differential scanning calorimetric examination was performed in all esophagus samples. Focal erosion of the esophagus segments was more explicit in the nitinol group at the histology. On the 7th week all of the biodegradable stent were in the stomach and on the 12th week these were completely solved. DSC examination showed significant alterations in the structure of the esophagus in both stented group compared to the healthy control. This experiment showed that the new self-expandable stents are safety and suitable procedure without deterioration of the
esophageal wall. According to our DSC results the thermal denaturation of intact esophagus, its mucosa and muscle fragments revealed significant differences compared to healthy sample in favour the new biodegradable stent. Safety and efficiency in the experimental model had encouraged us to apply this method successfully patients with bleeding esophagus varices. The long term goal is to show that stent placement could be an effective way of decreasing or stabilising the acute bleeding from ruptured esophagus varices in cirrhotic patients.
B2522 – DSC examination of intestinal tissue following warm ishemia and reperfusion injury
The fact that small bowel is extremely sensitive to ischemia/reperfusion (I/R) injury had encouraged us to compare the conventional histology and differential scanning calorimetry (DSC) methods in intestinal structural changes following experimental warm I/R models. Our histological findings showed that longer warm I/R period caused more severe damage in structure of mucosa and crypts, but there were no changes in the muscular layer. According to our DSC data (transition temperature, calorimetric enthalpy) suggest that the thermal destruction of mucosa, muscular layer and total intestinal wall following I/R injury revealed significant differences compared to normal bowel structure.
B2521 – Human uterus in pregnancy, as it can be monitored by DSC examination. A preliminary study
The myometrium is the main component in enlargement of uterus and plays essential role in labour contraction. Its physiologic and elastic state has a decisive role in the labour process. We have made the first attempt to find correlation between the thermal parameters of uterus and progress of gravidity. Significant differences were observed between cyclic, non-pregnant and postmenopausal state (0.69 and 0.98 J g–1 calorimetric enthalpy as well as 60.6 and 63.1°C for Tm), as well as among the different gestational stages and complications. In case of twin pregnancy and dystocia we have found extremities: 0.37 and 1.34 J g–1 calorimetric enthalpies, with 62 and 61.6°C melting temperatures. DSC method seems to be promising tool to follow and understand different molecular changes in the myometrium during pregnancy and its complication of human labour.
B2520 – DSC investigation of early pregnant uterus of the rat
The aim of the present work was the characterization of nonpregnant and early pregnant myometrium (days 3–6) of the rat by means of differential scanning calorimetry (DSC). The spontaneous motor activity as well as the KCl-evoked contractions of isolated uterine rings was additionally recorded. A relatively close correlation was found between calorimetric enthalpy (DH) and the contractility of the uterus samples. Our results indicate that DSC is a useful tool for the investigation of the functions of developing myometrium and it can be considered as supplementing the traditional structural and functional methods.
B2519 – DSC analysis of human fat tissue in steroid induced osteonecrosis. A preliminary study
Osteonecrosis (ON) of the femoral frequently occurs after steroid medication. One of the final pathways leading to steroid induced ON is thought to be pathologic fat metabolism. The pathobiological mechanism underlying the induction of fat metabolism outslides by steroids leading to ON has not been fully elucidated. The purpose of this study was to examine the intraoperative obtained gluteal fat tissue from ON patients with histology, gas chromatography (GC) and differential scanning calorimetry (DSC) and to compare them with otherwise healthy patient’s samples. The histological sections showed no significant differences compared with the control group. GC revealed that fraction of saturated fatty acids decreased in ON samples from mean values of controls of 24% to 21, the polyunsaturated fraction from 20 to 14%. The monounsaturated acids showed an increase from mean rate of 52% of the controls to 65% of steroid treated samples. DSC curves correlate with chromatographic analysis of the tissue fatty acids (Steroid treated, heating between 0–100°C: Tm=5.7°C, ?H= –15.8J/g–1; heating between –20–100°C: Tm= –9.96 and 5.85°C, ?H=–59.17 and –16.2 J g–1. Non-necrotic, heating between 0–100°C: two separable transition with Tm=5.7 and 9.9°C, total ?H= –20.8 J g–1; heating between –20–100°C: Tm= –10.9 and 4.95°C, total ?H= –75.8 J g–1.). Our preliminary findings are rather tendentious. Further investigations are needed with higher sample rate and under other anamnestic circumstances too.
B2518 – Differential scanning calorimetric examination of the ruptured Achilles tendon in human
The Achilles tendon rupture is a common injury of the foot in middle age and physically active population in Europe. The aetiology of the degenerative changes in the collagen structures of the tendon which could be disposed for the rupture is still not clear. Our hypothesis was that before the injury there is a clear pathological abnormality in the tissue elements building up the Achilles tendon, which is responsible for the disease, and could be monitored besides the classical histological methods by differential scanning calorimetry.
The thermal denaturation of human samples was monitored by a SETARAM Micro DSC-II calorimeter. All the experiments were performed between 0 and 100 ?C. The heating rate was 0.3 K/min. DSC scans clearly demonstrated significant differences between the control and ruptured samples (control:Tm = 59.7 ?C, T1/2 = 1.4 ?C and ?Hcal = 8.54 J/g; ruptured: Tm = 62.75 ?C, T1/2 = 2.6 ?C and ?Hcal = 1.54 J/g). These observations could be explained with the structural alterations caused by the biochemical and structural processes.
With our investigations we could demonstrate that DSC is a useful and well applicable method for the investigation of collagen tissue of the human Achilles tendon. We can prove with this method, that the earlier series of microtraumas which result a scar formation in the tendon tissue increases the thermal stability of collagen in ruptured tendon.
B2517 – Application of isotherm calorimetry in the development of foods containing probiotic live flora and enriched with bioavailable Ca2+
The development of functional foods of probiotic effect based on the slime-producing strains isolated in the 1980s, and that of enriched with Ca on the utilization of the high Ca-containing whey of the quarg production in the Carpathian basin using fermentation. The probiotic properties of the slime-producing microbe strains isolated have been proved by in vitro and in vivo examinations. We have used an isotherm DSC method to identify the probiotic microbes. The percentile ratio of probiotic and other microbes was determined in the product by this technique. By utilization of quarg whey a special additive food for Ca-enrichment has been developed which is suitable to complete or enrich different foods (dairy, meat and bakery products).
The products developed are: probiotic kefir (Synbiofir), probiotic sour cream, probiotic butter cream, poultry meat products completed with Ca, bakery products completed with Ca.
B2516 – DSC analysis of human fat tissue in alcohol-induced avascular necrosis of the femoral head. A preliminary study
The osteonecrosis or avascular necrosis of the antero-superior part of the human femoral head (ANFH) often causes incongruity in the hip joint and leads to severe pain and disability in middle aged patients. This abnormality often requires surgical intervention, mainly total hip arthroplasty (THA). The possible pathomechanisms of ANFH are fat embolism, arterial occlusion, fatty necrosis of osteocytes and intraosseous hypertension. Factors have been proposed in the literature that can lead to this condition are alcohol abuse, steroid therapy, metabolic changes, dyslipidaemia. In some case we can not verify any factor; these are the so called idiopathic ANFH cases. We observed macroscopic variation in colour and consistency of the subcutaneous fat tissue in patients with ANFH compared to osteoarthritis or hip fracture during THA procedures. Subcutaneous fat tissues during THA from a patient with ANFH due to alcohol
abuse were compared with an otherwise healthy patient who underwent surgery due to traumatic hip fracture. No histological changes were notified in the size; shape of adipocytes and in the cells of the septae of the connective tissue. Gas chromatography showed that the patient with alcoholic ANFH had less long chain fatty acids. DSC revealed, that in case of non-necrotic sample as a reference, during heating between 0–100°C two separable transitions are with Tm=5.7 and 9.9°C, total ?H= –20.8 J g–1. In –20–100°C range endotherms with Tm= –10.9 and 4.95°C, total ?H= –75.8 J g–1 could be detected. In case of alcohol-induced avascular necrosis we have found endotherms between 0–100°C with: Tm=7.3°C, total ?H= –26.9 J g–1, and heating between –20–100°C: Tm= –0.25°C, total ?H= –103.3 J g–1 thermal parameters. The alteration in the fatty acid profile did not cause histological changes, but we were able to detect it with analytical methods e.g. DSC and gas chromatography.
B2515 – DSC analysis of human fat tissue in idiopathic avascular necrosis of the femoral head. A preliminary study
Avascular necrosis of human femoral head (ANFH) causes incongruity in the joint that leads to disability in patients requires total hip arthroplasty (THA). Several etiological factors of ANFH have been proposed in the literature but there are cases of idiopathic origin. We observed macroscopic variation in quality of the subcutaneous fat tissue in patients with ANFH compared to patients with osteoarthritis or hip fracture during THA procedures. The samples were analysed by histology, gas chromatography (GC) and differential scanning calorimetry (DSC).
Conclusion: the alteration in the fatty acid profile did not cause histological changes, however we could detect biochemical changes using DSC and GC.
B2514 – Intermediate states of myosin head during ATP hydrolysis cycle in psoas muscle fibres by EPR and DSC. A review
Force generation in muscle during contraction arises from direct interaction of the two main protein components of the muscle, myosin and actin. The process is driven by the energy liberated from the hydrolysis of ATP. In the presence of CaATP the energy released from hydrolysis produces conformational changes in myosin and actin, which can be manifested as an internal motion of myosin head while bound to actin. It is suggested that myosin heads attached to actin produce conformational changes during the hydrolysis process of ATP, which results in a strain in the head portion of myosin in an ATP-dependent manner. These structural changes lead to a large rotation of myosin neck region relieving the strain. Paramagnetic probes and EPR spectroscopy provide direct method in which the rotation and orientation of specifically labelled proteins can be followed during muscle activity. In order to find correlation between local and global structural changes in the intermediate states of the ATPase cycle, the spectroscopic measurements were combined with DSC measurements that report domain stability and interactions. In the review a detailed description of the application of EPR and DSC techniques in muscle protein research will be given. The measurements show that the small local structural changes detected by EPR after nucleotide binding influence the global structure of protein system responsible for muscle contraction.
B2513 – Effect of phalloidin on the skeletal muscle ADP-actin filaments
The effect of phalloidin on the thermal stability of skeletal actin filaments polymerized from ADP-binding monomers was investigated with the method of differential scanning calorimetry. Phalloidin shifted the melting temperature of the ADP-F-actin from 59.1±1.0 to 80.0±1.2°C. The stabilizing effect of phalloidin propagated cooperatively along the filament. The cooperativity
factor according to the applied model was 1.07±0.11. With these measurements it was possible to demonstrate that the binding of phalloidin has lower influence on the adjacent protomers in ADP- (k=1) than in ATP-actin filaments (k=3).
B2512 – Differential scanning calorimetric examination of the human skeletal muscle in a compartment syndrome of the lower extremities
The compartment syndrome—conditions of elevated intramuscular pressure—is one of the most serious complications of the injuries of the lower extremities. Early diagnosis is important, as delayed treatment leads to significant complications. The diagnosis of compartment syndrome is most commonly made by clinical examination and direct measurement of the intra-compartmental pressure. Our hypothesis was that in different stages of compartment syndrome there is a clear pathological abnormality in the tissue elements of the affected muscles, which is responsible for seriousness of the disease, and could be monitored besides the classical histological methods by differential scanning calorimetry. The thermal denaturation of different
parts of human samples was monitored by a SETARAM Micro DSC-II calorimeter. All the experiments were performed between 0 and 100 °C. The heating rate was 0.3 K/min. DSC scans clearly demonstrated significant differences between the different types and conditions of samples (control: Tm = 55.5; 59.9 °Cand ?Hcal = 0.52 J/g, Gr. I.: Tm = 58.1; 62.2 °C and ?Hcal
= 0.28 J/g, Gr. II.: Tm = 57.45; 61.5 °C and ?Hcal = 0.24 J/g, Volkmann’s ischemic contracture Tm = 57.75; 61.8; 65.8 °C and ?Hcal = 0.74 J/g). These observations could be explained
with the structural alterations caused by the biochemical processes. The heat capacity change between native and denatured states of muscle samples was significant, indicating significant water loosing during denaturation, but independent from the structural alterations.
B2511 – Calorimetric examination of the human meniscus
Meniscus degeneration is a very frequent disease of human beings mainly in the developed countries. The ability of the meniscus to participate in load bearing, shock absorption, joint lubrication, and joint stability depends on the maintenance of its structural integrity. Therefore the pathology of the degeneration has been subject of many publications before. These studies all agreed that the grade of the degeneration correlated with the patient’s age, weight, profession, and athletic activity . These reviews also described the biochemical changes in the structure, too [2, 3]. In the current study authors examined degenerated human meniscus with differential scanning calorimetry and demonstrated thermal differences between healthy and intraoperatively removed pathological samples.
B2510 – Differential Scanning Calorimetry examination of transverse carpal ligament in carpal tunnel disease
The carpal tunnel syndrome – compression of the median nerve by the transverse ligament of the wrist – is a serious disease of the human hand. The electrophysiological changes in the median nerve can be easily followed by electroneurography (ENG). The degenerative changes in the collagen structures of the carpal ligament cause shrinking of the ligament and compression of the nerve. According to the present study we could demonstrate that DSC is a useful and well applicable method for the investigation of collagen tissue of the human carpal transverse ligament. DSC scans clearly demonstrated significant differences between the different types and conditions of samples (control: Tm=61.3°C and ?Hcal=4.04 J g–1, mild: Tm=62°C and ?Hcal=4.3 J g–1, middle: Tm=61.5°C and ?Hcal=5.17 J g–1 as well as severe: Tm=61.85°C and ?Hcal=8.44 J g–1). After these investigations we can choose the
optimal time of surgical therapy of different clinical level carpal tunnel syndrome too.
B2509 – Effect of phalloidin on filaments polymerized from heart muscle ADP-actin monomers
The effect of phalloidin on filaments polymerized from ADP-actin monomers of the heart muscle was investigated with differential scanning calorimetry. Heart muscle contains a-skeletal and a-cardiac actin isoforms. In the absence of phalloidin the melting temperature was 55°C for the a-cardiac actin isoform and 58°C for the a-skeletal one when the filaments were generated from ADP-actin monomers. After the binding of phalloidin the melting temperature was isoform independent (85.5°C). We concluded that phalloidin stabilized the actin filaments of a-skeletal and a-cardiac actin isoforms to the same extent when they were polymerized from ADP-actin monomers.
B2508 – Thermal transitions of actin
Actin is one of the main components in the eukaryote cells which plays significant role in many cellular processes, like force-generation, maintenance of the shape of cells, cell-division cycle and transport processes. In this study the thermal transitions of monomer and polymerized actins were studied to get information about the changes induced by polymerization and binding of myosin to actin using DSC and EPR techniques. The main thermal transition of F-actin was at 67.5°C by EPR using spin-labeled actin (the relative viscosity change was around 62°C), while the DSC denaturation Tms were at 60.3°C for G-actin and at 70.5°C for F-actin. Applying the Lumry–Eyring model to obtain the parameters of the kinetic process and calculate the activation energy, a ‘break’ was found for F-actin in the function of first-order kinetic constant vs. 1/T. This indicates that an altered interdomain interaction is present in F-actin. The addition of myosin or heavy meromyosin (HMM) in different molar ratio of myosin to actin has changed significantly the EPR spectrum of spin-labeled F-actin, indicating the presence of the supramolecular complex. Analyzing the DSC traces of the actomyosin complex it was possible to identify the different structural domains of myosin and actin.
B2456 – Dehydrated native biopolymers – a unique representative of glassy systems
The present work summarizes the results of our studies of the heat induced conformational and relaxation transitions in all the three main classes of biopolymers at different hydration degree. Detection of the excess heat capacity drop and absolute values of the heat capacity in a wide range of temperatures and water content allowed us to get a deeper insight in the nature
of the processes occurring in these biopolymers. As a result, we were able to distinguish the individual features of the thermal properties related to the peculiarities of the steric structure for each biopolymer studied and assess common features for every biopolymer system undergoing a glass transition.
B2394 – The Unfolding/Denaturation of Immunogammaglobulin of Isotype 2b and its Fab and Fc Fragments
The unfolding and further denaturation of IgG and its Fab and Fc fragments were studied both on a macroscopic and molecular level, using differential scanning calorimetry and circular dichroism spectroscopy, respectively. It was shown that the structural integrity of the Fab and Fc units was retained after fragmentation of the IgG. The Fab fragment denatured
at ;61°C and the Fc fragment at 71°C. The structural transitions observed in the whole IgG is the sum effect of those determined for the isolated Fab and Fc fragments.
B2393 – Porous three-dimensional scaffolds made of mineralised collagen: Preparation and properties of a biomimetic nanocomposite material for tissue engineering of bone
For healing of bone defects and as matrix for tissues engineering, porous scaffolds are required that can be easily pre-seeded with cells in the lab and invaded by tissue after implantation.We have developed porous 3D scaffolds consisting of mineralised collagen type I—a nanocomposite, which mimics the composition of extracellular matrix of healthy bone tissue. The steps of material processing as well as the physico-chemical, structural and mechanical properties of this biomaterial are described in detail. The technology of differential scanning calorimetry, which provides an excellent possibility to investigate collagen-based materials, was used to characterise the final scaffold and all its intermediates in situ. Furthermore, the interconnecting porosity of the scaffolds with pore diameters of about 200 m has been shown to be highly suitable for homogenous cell seeding, demonstrated with human marrow stromal cells.
B2387 – Heat of adsorption of cysteine enzymes on mesoporous silica with high specific surface area
The adsorption of a cysteine enzyme, on mesoporous silica with high specific surface area synthesized by the sol-gel method, was studied in a heat flow calorimeter, to determine the energy involved in the adsorption process of the protein. The adsorption was carried out at a constant temperature of 30?C to avoid the denaturation of the enzyme.
The observed results indicate that the obtained biomaterials (silica-enzyme) have possibilities for their application in several biotechnology processes.
The heat of papain adsorption and the solid-enzyme (SiO2-Papain) interactions at different pH are presented.
B2373 – Effect of acetonitrile on the hydration of human serum albumin films: a calorimetric and spectroscopic study
A new experimental approach based on the combination of calorimetric and FTIR spectroscopic measurements was proposed to study simultaneously the sorption of water and organic solvent, and corresponding changes in the structure of protein films in the water activity range from 0 to 1.0. Enthalpy changes ( Htot) on the interaction of water with the dried human serum albumin (HSA) in the presence and absence of acetonitrile (AN) have been measured using a Setaram BT-2.15 calorimeter at 298 K. Spectroscopic data on water and organic
solvent vapor sorption by the HSA films and the corresponding changes in the protein secondary structure were determined by means of a Bruker Vector-22 FTIR spectrometer. By using a water activity-based comparison we characterised the effect of acetonitrile on the hydration and structure of the HSA films. Acetonitrile (AN) sorption isotherm resembles a smooth curve. HSA film binds about 250 mol AN/mol protein at the lowest water activities. As the water activity increases from 0 to 0.8, the sorption of AN gradually decreases from 250 to 150 mol
AN/mol HSA. At aw > 0.8, the sorption of AN sharply decreases to zero. Acetonitrile decreases markedly the water content at a given aw. This behavior suggests that the suppression in the uptake of water is due to a competition for water-binding sites on the HSA films by acetonitrile. Changes in the secondary structure of HSA were determined from infrared spectra by analyzing the structure of amide I band. Acetonitrile increases the intensity of the 1654 cm?1 band that was assigned to the -helix structure. Changes in the intensity of the 1654 cm?1 band agree
well with the decrease in water uptake in the presence of AN. An explanation of the acetonitrile effect on the hydration and structure of the HSA films was provided on the basis of hypothesis on water-assisted disruption of polar contacts in the initially dried protein.
B2315 – Differential scanning calorimetric examination of the ruptured Achilles tendon in human
The Achilles tendon rupture is a common injury of the foot in middle age and physically active population
in Europe. The aetiology of the degenerative changes in the collagen structures of the tendon which could
be disposed for the rupture is still not clear. Our hypothesis was that before the injury there is a clear
pathological abnormality in the tissue elements building up the Achilles tendon, which is responsible for
the disease, and could be monitored besides the classical histological methods by differential scanning
The thermal denaturation of human samples was monitored by a SETARAM Micro DSC-II calorimeter.
All the experiments were performed between 0 and 100 ?C. The heating rate was 0.3 K/min. DSC
scans clearly demonstrated significant differences between the control and ruptured samples (control:
Tm = 59.7 ?C, T1/2 = 1.4 ?C and Hcal = 8.54 J/g; ruptured: Tm = 62.75 ?C, T1/2 = 2.6 ?C and Hcal = 1.54 J/g).
These observations could be explained with the structural alterations caused by the biochemical and
With our investigations we could demonstrate that DSC is a useful and well applicable method for the
investigation of collagen tissue of the human Achilles tendon. We can prove with this method, that the
earlier series of microtraumas which result a scar formation in the tendon tissue increases the thermal
stability of collagen in ruptured tendon.
B2314 – Analysis of skin penetration of phytosphingosine by fluorescence detection
and influence of the thermotropic behaviour of DPPC liposomes
Phytosphingosine (PS) is a promising compound in skin formulations, considering its application in the
treatment of acne and different inflammations as well as in the ‘anti age’ cosmetics. PS, as an active
substance was incorporated in DPPC liposomes intended to standard diffusion experiments, where dermatomed
porcine skin was mounted in FRANZ cells. The proved skin retention was about 5.5% (w/w)
after 24 h and about 6.8% (w/w) after 48 h of the applied PS amount, whereas only about 0.05% (w/w) and
about 0.07% (w/w) PS, respectively, could be observed in the acceptor medium. To increase analytical
sensitivity PS was derivatised by o-phtalaldehyde (OPA) reagent and analysed by HPLC with fluorescence
detection. The higher amount of PS within the skin symbolised an interaction with lipid structures in skin.
Further evaluation of this interaction was accomplished by applying microDSC studies of PS with DPPC
as a model membrane. For this purpose liposomes were prepared by increasing PS content. The characteristic
endothermic peak observed for the single system was shifted to a slightly higher temperature
and broadened as the mole fraction of PS increased. This might be the effect of mixing of PS with DPPC.
An addition of 10 mol% PS resulted in more than double sized particles pointing to a possible change in
the liposomal shape.
B2308 – Differential scanning calorimetry study of glycerinated rabbit psoas muscle fibres in intermediate state of ATP hydrolysis
Thermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins (actin and myosin) their native environment in striated muscle fibres. The interaction of actin with myosin in the highly organized muscle structure affected by internal forces; therefore their altered conformation and interaction may differ from those obtained in solution. energetics of long functioning intermediate states of ATP hydrolysis cycle was studied in muscle fibres by differential scanning
B2305 – Evaluation of Structural Changes Induced by High Hydrostatic Pressure in Leuconostoc mesenteroides
Scanning electron microcopy (SEM), transmission electron microscopy (TEM), and differential scanning
calorimetry (DSC) were used to evaluate structural changes in Leuconostoc mesenteroides cells as a function of
high-hydrostatic-pressure treatment. This bacterium usually grows in chains of cells, which were increasingly
dechained at elevated pressures. High-pressure treatments at 250 and 500 MPa also caused changes in the
external surface and internal structure of cells. Dechaining and blister formation on the surface of cells
increased with pressure, as observed in SEM micrographs. TEM studies showed that cytoplasmic components
of the cells were affected by high-pressure treatment. DSC studies of whole cells showed increasing denatur-
ation of ribosomes with pressure, in keeping with dense compacted regions in the cytoplasm of pressure-treated
cells observed in TEM micrographs. Apparent reduction of intact ribosomes observed in DSC thermograms
was related to the reduction in number of viable cells. The results indicate that inactivation of L. mesenteroides
cells is mainly due to ribosomal denaturation observed as a reduction of the corresponding peak in DSC
thermograms and condensed interior regions of cytoplasm in TEM micrographs
B2287 – Conformational change of adsorbed and desorbed bovine serum albumin on nano-sized magnetic particles
Adsorptions of bovine serum albumin (BSA) on nano-sized magnetic particles with and without the presence of carbodiimide were studied.
Desorption of BSA from magnetic particles were carried out in either NaOH or Na2HPO4 solutions. The structures of native BSA, adsorbed
BSA on magnetic particles, and desorbed BSA were studied by several methods, circular dichroism (CD), fluorescence spectroscopy and
differential scanning calorimetry (DSC). The magnitude of conformational changes of protein was determined by calculating the -helix
content from the circular dichroism (CD) spectra and by evaluating fluorescence spectrum and DSC thermograms. Adsorbed BSA on magnetic
particles shows no thermal transition with respect to the native BSA. The structural change of BSA when desorbed by Na2HPO4 solution is
much smaller in comparison to that when desorbed by NaOH solution. Hence, this indicates that BSA could be desorbed from nano-sized
magnetic particles using Na2HPO4 without much conformational change.
Aqueous concentrated lecithin mixtures (asolectin from soybean) show typical lamellar liquid crystalline behavior and the individual
lamellae tend to form spherical supramolecular structures, i.e., multilamellar vesicles. When part of the lecithin is replaced by the
anionic surfactant sodium dodecyl sulfate (SDS), the compact multilamellar vesicles disappear and the viscosity decreases. By adding
poly(diallyldimethylammonium chloride) (PDADMAC) to the lecithin/SDS system, the formation of multilamellar vesicles can be induced
again and the viscosity increases. However, one characteristic feature of these polymer-modified systems is a temperature-dependent phase
transition from a compact multilamellar vesicle phase to a more swollen liquid crystalline phase. The polymer-modified multilamellar compact
vesicles are of interest for utilization as new thermosensitive drug delivery systems.
B2270 – Immobilization of alpha-chymotrypsin in kappa-carrageenan beads prepared with the static mixer
The alpha-chymotrypsin-loaded kappa-carrageenan beads preparation and properties are described. The beads were obtained by emulsification/
thermal gelation with a sunflower oil at ambient temperature as the continuous phase using a Sulzer SMX static mixer. The mean
Sauter bead diameter was about 300 µm. The alpha-chymotrypsin encapsulation efficiency may be increased in two times by preliminary enzyme
cross-linking by glutaraldehyde. The stability upon storage is higher for beads-containing cross-linked alpha-chymotrypsin (no activity
loss during at least 2 weeks). The alpha-chymotrypsin-loaded kappa-carrageenan beads may be used in repeated batch runs and the operational
biocatalyst stability may be enhanced by kappa-carrageenan beads the coating by chitosan (no significant activity decrease in seven runs).
B2259 – Cutin and suberin monomers are membrane perturbants
The interaction between cutin and suberin monomers, i.e., omega-hydroxylpalmitic acid, alpha,omega-hexadecanedioic acid, alpha,omega-hexadecanediol, 12-
hydroxylstearic acid, and phospholipid vesicles biomimicking the lipid structure of plant cell membranes has been studied by optical and
transmission electron microscopy, quasielastic light scattering, differential scanning calorimetry, and 31P solid-state NMR. Monomers were
shown to penetrate model membranes until a molar ratio of 30%, modulating their gel to fluid-phase transition, after which monomer crystals
also formed in solution. These monomers induced a decrease of the phospholipid vesicle size from several micrometers to about 300 nm.
The biological implications of these findings are discussed.
B2250 – 4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization
The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol.
The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol,
irreversible in the intermediate (50mM < c < 250 mM) and again reversible in the presence of 250mM and higher concentrations
of 4-chlorobutan-1-ol. In the presence of 50mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding
at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible
thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model,
rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism
and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary
structure and high content of secondary structure in the presence of 50mM4-chlorobutan-1-ol at a temperature corresponding to the second
transition. Similar results have been observed at pH 3.9.
B2248 – Effect of polycyclic aromatic hydrocarbons on erythrocyte membranes by DSC and EPR
Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) experiments were performed on human
erythrocyte membranes and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model systems in order to study the effect the polycyclic
aromatic hydrocarbons on lipid structure and dynamics. Eight different compounds among others naphthalene and pyrene were compared,
which occur in significant concentrations in dust collected from the air in large cities.
Experiments using spin label technique showed that the compounds induced mobility changes in the lipid region in the environment of
the fatty acid probe molecules incorporated into the membranes. The effects depended on the structure and concentration of the different
compounds. Similarly to EPR observations, DSC measurements reported decrease of transition temperature in comparison to control DPPC
vesicles. These results suggest that polycyclic aromatic hydrocarbons were able to modify the internal dynamics of erythrocyte membranes
which might lead to damage of the biological functions.
B2247 – Cloud-point temperature and liquid-liquid phase separation of supersaturated lysozyme solution
The detailed understanding of the structure of biological macromolecules reveals their functions, and is thus
important in the design of new medicines and for engineering molecules with improved properties for industrial
applications.Although techniques used for protein crystallization have been progressing greatly, protein crystallization
may still be considered an art rather than a science, and successful crystallization remains largely empirical and
operator-dependent.In this work, a microcalorimetric technique has been utilized to investigate liquid-liquid phase
separation through measuring cloud-point temperature Tcloud for supersaturated lysozyme solution.The effects of
ionic strength and glycerol on the cloud-point temperature are studied in detail.Over the entire range of salt
concentrations studied, the cloud-point temperature increases monotonically with the concentration of sodium chloride.
When glycerol is added as additive, the solubility of lysozyme is increased, whereas the cloud-point temperature is
B2232 – The effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin filaments
In this work the effect of phalloidin and jasplakinolide
on the dynamic properties and thermal stability of actin filaments
was studied. Temperature dependent fluorescence resonance
energy transfer measurements showed that filaments of Ca-actin
became more rigid in the presence of phalloidin or jasplakinolide.
Differential scanning calorimetric data implied that the stiffer
filaments also had greater thermal stability in the presence of
phalloidin or jasplakinolide. The fluorescence and calorimetric
measurements provided evidences that the extent of stabilization
by jasplakinolide was greater than that by phalloidin.
B2225 – Adsorption and desorption of lysozyme on nano-sized magnetic particles and its conformational changes
Adsorption and desorption of lysozyme on nano-sized magnetic particles and its conformational change were studied in thiswork. Adsorption
of lysozyme on nano-sized magnetic particles (Fe3O4)was carried out at different pH. Maximum adsorption of lysozyme (4.65 mg/m2) occurred
at its isoelectric point (pI = 11.1). Differential scanning calorimetry (DSC) results show that the lysozyme adsorbed on magnetic particles
did not show any thermal transition over the range 20-100°C. High desorption of lysozyme from magnetic particles was achieved using
NaH2PO4 (pH4.0) (90%) and NaSCN (pH6.0) (97%) as desorbents. The conformational change of the lysozyme desorbed by NaH2PO4 was
small, while the lysozyme desorbed by NaSCN underwent a significant conformational change as measured by the intrinsic fluorescence.
Eighty-eight and 82% activity was retained in the desorbed enzyme for desorption by NaH2PO4 and NaSCN, respectively.
B2224 – Interaction of 2,2,2-trifluoroethanol with proteins: calorimetric, densimetric and surface tension approach
The thermal denaturation of hen egg-white lysozyme was studied in the presence of 2,2,2-trifluoroethanol (TFE)
at various pH values using micro differential scanning calorimetry.Quantitative thermodynamic parameters accompanying
the thermal transitions were evaluated.It is observed that thermal unfolding of lysozyme in the presence of
TFE upto a concentration of 4.0 mol dm-3 follows a two-state denaturation mechanism as indicated by the equality
of van't Hoff and calorimetric enthalpies.The finer details of interaction were studied by measuring the partial molar
volume of some constituent amino acids and glycine peptides from water to aqueous TFE at 298.15 K. The physicochemical
properties of aqueous TFE: apparent molar heat capacities, apparent molar volumes and surface tension
were measured to understand the intrinsic properties of the cosolvent as well.Fr om the correlation among the thermal
unfolding data on lysozyme in aqueous TFE, calculated preferential interaction parameters, physico chemical
properties of aqueous TFE and partial molar volumes of transfer, it is concluded that both solvent mediated effect
and direct interaction constitute the mechanism of TFE-protein interactions.
B2223 – The tryptophane residues of dimeric arginine kinase: roles of Trp-208 and Trp-218 in active site and conformation stability
Roles of the two tryptophane residues of dimeric arginine kinase (AK) were individually investigated by site-directed mutagenesis. Both
residues were fully conserved in the phosphogen kinase family and the mutant proteins were analyzed by enzyme kinetics, fluorescence
spectroscopy, fluorescence quenching experiments, thermal stability and conformational stability. Our studies revealed that Trp-218 was
located at the active site of AK and was the major fluorescence contributor (96.9%). Single replacement of this residue by alanine led to almost
complete inactivation of the enzyme. In addition, a decrease in the melting temperature in differential scanning calorimetry (DSC) profiles and
the equilibrium studies in guanidine hydrochloride (GdnHCl) denaturation after mutagenesis also suggested that Trp-218 takes part in
stabilizing the conformational structure of AK. Although another tryptophane, Trp-208 was not located at the active sites, it may take part in
maintaining the correct dimer conformation for catalysis. Replacement of this tryptophane by alanine decreased the activity to 70.3% and
made it susceptible to heat and denaturants, such as GdnHCl. In addition, Trp-208 also seemed to play an important role in correct protein
B2211 – Linkage of proton binding to the thermal dissociation of triple helix complex
The effects of cytosine protonation on the thermodynamic properties of parallel pyrimidine motif DNA triplex were
investigated and characterized by different techniques, such as circular dichroism (CD), ultraviolet spectroscopy (UV) and
differential scanning calorimetry (DSC). A thermodynamic model was developed which, by linking the cytosine ionization
equilibrium to the dissociation process of the triplex, is able to rationalize the experimental data and to reproduce the pH
dependence of the free energy, enthalpy and entropy changes associated with the triplex formation. The results are useful to
systematically introduce the effect of pH in a more general model able to predict the stability of DNA triplexes on the basis of
the sequence alone.
B2177 – Thermal analysis of the human intervertebral disc
Intervertebral disc (IVD) degeneration is one of the most common musculuskeletal disorders
affecting western society. Degeneration alters the morphology and the mechanical properties of the
discs. According to previous reports, DSC proved to be a suitable method for the demonstration of
thermal consequences of local as well as global conformational changes in the structure of the human
intervertebral discs. In the present study, a wide spectrum of degenerated IVD was examined by
DSC. The results suggest that definitive differences exist between the stages of disc degeneration in
B2154 – Chitosan-based ferrimagnetic membrane
A chitosan-based ferrimagnetic membrane (Chitosan/NiFe2O4) was prepared and it showed a typical magnetization
of soft ferrimagnetic material with Ms=16emu/g at 50 kOe and a high capacity to extract Hg(II), Cu(II) and Zn(II)
ions from aqueous solutions. These results indicate that the Chitosan/NiFe2O4 is a good candidate to remove heavy
metals from polluted water with the possibility that its recovery can be done by an electromagnet.
B2146 – Differential scanning calorimetric and histological examinations of the long head of the biceps in cadavers
Shoulder pain is a common presentation of the dysfunction of the glenohumeral joint. The long head of the biceps tendon has been
proposed as a source of pain in rotator cuff pathologies. The rotator cuff is a dynamic stabilizer of the glenohumeral joint, and its tear can
create different shoulder dysfunctions. The long head of the biceps has a special intraarticular localisation, so the arthricular destruction
affects its tendon too. In the process of the rotator cuff degeneration and tear the structure of the biceps tendon pathological transforms.
With foregoing studies authors have demonstrated the feasibility of DSC in the investigation of the musculoskeletal system.
The aim of this study was to establish the curves and the histological properties of the tendon of the long head of the biceps in
different magnitudes of the rotator cuff tear on cadavers. The DSC results clearly proved that definitive differences are present between
the structural state of the tendons in different magnitudes of the rotator cuff tears, which have also been demonstrated by the
B2143 – Optimisation of production and storage stability of the starter bacteria Streptococcus thermophilus and Lactobacillus plantarum
The growth of Lactobacillus plantarum and Streptococcus thermophilus was modelled by fitting
isothermal calorimetric traces to arbitrary mathematical functions. There was a correlation between certain
features on the thermal trace and parameters derived from a Baranyi fit to viable count data. In the presence
of 75 g L-1 lactose, S. thermophilus showed a slower rate of growth than in the presence of 5 g L-1 lactose when
measured by both viable count and heat flow. A simple equation described the variation in glass transition
temperature with water activity for all preparations. A master curve fit for all mortality data for S. thermophilus
showed the importance of a transition temperature for the survival of freeze-dried preparations of bacteria.
The temperature at which survival began to be impaired was 15-20°C below the glass transition temperature
determined by differential scanning calorimetry.
B2085 – cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei
A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was
determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-
alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on
denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the
number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of
the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a
precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain
in an elasmobranch.
B2057 – Adsorption-induced conformational changes of proteins onto ceramic particles: Differential scanning calorimetry and FTIR analysis
Three model proteins, bovine serum albumin, hen's egg lysozyme and bovine serum fibrinogen, were adsorbed from aqueous solution onto finely
dispersed ceramic particles, namely different kinds of alumina and hydroxyapatite particles. The influence of adsorption on protein secondary
structure was investigated. The FTIR spectroscopic findings were compared with the results of DSC measurements. In almost all cases it was
found that adsorption results in destabilisation and structural loss of the bound protein. A decrease in transition enthalpy is correlated with a
loss in alpha-helical structure, which seems to be the most sensitive structure on adsorption-induced rearrangements. A total collapse of structure
in the adsorbed proteins was not determined on any ceramic surface. Some residual structure is always retained. Structural changes in the Dor
E-domains of fibrinogen could be independently observed by two different calorimetric signals. The two techniques applied in the present
study - micro-DSC and FTIR spectroscopy - can be concluded to provide complementary information on adsorption-induced structural changes
on both the molecular (thermal stability, overall structure) and the sub-molecular level (secondary structure).
B2036 – A calorimetric study of dimyristoylphosphatidylcholine phase transitions and steroid-liposome interactions for liposomes prepared by thin film and proliposome methods
Using high sensitivity differential scanning calorimetry (HSDSC), the phase transitions of dimyristoylphosphatidylcholine (DMPC) liposomal
bilayers and their interaction with the model steroid beclometasone dipropionate (BDP) were found to be dependent on the method of liposome
manufacture. Ethanol-based proliposomes produced liposomes having no phospholipid pretransition, a main transition of high enthalpy and a low
onset temperature, and a very low incorporation of the steroid (maximum 1 mol%). This was attributed to an alcohol-induced interdigitation of
the bilayers, which was not apparently reversed by flushing the liposome dispersion with nitrogen in an attempt to remove ethanol. For liposomes
manufactured by thin film or particulate-based proliposome methods, 1-2.5 mol% steroid was optimal for incorporation within bilayers, although
the nature of the steroid interaction with the bilayers differed between the two methods. For liposomes manufactured by the thin film method, a
higher steroid concentration resulted in a broadened main transition and a reduced melting cooperativity. This suggests that BDP formed separate
domains within the bilayers which caused non-ideal mixing and phase separation at 5 mol% steroid. This observation was absent for liposomes
generated from particulate-based proliposomes, indicating separate steroid domains were not formed and subsequent non-ideal mixing and phase
separation did not occur. In addition, liposomes generated from particulate-based proliposomes showed reduced pretransition and main transition
enthalpies. These differences were attributed to the employment of sucrose to manufacture the particulate-based proliposomes. This study has
shown that the thermal behaviour of liposomes and their interaction with beclometasone dipropionate were dependent on the method of liposome
manufacture. Moreover, particulate-based proliposomes may provide a reasonable alternative to the conventional thin film method in producing
liposomes incorporating this steroid.
B2020 – High sensitivity differential scanning calorimetry investigation of the interaction between liposomes, lactate dehydrogenase and tyrosinase
High sensitivity differential scanning calorimetry (HSDSC) has been used to study the interaction of the model proteins lactate dehydrogenase
(LDH) and tyrosinase with dimyristoylphosphatidylcholine (DMPC) liposomes, and relate this to the thermal and physical stability of the proteins.
On heating, both LDH and tyrosinase denatured irreversibly in a time-dependent manner and modified the phase transition behaviour of DMPC
liposomes at all concentrations investigated. The most marked effects occurred for the pretransition rather than the main phospholipid phase
transition. The effects on the bilayer are likely to result from electrostatic interactions of the hydrophilic proteins with the head-groups of DMPC
molecules, whilst due to their hydrophilic nature they do not penetrate into the bilayer. Tyrosinase is more highly ionised than LDH at the pH of
the investigation, which may explain why tyrosinase has a greater effect than LDH on the HSDSC scans at mg/ml protein concentrations.
B1976 – In-plane miscibility and mixed bilayer microstructure in mixtures of catanionic glycolipids and zwitterionic phospholipids
SAXS/WAXS studies were performed in combination with freeze fracture electron microscopy using mixtures of a new Gemini catanionic
surfactant (Gem16-12, formed by two sugar groups bound by a hydrocarbon spacer with 12 carbons and two 16-carbon chains) and the zwitterionic
phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) to establish the phase diagram. Gem16-12 in water forms bilayers with the same
amount of hydration water as DPPC. A frozen interdigitated phase with a low hydration number is observed below room temperature. The kinetics of
the formation of this crystalline phase is very slow. Above the chain melting temperature, multilayered vesicles are formed. Mixing with DPPC
produces mixed bilayers above the corresponding chain melting temperature. At room temperature, partially lamellar aggregates with local nematic
order are observed. Splitting of infinite lamellae into discs is linked to immiscibility in frozen state. The ordering process is always accompanied by
dehydration of the system. As a consequence, an unusual order-disorder phase transition upon cooling is observed.
B1864 – A simple model for the cooperative stabilisation of actin filaments by phalloidin and jasplakinolide
The stabilisation of magnesium actin filaments by
phalloidin and jasplakinolide was studied using the method of differential
scanning calorimetry. The results showed that actin
could adapt three conformations in the presence of drugs. One
conformation was adapted in direct interaction with the drug,
while another conformation was identical to that observed in
the absence of drugs. A third conformation was induced through
allosteric inter-protomer interactions. The effect of both drugs
propagated cooperatively along the actin filaments. The number
of the cooperative units determined by using a quantitative model
was larger for jasplakinolide (15 actin protomers) than for phalloidin
B1841 – Thermodynamic properties of aqueous solutions of maltodextrins from laser-light scattering, calorimetry and isopiestic investigations
The thermodynamic properties of maltodextrins in aqueous solutions were studied by the isopiestic method, laser-light scattering and
calorimetry. Three different maltodextrin samples with molar masses between 1000 and 3000 were investigated. The second and third
osmotic virial coefficients of oligomers were determined. It was found that an influence of the molar mass on the second and third osmotic
virial coefficients has to be considered only for the low molecular oligomers (up to maltotriose), whereas it can be neglected for oligomers
with higher molecular mass. Furthermore, the Virial Equation with Relative Surface Fractions (VERS) model was used to correlate the
experimental data for the activity of water and the heat of dilution. The results of that investigation were used to predict the activity of water
and some other thermodynamic properties of some other low molecular saccharides for which experimental results are available in the
B1836 – Molecular mechanism of the inhibition of cytochrome c aggregation by Phe-Gly
Experimental and computational studies suggest that few general principles govern protein/protein interactions and aggregation.
The knowledge of these rules may be exploited to design peptides that are able to interfere with the self-assembly and aggregation of
proteins. This work is aimed to verify the validity of this hypothesis by investigating the interaction of cytochrome c with Phe and
Gly amino acids, Ala-His (carnosine), and two water-soluble dipeptides Phe-Gly and Gly-Phe. The combined use of 1H NMR, MD,
and DSC has shown that: (i) at neutral pH, only Phe-Gly is able to prevent the thermally induced aggregation of cytochrome c; (ii)
Phe-Gly interacts with Gly45 and Phe46 residues of the protein, either when the protein is in the folded or in the unfolded state; and
(iii) the interaction of Phe-Gly with cytochrome c is sequence-dependent. These results support the hypothesis that the basic principles
that describe protein aggregation can be used for the design of peptides with antiaggregating properties.
B1830 – Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability
Crystalline amylose complexes were prepared with decanal, 1-butanol, menthone and alpha-naphtol. Their crystalline structure and the related
helical conformation, determined by wide angle X-ray diffraction (WAXD) and 13C CPMAS solid state NMR, were assigned to V6I, V6II,
V6III and V8 types, respectively. It was possible to propose some hypotheses on the possible nature of interactions and especially intra-/interhelical
inclusion. Some shifts in the NMR C1 carbon signals were attributed to the presence of ligand in specific sites inside the structure for a
same type of V6 helical conformation. Moreover, the crystallinity and polymorphic changes induced by desorption/rehydration were studied.
A general increase of the carbon resonances sharpness upon rehydration has been observed, but also a V6II-V6I transition when decreasing
the water content. Differential scanning calorimetry (DSC) experiments were also performed to approach the thermostability of the four types
of complex and also the way they form again after melting/cooling sequences.
B1826 – Recovery of nanoparticles produced in phosphatidylcholine-based template phases
This paper focuses on the characterization and use of polymer-modified phosphatidylcholine (PC)/sodium dodecyl sulfate (SDS)-based
inverse microemulsions as a template phase for BaSO4 nanoparticle formation. The area of the optically clear inverse microemulsion phase
in the isooctane/hexanol/water/PC/SDS system is not significantly changed by adding polyelectrolytes, i.e., poly(diallyldimethylammonium
chloride) (PDADMAC), or amphoteric copolymers of diallyldimethylammonium chloride and maleamid acid to the SDS-modified inverse
microemulsion. Shear experiments show non-Newtonian flow behavior and oscillation experiments show a frequency-dependent viscosity
increase (dilatant behavior) of the microemulsions. Small amounts of bulk water were identified by means of differential scanning calorimetry.
One can conclude that the macromolecules are incorporated into the individual droplets, and polymer-filled microemulsions are formed.
The polymer-filled microemulsions were used as a template phase for the synthesis of BaSO4 nanoparticles. After solvent evaporation the
nanoparticles were redispersed in water and isooctane, respectively. The polymers incorporated into the microemulsion are involved in the
redispersion process and influence the size and shape of the redispersed BaSO4 particles in a specific way. The crystallization process
mainly depends on the type of solvent and the polymer component added. In the presence of the cationic polyelectrolyte PDADMAC the
crystallization to larger cubic crystals is inhibited, and layers consisting of polymer-stabilized spherical nanoparticles of BaSO4 (6 nm in
size) will be observed.
B1816 – Thermodynamics of alpha-lactalbumin-dl-a-dipalmitoylphosphatidylcholine interactions and effect of the antioxidant nicotinamide on these interactions
Differential scanning calorimetry has been used to understand the thermodynamics of the interactions of dl-alpha-dipalmitoylphosphatidylcholine
(DPPC) with alpha-lactalbumin and the effect of the antioxidant nicotinamide on these interactions. Nicotinamide decreases the
thermal transition temperature of both the lipid and the protein at high concentrations. The thermal unfolding transitions of the protein were
two state and calorimetrically reversible. There was no significant change in the shape and thermodynamic parameters accompanying the
lipid endotherms, suggesting that nicotinamide did not penetrate the lipid bilayer. The thermal unfoldings of alpha-lactalbumin in the presence of
DPPC as cosolute also adhered to two-state reversible mechanism. The changes in the thermodynamic parameters accompanying the thermal
transitions were small, indicating no significant interaction of alpha-lactalbumin with DPPC. The changes in the thermodynamic parameters
indicate that the lipid bilayer organization, as well as the partitioning of the extrinsic protein alpha-lactalbumin into the bilayer, is not affected in
the entire studied concentration range of the lipid. It is observed that the presence of increasing concentration of nicotinamide (as high as 1.0
mol dm 3) in the lipid-protein mixture does not affect its partitioning into the lipid bilayer, although nicotinamide preferentially interacts
with a-lactalbumin. The change in the effect of nicotinamide on lipid transition temperature in the mixture and literature report suggests that
nicotinamide may be forming a hydrogen-bonded complex with the protein through its amide functionality. The surface tension data of
aqueous nicotinamide in combination with the thermal denaturation results of protein in presence of nicotinamide confirmed that surface
tension effect does not have any significant contribution to the effect of nicotinamide on protein.
B1809 – Heat capacities of Uracil, Thymine, and its alklated, cyclooligomethylenated, and halogenated derivatives by Differential Scanning Calorimeter
The molar heat capacity (Cp) of solid uracil, its alkylated and halogenated derivatives, and cyclooligomethylenouracils
in the temperature range of (298.15 to 343.15) K by a differential scanning calorimeter (SETARAM
TG-DSC 111) were determined. It was demonstrated that the Cp value increases by increasing the number of
methylene groups attached to the diketopyrimidine ring. The correlations Cp ) f(T) are given. The contributions
of C-CH3, N-CH3, and C-NO2 groups as well as F, Cl, Br, and I atoms in the value of Cp for the temperature
of 298.15 K are presented. The Chickos method for calculation of Cp values is discussed.
B1799 – DSC and Raman studies of the side chain length effect of ubiquinones on the thermotropic phase behavior of liposomes
Differential scanning calorimetry and Raman spectroscopy have been used to examine the effects of ubiquinones (UQn) on the thermotropic
phase behavior of dipalmitoylphosphatidylcholine (DPPC) in multilamellar vesicles, for UQ/DPPC molar ratios ranging from 0.01 to 0.1. The
influence of the side chain length has been investigated by comparing the effect of a series of UQ with 2 (UQ2), 4 (UQ4), 6 (UQ6) and 10 isoprene
In the presence of increasing amount of UQ2 or UQ4, concomitant shift of the gel to liquid crystalline phase transition towards lower temperatures
and vanishing of the pretransition are observed. Short-chain ubiquinones are thus inserted parallel to phospholipid chains, their benzoquinone ring
being close to the DPPC polar headgroups. In addition, broadening and skewing of the main transition peak support the fact that UQ2 and UQ4
are laterally self-organized in highly concentrated regions located at the boundary of lipid domains. The lipid thermotropic behavior is not affected
by the presence of other analogues of the series, UQ6 and UQ10. They remain homogeneously dispersed within the midplane of the phospholipid
bilayer. Such a chain length dependence on the location and the organization of ubiquinones analogues may be correlated with their biological
activities in biomembranes.
B1798 – Thermal analysis on parchments I: DSC and TGA combined approach for heat damage assement
Ancient, newand artificially aged parchments were investigated with both differential scanning calorimetry (DSC) and thermogravimetry (TGA).
Criteria to define a quantitative ranking of the damage experienced by the bulk collagen of historical parchments were assessed. A damage-related
correlation was found between the collagen denaturation temperature and the moisture content of the parchment. Qualitative rules for the evaluation
of the damage at the nano-and mesoscopic level were achieved on the basis of peculiarities of the shape and width of the DSC signals and confirmed
by small angle X-ray scattering patterns.
B1773 – On the thermal stability of the two dimeric forms of ribonuclease A
The thermal stability of the two dimers of RNase Awith N- or C-terminal swapped ends is investigated by means of dissociation kinetics,
differential scanning calorimetry, and circular dichroism measurements. The data indicate that the dimer characterized by the swapping of the
N-terminal alpha-helices is less prone to monomerize when compared to the dimer characterized by the swapping of the C-terminal h-strands.
This finding is correlated to the structural features of the so-called open interface of the dimeric forms.
B1748 – Interaction of Ag(I), Hg(II), and Cu(II) with 1,2-ethanedithiol immobilized on chitosan: Thermochemical data from isothermal calorimetry
The nature of interactions between metal ions Ag(I), Hg(II), Cu(II) and chitosan derivative of 1,2-ethanedithiol, QTDT, was investigated by
isothermal calorimetry using the membrane breaking technique. Simultaneous determination of thermal effects, Qint, and amount of cation
that interacts, nint, are described. The experimental data have been interpreted in terms of the Langmuir equation to determine the maximum
adsorption capacity to form a monolayer, Nmon, and the energy of interaction for a saturated monolayer per gram of QTDT, Qmon. With
Nmon and Qmon, the molar enthalpy of interaction for formation of a monolayer of anchored cations per gram of QTDT, monHm, was
determined. The monHm values for Ag(I), Hg(II), and Cu(II) were -60.56, -58.05, and -84.36 kJ mol-1, respectively. Negative values
of G show the spontaneity of the interaction processes. The least entropically favourable processes, i.e., those which present more negative
S values, seem to be compensated by the more favourable enthalpic parameter.
B1738 – Neat DODAB vesicles: Effect of sonication time on the phase transition thermodynamic parameters and its relation with incomplete chain freezing
"Vesicle solutions formed by the cationic lipid dioctadecyldimethylammonium bromide have been extensively used in the past
20 years as model membrane systems. Often, discrepancies are found in the literature for the thermodynamic parameters of the
gel-to-liquid crystal (gel-lc) phase transition of these vesicles. In this work we present a systematic DSC investigation on the
influence of the sonication method on the main temperature and enthalpy of the transition for DODAB vesicles, prepared both
in normal and deuterated water. It is shown that as the sonication time increases, the Tm and enthalpy values decrease until a
plateau value is reached. This effect is associated with incomplete chain crystallization in the highly curved vesicles (produced
by sonication) when they are cooled below the transition temperature. The effect of aging on the sonicated solutions has also
been monitored by DSC."
B1733 – Characterization of a putative fusogenic sequence in the E2 hepatitis G virus protein
With the aim of better understanding the fusion process mediated by the envelope proteins of the hepatitis G virus (HGV/GBVC),
we have investigated the interaction with model membranes of two overlapping peptides [(267-284) and (279-298)] belonging to
the E2 structural protein. The peptides were compared for their ability to perturb lipid bilayers by means of different techniques such
as differential scanning calorimetry and fluorescence spectroscopy. Furthermore, the conformational behaviour of the peptides in
different membrane environments was studied by Fourier-transform infrared spectroscopy and circular dichroism. The results
showed that only the E2(279-298) peptide sequence was able to bind with high affinity to negatively charged membranes, to
permeabilize efficiently negative lipid bilayers, to induce haemolysis, and to promote inter-vesicle fusion. This fusogenic activity
could be related to the induced peptide conformation upon interaction with the target membrane.
B1731 – Interaction of fluoxetine with phosphatidylcholine liposomes
Fluoxetine (Prozac) is one of the latest of a new generation of antidepressants, approved by FDA in 2002.
The interactions of fluoxetine with multilamellar liposomes of pure phosphatidylcholine (PC) or containing cholesterol 10% molar were
studied as a function of the lipid chain lengths, using differential scanning calorimetry and spin labelling EPR techniques.
The DSC profiles of the gel-to-fluid state transition of liposomes of DMPC (C14:0) are broadened and shifted towards lower temperatures
at increasing dopant concentrations and, with less than 10% fluoxetine, any detectable transition is destroyed. The broadened profiles and the
lowered transition temperatures demonstrate that both the size and the packing of the cooperative units undergoing the transition are modified
by fluoxetine, leading to a looser and more flexible bilayer. No phase separation was observed.
The effects of fluoxetine on the thermotropic phase behaviour of DPPC (C16:0) and, even more, of DSPC (C18:0) are different from that
of DMPC. In fact, in the former cases, two peaks appeared at increasing dopant concentrations, suggesting the occurrence of a phase
separation phenomenon, which is a sign of a binding of fluoxetine in the phosphate region.
In cholesterol containing membranes, fluoxetine, even at low concentrations, leads to a general corruption of the membrane, both in terms
of packing and cooperativity, and the formation of any new phase is no longer observable.
EPR spectra reflect the disordered motion of acyl chains in the bilayer. It was found that fluoxetine lowers the order of the lipid chains
mainly in correspondence of the fifth carbon position of SASL, indicating a possible accumulation near the interfacial region.
B1724 – Identification of pitfalls in the analysis of heat capacity changes of -lactoglobulin A
Information on changes in heat capacity ( Cp) of proteins upon unfolding is used frequently in literature to understand possible followup
reactions of protein denaturation, like their aggregation propensity. This thermodynamic property is intrinsic to the protein's architecture and
unfolding and should be independent of the approach used to evaluate it. However, for many proteins, the reported values for Cp vary considerably.
To identify whether the origin of these discrepancies lies within the experimental approach chosen and/or in the too simplified unfolding models
used in the analysis of the data, we choose -lactoglobulin A, a relatively small protein, but disputed for its two-state unfolding, and established its
Cp from tryptophan fluorescence, near-UV circular dichroism and differential scanning calorimetric measurements. In view of the large variation
for the obtained Cp (between 3.2 and 10.1±0.8 kJ/(mol K)), it is evident that: (1) the sensitivity of different approaches to the structural changes;
(2) irreversibility of unfolding; (3) non-ideal two-state unfolding behaviour need to be considered prior to interpretation. While the first two points
can be addressed by using multiple approaches, the applicability of the selected unfolding behaviour for the analysis is often less easy to establish.
In this work, we illustrate that by checking the wavelength-dependence used to detect protein conformational changes a tool is provided that gives
a direct insight in the validity of the interpretation in these studies. An experimentally validated determination of Cp allows a more proper use
for the mechanistic understanding of protein denaturation and its follow-up reactions, avoiding pitfalls in the interpretation.
B1713 – Selected physical properties of chitin prepared from shiitake stipes
Fungal chitins were prepared from air-dried shiitake stipes by alkaline treatment and three decolorization methods. Yields of chitins A,
B and C were 283.2, 367.2 and 250.8 mg/g, respectively. Three chitins were similar in the element profile due to their related N/C ratios.
However, it seemed that the nitrogen content in chitin C was the highest (35.4 mg/g). Three chitins showed a melting endothermic peak at
248.65-248.92°C, higher than stipes at 243.53°C. The thermal decomposition enthalpies (DH) of chitins A and B were the same and
higher than DH of chitin C and stipes. Under electron microscopic examination, shiitake stipes showed the aggregated flakes with dense
and firm structure and without porosity whereas chitins were more compact than stipes. The major crystalline peak for three chitins was
at 19.3-19.61 and three WAXD patterns were similar. Generally, three chitins prepared showed similar physical characteristics as
evidenced by the element analysis, DSC, SEM and WAXD patterns.
B1712 – Structural studies of E. coli ribosomes by spectroscopic techniques: A specialized review
We present a review on our interdisciplinary line of research based on strategies of molecular biology and biophysics. These have been
applied to the study of the prokaryotic ribosome of the bacterium Escherichia coli. Our investigations on this organelle have continued for more
than a decade and we have adopted different spectroscopic biophysical techniques such as: dielectric and fluorescence spectroscopy as well as
light scattering (photon correlation spectroscopy). Here we report studies on the whole 70S ribosomes and on the separated subunits 30S and
50S. Our results evidence intrinsic structural features of the subunits: the small shows a more "floppy" structure, while the large one appears
to be more rigid. Also, an inner "kernel" formed by the RNA/protein association is found within the ribosome. This kernel is surrounded by a
ribonucleoprotein complex more exposed to the solvent. Initial analyses were done on the so called Kaldtschmit-Wittmann ribosome: more
recently we have extended the studies to the "tight couple" ribosome known for its better functional performance in vitro. Data evidence a
phenomenological correlation between the differential biological activity and the intrinsic structural properties of the two-ribosome species.
Finally, investigations were also conducted on particles treated at sub-denaturing temperatures and on ribosomes partially deproteinized by
salt treatment (ribosomal cores). Results suggest that the thermal treatment and the selective removal of proteins cause analogous structural
B1697 – Microcalorimetric determination of displacement adsorption enthalpies of protein refolding on a moderately hydrophobic surface at 308 K
Both microcalorimetric determination of displacement adsorption enthalpies ?H and measurement of adsorbed amounts of guanidine - denatured lysozyme (Lys) refolding on the surface of hydrophobic interaction chromatography (HIC) packings at 308 K were carried out and compared with that at 298 K. Study shows that both temperature and concentration of guanidine hydrochloride (GuHCl) affect the molecular mechanism of hydrophobic interaction of protein with adsorbent based on the analysis of dividing ?H values into three kinds of enthalpy fractions. The adsorption in higher concentrations of GuHCl (>1.3 mol L-1) at 308 K is an enthalpy-driving process, and the adsorption under other GuHCl concentrations is an entropy-driving process. The fact that the Lys denatured by 1.8 mol L-1 GuHCl forms a relatively stable intermediate state under the studied conditions will not be changed by temperature.
B1696 – Effect of salt concentrations on the displacement adsorption enthalpies of denatured protein folding at a moderately hydrophobic surface
The displacement adsorption enthalpies (?H) of the refolding of lysozyme (Lys) denatured by 1.8 mol L-1 guanidine hydrochloride (GuHCl) on a moderately hydrophobic surface at 298 K, pH 7.0 and various (NH4)2SO4 concentrations were determined by using a Micro DSC-III calorimeter. The study shows that the effect of salt concentrations on the three fractions of the enthalpy is that with increasing (NH4)2SO4 concentrations, the molecular conformation enthalpy of the adsorbed Lys has probably no distinct change at 1.8 mol L-1 GuHCl; the adsorption affinity enthalpy (exothermic) becomes more negative; and the dehydration enthalpy (endothermic) decreases. At lower salt concentrations, the dehydration, especially squeezing water molecules led by molecular conformation, which leads to an entropy-driving process, predominates over the adsorption affinity (also including the orderly orientation of molecular conformation), while at higher salt concentrations, the latter is prior to the former for contribution to ?H and induces an enthalpy-driving process. Also, the optimal NH4)2SO4 concentration favoring refolding and renaturing of Lys denatured by 1.8 mol L-1 GuHCl was found.
B1680 – Vesicles with superior stability at high temperature
The formation of vesicles in the synthetic bolaamphiphiles SEDA/DEAB mixed systems in the temperature range of (25-80°C) was found by FF-TEM. No phase transition temperature was detected in this temperature range by both DSC and fluorescent probe measurements. In addition, the results of the IR spectra showed that the hydrocarbon chains in the mixed systems are in highly ordered arranging state as the temperature increased to higher temperature. All these results showed the superior thermal stability of the vesicles in the SEDA/DEAB mixed systems.
B1675 – Thermal characterisation of actin filaments prepared from adp-actin monomers
The thermodynamic properties of the ADP- and ATP-actin filaments were compared by the method of differential scanning calorimetry. The lower melting point for the ADP-F-actin filament (58.4 vs. 64.5°C for ATP-F-actin) indicated that compared to the ATP-actin filaments its structure was less resistant to heat denaturation. The detailed thermodynamic characterisation of the proteins was carried out by the analysis of the calorimetric enthalpy, the entropy and the free enthalpy changes. All of the determined parameters gave lower values to the ADP-actin filaments than to the ATP-actin filaments. The calculated values of the activation energy also demonstrated that compared to the ADP-F-actin the ATP-F-actin was thermodynamically more resistant to the denaturing effect of heat.
Based on all of this information we have concluded that the actin filament prepared from ADP containing magnesium saturated actin monomers at pH 8.0 is thermodynamically less stable than the ones obtained from ATP-actin monomers
B1672 – DSC examination of the oesophagus after two different self-expandable stents implantation
An experimental study
Massive bleeding from oesophagus varices presents a life threatening complication of liver cirrhosis. No effective method of treatment is available until now, that would guarantee high grade of patient wellness during the conditioning and investigation phase until the definitive treatment could be introduced.
The fact that we have not found any report in the literature about self-expandable metal stents (SEMS) application in acute variceal bleeding had encouraged us to use stents usually used for oesophageal malignancy and furthermore develop a special stent for this individual indication.
The aim of this study was to evaluate the tissue response to oesophagus stent designed for stop acute variceal bleeding in animal experiment in compare with another stent used for iatrogenic treatment of different strictures of the oesophagus. Tissue oxygen saturation (StO2) measurement was performed before and after the implantation of the stents. Macroscopic and histological investigations of the stented oesophagus segments were observed after 10 days.
Differential scanning calorimeter (DSC) is a well-established method for the demonstration of thermal consequences of local and global conformational changes in biological systems, but it has never been used for the investigation of the oesophagus. According to our results the thermal denaturation of intact oesophagus, its mucosa and muscle fragments revealed significant differences compared to healthy sample in favour of the new stent.
B1669 – Effect of acetonitrile on the hydration of human serum albumin films: a calorimetric and spectroscopic study
A new experimental approach based on the combination of calorimetric and FTIR spectroscopic measurements was proposed to study simultaneously the sorption of water and organic solvent, and corresponding changes in the structure of protein films in the water activity range from 0 to 1.0. Enthalpy changes (?Htot) on the interaction of water with the dried human serum albumin (HSA) in the presence and absence of acetonitrile (AN) have been measured using a Setaram BT-2.15 calorimeter at 298 K. Spectroscopic data on water and organic solvent vapor sorption by the HSA films and the corresponding changes in the protein secondary structure were determined by means of a Bruker Vector-22 FTIR spectrometer. By using a water activity-based comparison we characterised the effect of acetonitrile on the hydration and structure of the HSA films. Acetonitrile (AN) sorption isotherm resembles a smooth curve. HSA film binds about 250 mol AN/mol protein at the lowest water activities. As the water activity increases from 0 to 0.8, the sorption of AN gradually decreases from 250 to 150 mol AN/mol HSA. At aw > 0.8, the sorption of AN sharply decreases to zero. Acetonitrile decreases markedly the water content at a given aw. This behavior suggests that the suppression in the uptake of water is due to a competition for water-binding sites on the HSA films by acetonitrile. Changes in the secondary structure of HSA were determined from infrared spectra by analyzing the structure of amide I band. Acetonitrile increases the intensity of the 1654 cm-1 band that was assigned to the alpha-helix structure. Changes in the intensity of the 1654 cm-1 band agree well with the decrease in water uptake in the presence of AN. An explanation of the acetonitrile effect on the hydration and structure of the HSA films was provided on the basis of hypothesis on water-assisted disruption of polar contacts in the initially dried protein
B1665 – Assessment of damage in old parchments by DSC and SEM
Environmental impact on parchment was investigated by differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Parchments subjected to accelerated ageing and old parchments were compared to evaluate quality and extent of deterioration. Stability of fibrillar collagen within parchment was determined from the changes in thermodynamic parameters associated with thermal denaturation. Parchment surface was characterised, and specific morphological criteria were selected for damage assessment. The thermodynamic and morphological changes of collagen induced by deterioration are discussed, and their correlations are proposed as a means of ranking damage in old parchments
B1663 – DSC study on the motor protein myosin in fibre system
We have examined by DSC the complexes of myosin with actin in fibre system in the absence of nucleotides and the intermediate state of ATP hydrolysis by mimicking stable complex with myosin and ADP and beryllium fluoride in muscle fibres. Comparing the DSC results with other structural analogues of phosphate Pi leads the conclusion that the AM.ADP.BeFx complex favours the AM.ADP.Pi complex in fibre system. The deconvolution of DSC scans resulted in four transitions, the first three transition temperatures were almost independent of the intermediate state of the muscle, the last transition temperature was shifted to higher temperature, depending on the actual intermediate states of ATP hydrolysis. In AM.ADP.Vi state the transition temperature at the second and third transitions (actin binding domain and myosin rod) varied only slightly, whereas the last one (the fourth transition) shifted markedly to higher temperature depending on the ternary complex, e.g. in case of ADP plus BeFx it was 77.7°C, the highest value in weakly binding state of myosin to actin. The sum of calorimetric enthalpies of the first and last curves was practically constant, but their fractions depended on the state of the muscle. In strongly binding state of myosin to actin (rigor, ADP state) the fraction of the first transition was much larger, than the last one, whereas in weakly binding state of myosin to actin, the fraction of the first transition decreased at the expense of the last one. It supports also the view that these transitions are parts of the same portion of the myosin molecule.
B1662 – Differential scanning calorimetric examination of the tracheal cartilage after primary reconstruction with differential suturing techniques
Long segmental tracheal defects often lead to life threatening clinical conditions. Treatment of these lesions still represents an unsolved surgical challenge. The aim of the study was to test the effect of continuous and simple interrupted suturing in the replacement of long segment tracheal defects using polytetrafluoroethylene (PTFE) vascular prosthesis in a rabbit model.
2 cm long segment of the cervical trachea was resected in 20 New Zealand rabbits. The trachea was replaced with reinforced polytetrafluoroethylene vascular graft. The anastomoses were performed telescopically using continuous (group I, n = 10) or simple interrupted sutures (group II, n = 10). Laser Doppler measurements were taken before the resection and following the anastomoses. Length of survivals were noted, the patency and microscopical pattern of the anastomoses were evaluated. Calorimetric examinations were performed to detect possible structural changes in the tracheal cartilage.
Following the resection local microcirculation decreased by 9 ± 4%. The anastomoses caused a significant decrease of 29% in group I (p = 0.02) and 13% in group II. The mean survival was 58 ± 17 and 135 ± 25 days, respectively. Calorimetric results showed no change after the resection, but significant shift in melting temperature and calorimetric enthalpy proved the presence of structural changes of the cartilage in group II.
We saw significant lowering of microcirculation following continuous sutures, while simple interrupted stitches produced only moderate decrease. We found that interrupted suture technique is superior to the continuous technique causing only moderate damage to the tracheal anastomosis.
B1661 – The effect of pyrene labelling on the thermal stability of actin filaments
The ability of actin to form filaments is fundamental to its biological function and often characterised by various methods in vitro. One of the most frequently used methods capitalises on the observation that the fluorescence emission of a pyrene label on the Cys-374 residue of actin is enhanced by a factor of 20 during polymerisation. This method inherently involves the chemical modification of actin monomers with pyrene. It was reported earlier that the pyrene labelling of actin monomers has only small effect on the polymerisation and depolymerisation rates of actin, indicating that the method is suitable to characterise the effect of actin-binding proteins or peptides on the polymerisation kinetics.
In our present work we tested the effect of the pyrene labelling on the thermal denaturation of actin filaments by using the method of differential scanning calorimetry (DSC). By recording the heat denaturation profiles of unlabelled and pyrene labelled actin filaments we observed that pyrene labelling shifted the melting point (Tm) of actin filaments from 66 to 68°C. A similar effect was detected in the presence of equimolar concentration of phalloidin where the Tm shifted from 79 to 82°C. We concluded that the observed pyrene labelling induced differences of the thermal denaturation of actin filaments were small. The DSC results, therefore, confirmed that the methods based on the measurements of pyrene intensity during actin polymerisation are suitable to characterise the polymerisation kinetics of actin under in vitro conditions.
B1660 – DSC examination of the rotator cuff muscles in rabbits
Rotator cuff tear is a common musculoskeletal disorder with pathological changes occurring in the structure of the rotator cuff musculature (fatty infiltration). Severe fatty infiltration, observed on MRI or CT scan, negatively influences the result of rotator cuff reconstruction in human beings. The basic histological and biochemical alterations in fatty infiltration of the rotator cuff muscle with torn tendon are still not clear. Differential scanning calorimetric (DSC) examination is a well-established method for the demonstration of thermal consequences of local and global conformational changes in biological systems. With foregoing studies, authors have demonstrated the feasibility of DSC in the investigation of the musculoskeletal system. The aim of this study was to establish the thermograms of the rotator cuff muscles with normal and torn tendons, experimentally induced in rabbits. The DSC results clearly proved that definitive differences are present between the muscles with normal and torn tendons, which have also been demonstrated by MRI and CT scans.
B1649 – Temperature-induced denaturation of Aes acetyl-esterase from Escherichia coli
The thermal stability of the Aes acetyl esterase from Escherichia coli has been investigated by means of differential scanning calorimetry and circular dichroism measurements. The calorimetric curves show a denaturation temperature of 68°C for Aes and 61°C for the single point mutant V20D-Aes. The same values are obtained from CD denaturation curves of the two proteins recorded in both the far-UV and near-UV regions. Even if the denaturation process is irreversible and characterized by a single calorimetric peak and a single inflection point in both far- and near-UV CD curves, the overall data indicate that the process is more complex than a two-state transition. This is in line with the presence of two structural domains in the 3D model of Aes, according to homology modelling. A comparison of the thermal stability of Aes with those of the homologous thermophilic EST2 and hyperthermophilic AFEST suggests that the optimization of charge-charge interactions should not be so effective in the case of the mesophilic enzyme.
B1623 – Detoxification of phenanthrene by C. elegans evaluated by calorimetry
The filamentous fungus Cunninghamella elegans IM 1785/21Gp has an ability to remove phenanthrene to hydroxylated products with high efficiency. The sensitivity of C. elegans spores to phenanthrene derived metabolic products, 9-phenanthrols, as a model example of hydroxyderivatives produced by this fungus, was determined in this work. Calorimetric measurements showed that 9-phenanthrol disturbed the metabolic activity and spore germination of C. elegans less than phenanthrene did. However, at concentrations exceeding 60-70 mg L-1, phenanthrol strongly affected the intracellular processes of this fungus.
B1616 – Thermodynamic characterization of different actin isoforms
The thermodynamic properties of the cardiac and skeletal alpha-actin isoforms were studied to characterize the molecular bases of the functional differences between them with the method of differential scanning calorimetry (DSC). The thermal properties of the actin filaments were described in the presence of calcium and magnesium ions as well. Based on the calculated free energy changes the alpha-cardiac actin filaments appeared to be more stable in its physiologically more relevant, magnesium saturated form. The magnesium saturated form of the alpha-cardiac actin filaments seemed to be more stable compared to the calcium saturated form of it. The enthalpy and entropy changes could differentiate between the alpha-cardiac and alpha-skeletal actin isoforms and between the calcium and magnesium saturated cardiac actin isoforms as well. Our results can demonstrate that the few differences between the amino acid sequences of the alpha-actin isoforms have an influence on the thermal properties and maybe on the function of these proteins as well.
B1615 – The effect of pH on the thermal stability of alfa-actin isoforms
The effect of pH was characterised on the thermal stability of magnesium saturated skeletal and cardiac alpha-actin isoforms with differential scanning calorimetry (DSC) at pH 7.0 and 8.0. The calorimetric curves were further analysed to calculate the enthalpy and transition entropy changes. The activation energy was also determined to describe the energy consumption of the initiation of the thermal denaturation process. Although the difference in Tmvalues is too small to interpret the difference between the alpha-actin isoforms, the values of the activation energy indicated that the alpha-skeletal actin is probably more stable compared to the alpha-cardiac actin. The difference in the activation energies indicated that lowering the pH can produce a more stable protein matrix in both cases of the isoforms. The larger range of the difference in the values of the activation energies suggested that the alpha-cardiac actin is probably more sensitive to the change of the pH compared to the alpha -skeletal actin.
B1614 – The effect of toxins on the thermal stability of actin filaments by differential scanning calorimetry
In our present study we performed the detailed characterisation of jasplakinolide and phalloidin on the thermal stability of actin filaments. The heat absorption curves were analysed by using the model established by Sanchez-Ruiz et al. . The analysis provided the activation energies attributed to the heat denaturation of actin filaments in the absence and in the presence of toxins. The results indicated that there are kinetic differences between the toxin-mediated stabilization of the Ca2+-and Mg2+-actin filaments. The effect of toxins appeared to be cation dependent.
B1613 – Examination of the swelling of heat protective hydrocolloids by DSC
While the basic fermented (sour) milk products, such as yogurt and kefir can be produced only in live flora version, the post heat-treatment is preferred in their flavored variations to increase the storage time. Casein being in sour coagulum precipitates during heat-treatment; therefore protective colloids surrounding the protein should be used to prevent it. Protective colloids are plant extracts, the most known of them are pectin and amylopectin. Basic requirement of protective colloid effect is the lower swelling temperature of hydrocolloid than the temperature of precipitation of sour coagulum. In this work we have examined the precipitation of sour coagulum as a function of the type of lactic acid bacteria cultures applied during fermentation as well as the swelling of heat protective plant hydrocolloids as a function of the composition (mainly of sugar content) of medium. To investigate the precipitation of fermented coagulum skimmed milk was fermented with mesophilic butter culture, thermophilic yogurt culture as well as with exopolysaccharide (EPS)-producing Prebiolact-2 culture. Precipitation was indicated in the increase of great extent of viscosity. Amylopectin was dispersed into aqueous solution of pH 4.5, the saccharose concentration of which was changed during the investigation of the swelling of heat protective hydrocolloids. A definite exothermic peak was assigned to the swelling of hydrocolloids during the DSC experiments. We could conclude that the precipitation temperature was increasing in the mesophilic-thermophilic-EPS producing microbes line, i.e. the heat stability and swelling temperature of hydrocolloids depend on the saccharose content of aqueous medium and they increase with rising the concentration of saccharose.
B1612 – Indication of proliferation of lactic acid bacteria in mixed cultures Estimation of their ratios by isoperibolic batch calorimetry
The supply and consumption of probiotic foods, and particularly probiotic dairy products, has grown steadily in recent years. In the production of dairy products of this type other microbes must also be used in addition to the microbes which provide the probiotic effect and which generally have a proliferation optimum at 37°C. The probiotic microbes have a neutral taste in dairy products consequently the taste of fermented dairy products is supplied by other microbes. These microbes are likewise lactic acid bacteria, and their proliferation optima are either below (mesophilic) or above (thermophilic) that of the probiotic microbes. It is imperative to have an indication of whether the probiotic bacteria have multiplied at the fermentation temperature used during the technology, since they provide the beneficial physiological effect of the product. Isothermic calorimetry appeared a suitable method for the indication of this process, because the amount of heat released during lactic acid bacterial proliferation differs from the probiotic one. In order to analyze the heat flow curves a deconvolutional program was developed which decomposed them into Gaussian curves, because the proliferation of individual microbes follows a lognormal distribution. The Gaussian curve characteristic for the culture was determined, and from the area under the curve the heat liberated during the creation of one microbe was calculated.
B1611 – Detection of probiotic microbes by DSC (isothermal) and that of the structure by electron microscope
Spreads with a fat content of 30-50% (these are called 'butter creams' in Hungarian and currently outsell butter) are generally made by post-heat treatment, and consequently contain no probiotic lactic acid bacteria. The Hungarian Dairy Research Institute (HDRI) has developed a spread that contains live lactic acid bacteria on the order of 10/g, some part of that are probiotic. The major advantage of the probiotic spread compared to the post-heat treated one is that it spreads easily and without sticking both at cold (0-5°C) and room temperature (20-24°C). This good spread ability arises from its structure, while its probiotic quality derives from the proliferation of probiotic microbes. During recent experiments the extent of probiotic microbe proliferation was examined by isotherm calorimetry, the microstructure was studied by electron microscope, and nutritional biological characteristics were measured (according to the ethical codex) by human clinical studies. By deconvolution of the heat flow curves it was determined that the probiotic cell count is in the order of 10/g, far in excess of the internationally accepted cutoff, 10/g. Structural research revealed that the fat globules in probiotic spread are distributed homogeneously, with an average diameter of less than 0.5 ?m. The findings from the human clinical examinations showed that the reduction in the blood cholesterol level occurring after consumption did not differ in the total cholesterol. The probiotic spread decreased significantly greater reduction in the supposedly harmful LDL cholesterol in the third hour following consumption, with less of a reduction in the HDL cholesterol level.
B1609 – Thermal stability of rat uterus during development DSC approach
The age dependence of thermal denaturation was monitored in rats anaesthetized after they're born at 7th, 14th, 21st, 28th, 35thand 42nddays. The samples were stored in rigor or physiological saline solution. The DSC scans in the early age groups show a low temperature exotherm (connective tissues: from gel to liquid crystal transition) and one endotherm (it is very probably the myosin). During further development the endotherms became more and more complex (due to the development of contractile system). At 42 days the scans seem to be similar to the adult ones. In the two buffers the endotherms markedly differ showing that the ATPase activity is present. In adult uterus, treated with nucleotides or estrogen, this activity significantly differs from the skeletal muscle. On the basis of our results we suppose, that the age dependent changes are decisive processes in the development of rat uterus.
B1608 – DSC measurement of cartilage destruction caused by septic arthritis
Treatment of a bacterial arthritis is a challenging task for a clinician as inadequate therapy can cause cartilage destruction and can result in severe osteoarthritis of the affected joint. The development of cartilage destruction in septic arthritis is not known in details. The aim of this study was to follow this process by calorimetric method. We induced experimental septic arthritis in knee joints of seven New Zealand rabbits by single inocculation of Staphylococcus aureusOKI 112001 culture (1.5 mL 8.108±5% c.f.u.). The first rabbit died on the 11thday. At that time all the other subjects were made overslept and samples were isolated from the cartilage of the femurs for calorimetric measurement. The DSC scans clearly demonstrated the development of infective structural destruction in cartilage from the first to the tenth day of incubation. In case of healthy control the melting temperatures (Tm) were: 49.7, 55 and 63.4°C and the total calorimetric enthalpy change (?H) was 0.55 J g-1. After the first day the enthalpy decreased (0.375 J g-1), the first two transition temperature shifted towards higher temperature: 57 and 63.15°C. Up to the fourth day the effect of infection culminated with Tmof 49.3, 55.9, 59.4, 62.8°C and further decrease of the ?H. At the fifth day the effect of infection is culminated in two separable thermal denaturation events (with 55 and 63.3°C Tms) with high jump in ?Hindicating the dramatic change of the structure of rabbit cartilage, so this time elapsed seems to be critical from the point of view of practical clinical relevance too. Between the 7thand 11thdays practically we had same melting temperatures (50 and 63°C) with low (~0.24 J g-1) enthalpy.
B1607 – Differential scanning calorimetric examination of the tracheal cartilage after primary reconstruction with differential suturing techniques
Resection and subsequent end-to-end anastomosis of the windpipe is a tried-and-tested acceptable method for the surgical treatment of segmental defects. There are a variety of different techniques for tracheal end-to-end anastomosis, but controversial reports highlight the fact that the suturing technique of the anastomosis is still subject of debate. We aimed to show the postoperative effects of the continuous and simple interrupted suturing technique respectively on the tracheal cartilage using differential scanning calorimetry. Transsection and subsequent reanastomosis of the cervical trachea was performed in 14 adult beagle dogs. The trachea was anastomized with continuous or simple interrupted sutures respectively depict no change in microcirculation after the resection of the trachea, but significant decrease following the completion the anastomosis with continuous sutures. Conventional histological analysis did not show any marked postoperative change in the tracheal cartilage but our DSC scans clearly demonstrated the differences between the intact cartilages and the ones involved in the anastomosis.
B1606 – Thermal analysis of the osteoarthritic human hyaline cartilage
Arthritis of major joints, especially osteoarthritis of the knee is a very frequent disease of human beings mainly in the developed countries. The pathology of osteoarthritis has been subject of many publications before, using a wide spectrum of different methods to evaluate degenerative changes of hyaline cartilage. The authors examined osteoarthritic human knee joint hyaline cartilage with differential scanning calorimetry. The different stages of cartilage degeneration have been verified by histological examinations. The research group demonstrated thermal differences between various stages of osteoarthritis. Besides explaining possible causes for experienced thermodynamic effects, the authors reflect upon future research ways and the possibilities of applying the method in practice.
B1605 – Effect of tetracaine on model and erythrocyte membranes by DSC and EPR
DSC and EPR experiments were performed on human erythrocyte membranes and DPPC vesicles in order to study the effect of the anaesthetic drug tetracaine on structure and dynamics of the lipid region. Experiments using spin label technique showed that tetracaine induced fluidity changes of the lipid region in the environment of the fatty acid probe molecules incorporated into the membranes in the vicinity of the lipid-water interface. Similarly to EPR observations, DSC measurements reported decrease of the main melting and the pretransition temperature in comparison to control DPPC vesicles, which is the sign of destabilisation of the structure in the head group region of the lipids. Similar effect was observed in the case of erythrocytes where the protein conformation was also controlled in the presence of drug. A separated membrane melting with well distinguished membrane protein phase transition was found that was affected significantly by tetracaine. These results suggest that tetracaine is able to modify not only the internal dynamics of erythrocyte membranes and produce destabilisation of the lipid structure, but the protein system as well. These might lead to further damage of the biological functions.
B1604 – Calorimetric determination of enthalpies of lysozyme folding at a liquid-solid interface
Calorimetric determination of the total enthalpy changes (?Hi) of guanidine-denatured lysozyme (Lys) during the adsorption with simultaneously refolding on the surface of hydrophobic interaction chromatography packings was carried out at 25±0.001°C. The measured ?Hiin the circumstances should include the changes in the three fractions: adsorption, dehydration and molecular conformation. It was found that when the unfolded Lys molecules are adsorbed and refold on the surface, entropy-driving caused by the dehydration of Lys mainly dominates the foregoing process. The refolding enthalpies of Lys, ??Hiwere found to be 10~100 folds higher than that measured in usual solutions.
B1596 – Interaction of phloretin and 6-ketocholestanol with DPPC-liposomes as phospholipid model membranes
Phloretin and 6-ketocholestanol are penetration enhancers for percutaneous delivery of certain topically applied drugs. In the present study some physicochemical experiments have been performed to elucidate the mechanism of action of phloretin and 6-ketocholestanol. The penetration enhancing effect of phloretin and 6-ketocholestanol is believed to be due to their increase of the fluidity of the intercellular lipid bilayers of the stratum corneum. Phospholipid vesicles were chosen as a simple model to represent these bilayers. The effect of phloretin and 6-ketocholestanol on phase transition temperature and enthalpy was studied using differential scanning calorimetry. Beside of that the size of liposomes was monitored when the amount of penetration enhancer in the liposome preparation was changed. Addition of increasing amounts of phloretin and 6-ketocholestanol to the bilayer resulted in lowering of phase transition temperatures and increasing the enthalpy. Additionally the size of the liposomes was increased when penetration enhancer was added. The results suggest that phloretin as well as 6-ketocholestanol would interact with stratum corneum lipids in a similar manner, both reduce the diffusional resistance of the stratum corneum to drugs with balanced hydrophilic-lipophilic characteristics.
B1569 – DSC study of gycerol-extracted muscle fibers in intermediate states of ATP hydrolysis
The heat capacity of contractile proteins actin and myosin was studied in psoas muscle of rabbit in strongly and weakly binding state of myosin to actin as a function of temperature by DSC. Deconvolution of the unfolding scans makes possible to characterize the structural domains of the macromolecules. We tried to approach the unfolding process in different intermediate state of ATP hydrolysis. The thermal transitions were calorimetrically irreversible, therefore the two-state irreversible model that describes fairly well the denaturation of different proteins was used for evaluation of the denaturation processes in muscle fibers in strongly (rigor, ADP) and weakly binding states (ATP·Vi, ADP·AlF4) of myosin to actin. Deconvolution resulted in four transitions, the first three transition temperatures were almost independent of the intermediate states of muscle, the last transition temperature was shifted to higher temperature, when the buffer solution was manipulated. The mean values in strongly binding states were Tm1=52.9±0.7°C, Tm2=57.9±0.7°C, Tm3=63.7±1.0°C and Tm4=67.8±0.7°C, but the last transition increased to higher temperature depending on the Pi analogue.
B1567 – Phase behaviour of polymer-grafted DPPC membranes for drug delivery systems design
DPPC dispersions containing DPPE with attached PEG of molecular masses 350, 2000 and 5000 were investigated by DSC in order to determine their phase behaviour and potential use as drug delivery systems. In comparison with previously obtained ESR data, DSC provided a definition of the lipid composition and temperature at which the vesicles are in a liquid crystalline phase. For DPPC DPPE-PEG 350 the composition range is at molar fractions 0<&khgr;PEG350<0.5.For DPPC DPPE-PEG 2000 the range of applicability is 0<&khgr;PEG2000<0.07 and for DPPC/DPPE-PEG 5000 system it is 0<&khgr;PEG5000<0.05.
B1546 – Thermal analysis of the cruciate ligaments of the human knee
Cruciate ligaments of the knee joint are important structures very often affected by the degenerative process in case of osteoarthritis. One of the most controversial issues in knee arthroplasty practice nowadays is the role of the posterior cruciate ligament. With foregoing studies authors have demonstrated the feasibility of DSC in the investigation of the musculoskeletal system. With current study authors established the thermal behaviour of healthy cruciate ligaments and detected the alterations in case of osteoarthritis. By establishing the DSC scans of the normal ligaments authors demonstrated clear differences between the posterior and anterior ligament. In addition alterations between normal and arthritic samples could be detected both in terms of changes in total enthalpy and heat capacity. Calorimetric findings have been verified by histological examinations as well.
B1535 – Thermal stability and internal dynamics of lysozyme as affected by hydration
A differential scanning micro-calorimetric (DSC) investigation has been performed to study the thermal stability of lysozyme solvated in glycerol as a function of the water content h(grams of water/grams of lysozyme). The unfolding process is strongly dependent on h, as it is witnessed by the behavior of the melting temperature Tm which shows a significant drop in the hydration range from 0 h to 0.4/0.5 h. The specific heat difference Cp between denatured and native state also displays an important variation for water amounts lower than ca. 0.5 h. For higher water amounts, both Tm and Cp seem to attain constant values. Such hydration dependent behavior is reminiscent of the trend exhibited by some quantities characterizing the protein internal dynamics on the pico- and nano-second timescale, as estimated by elastic neutron scattering. Also the protein mobility, which is measured through mean square displacements (MSD), increases mainly for water amounts lower than ca. 0.5 h, in the same range where the protein molecular rigidity rapidly drops down. This behaviour emphasizes the crucial role played by hydration water in determining both the protein thermodynamic and dynamic quantities.
B1534 – Solvent isotope effects on the phase-transition properties of lipid bilayers
Highly sensitive differential scanning microcalorimetry (DSC) has been used to investigate the phase transition properties of lipid vesicles prepared from 1,2-distearoyl--3-glyceryl-phosphatidylcholine (DSPC) in H2O and D2O. The data show that the response of pre-transition properties to D2O=>H2O substitution is stronger than the main transition properties. We find that there is a small increase in the phase transition temperature (?T0.5 K) and in the co-operative unit in the main transition. The increase in enthalpy (?H1 kJ.mol-1) and in transition temperature (?T2 K) observed in the pre-transition is comparable with that observed in quite different processes and systems, i.e. melting of nucleic acids and proteins and gel formation. It is suggested that D2O=>H2O substitution affects the thermal transition in these systems in such a way that the contributions of enthalpy and entropy to structural reorganization of water in these processes is modified.
B1533 – Effect of ethanol on the main phase transition of distearoylphosphatidylcholine
A highly sensitive scanning microcalorimeter and a low scanning rate (0.1 K/min) has been used to determine the thermodynamic properties of the main phase transition of distearoylphosphatidylcholine (DSPC) in presence of ethanol in the 0-0.20 ethanol mole fraction (x2) range. These measurements revealed previously unreported features of DSPC/water/ethanol phase behavior and visualized distinct changes in the main phase transition mechanism induced by addition of ethanol. It is found that the well known interdigitated phase in DSPC induced by ethanol above x2=0.01 abruptly disappears at x2=0.12. The abrupt change in the DSPC phase behavior at x2=0.12 shows some analogies with the observed one relative to the conformation response of DNA molecule to solvent condition altered by varying amounts of ethanol. As inferred from previous adiabatic compressibility and IR data in water/ethanol mixtures, a correlation between changes in properties of the solvent, and the effect of ethanol on both the conformational properties of DNA and the main phase transition of DSPC clearly appears from the data.
B1532 – Differential scanning calorimetry of chromatin at different levels of condensation
The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83°C, while the other two occur at 63°C and 74°C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63°C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.
B1531 – Structural stability of ribosomes subjected to RNase treatment evidenced by dielectric spectroscopy and differential scanning microcalorimetry
Previous studies from our laboratory demonstrated the existence of at least two levels of structural complexity in E. coli 70S ribosomes. Ribosomal RNA seems to be principally involved in the overall stability of these structures. In this paper we present an investigation of ribosomes subjected to treatment with RNase. The study is based on both differential scanning microcalorimetry and dielectric spectroscopy. In the thermograms obtained on treated ribosomes only the low temperature peak of the two typical denaturation events observed in native ribosomes, is promptly eliminated by the enzyme treatment. Dielectric spectroscopy measurements carried out on the same samples indicate an alteration of the dielectric behavior previously shown to consist of two subsequent relaxation processes. In fact, only the low frequency relaxation is affected by the treatment. The second one, observed at higher frequency, remains unaltered. The same effect on the dielectric parameters is observed if the ribosome particles are heated and then cooled prior to measurement. These results are consistent with the idea that two different structures are present within the ribosome. One is very stable and withstands both temperature and RNase treatment while the second is promptly abolished by both treatments. Data presented here strongly suggest that the RNA domains exposed to the solvent play a fundamental role in the stability of the 3-D structure of the ribosome particle.
B1530 – Instability of three-dimensional structures in ribosomal cores evidenced by microcalorimetric studies
In this paper we show a microcalorimetric investigation carried out on the so-called cores,
i. e. ribosomes deprived of select proteins by LiCl treatment. Thermal degradation of native
ribosomes gives rise to two thermal transitions occurring at different temperatures. In the
cores the high temperature peak persists even after treatment at very high ion strength (2 m
LiCl). This strongly suggests the existence of a very stable structure that was previously
observed also in particles treated with agents that hydrolyze the RNA moiety. The low temperature
peak gradually but dramatically decreases even though it never disappears completely.
This indicates that the treatment to obtain ribosomal cores does not cause complete
unfolding of the particle but only the destabilization of a structural three-dimensional domain
present in native ribosomes. These data are discussed in the light of previous results obtained
by dielectric spectroscopy and microcalorimetric studies on ribosomal particles.
B1495 – Native and hydrophobized human IgG. Enthalpies of heat-induced structural changes and adsorption onto silica
Differential scanning calorimetry (DSC) and isothermal calorimetric batch technique were used to monitor the heat-induced structural changes and adsorption properties of human immunoglobulin G (IgG), in native and hydrophobized states. The transition temperature (T max) and enthalpy of heat-induced conformational changes (cal H) of IgG in solution as well as the enthalpy change accompanying the adsorption of IgG onto hydrophilic silica (ads H), were shown to depend on the degree of the protein hydrophobicity (number of covalently attached alkyl chains). The adsorption enthalpy for all forms of IgG at all surface concentrations was found to be endothermic, that is the process is entropy driven. Factors affecting the IgG adsorption onto silica are discussed.
B1492 – Simultaneous effect of cadaverine and osmolytes on ct-DNA thermal stability
In the real cellular conditions, the biological macromolecules, as DNA and proteins, live in high concentrations of biomolecules, salts and so on. In particular organisms and cellular systems, which have adapted to extreme environmental conditions, accumulate particular organic solutes, known as osmolytes, at moderate to high concentrations to raise the osmotic pressure in the cytoplasm. This molecular crowding affects stability and activity of biological macromolecules. The goal of this work is to study the simultaneous effect of a natural osmolyte and polyamine on the stability of DNA. Particularly, we report a calorimetric study of the dependence of the thermal stability of a calf-thymus DNA (ct-DNA) on changing the osmolyte concentration in the presence of a constant concentration of a 1,5-diaminopentane, commonly known as cadaverine, which belongs to the class of natural polyamines. The osmolytes utilised were betaine, -arabitol, -arabitol and -sorbitol. The effect on the DNA thermal stability of cadaverine and of each osmolyte separately was also reported for comparison. Our data show that cadaverine does not lose its own ability to protect DNA by the destabilising action of high concentration of osmolyte molecules synthesised in conditions of environmental stress.
B1485 – Differential scanning calorimetric examination of the tracheal cartilage after primary reconstruction with continuos sutures. A preliminary study
Acquired upper airway stenosis is usually associated with a complex of pathological conditions at the high tracheal and the subglottic levels. Reported reconstructive techniques include widening of the airways by incorporation of grafts, segmental resection, and anastomosis or combined procedures. Progress in anaesthesia, surgical techniques, and understanding of the pathophysiology of the trachea has made primary tracheal reconstruction a safe operative procedure, although there are no reports observing its acute effect on the tracheal cartilage. Differential scanning calorimetry (DSC) is a well-established method for the demonstration of thermal consequences of local and global conformational changes in biological systems, including hyaline cartilage, but it has never been applied for the investigation of tracheal cartilage. According to the present study, the thermograms may prove the presence of structural changes of the cartilage after primary reconstruction in the short-term follow up (smaller melting temperature and calorimetric enthalpy in the operated dog). The differences were clearly demonstrated between the intact cartilages and the ones involved in the anastomosis.
B1482 – Interaction enthalpies of solid human serum albumin with water-dioxane mixtures : comparison with water and organic solvent vapor sorption
Enthalpy changes (?Htot) on the immersion of dehydrated human serum albumin (HSA) into water-dioxane mixtures have been measured using a Setaram BT-2.15 calorimeter at 298 K. Thermodynamic activity of water was varied from 0 to 1. Calorimetric results are discussed together with the FTIR-spectroscopic data on water and organic solvent vapor adsorption/desorption isotherms on solid HSA. Dioxane sorption exhibits a pronounced hysteresis. Calorimetric and dioxane desorption dependencies consist of two parts. No dioxane sorption was observed in low water activity region (aw<0.5). At low water activities, the ?Htot values are close to zero. At water activity about 0.5 the sharp exothermic drop of the interaction enthalpy values was observed. This exothermic drop is accompanied by the sharp increase in the amount of sorbed dioxane and additional water sorption (compared with that for pure water). Dioxane adsorption branch resembles a smooth curve. In this case, solid HSA binds more than 300 mol dioxane/mol HSA at low water activities. By using a water activity-based comparison we distinguished between dioxane-assisted and dioxane-competitive effect on water sorption. The obtained results demonstrate that the hydration "history" of solid protein is an important factor that controls as the state of protein macromolecule as well as the sorption of low-molecular organic molecules.
B1479 – Differential scanning calorimetry in the research of degenerative musculoskeletal disorders
Degenerative musculoskeletal disorders are very frequent diseases in human beings; the pathology of these has been a subject of much research before, using a wide spectrum of examining methods. DSC is a well-established method for the investigations of thermal consequences of alterations in biological systems. With foregoing studies, the authors have demonstrated the feasibility of DSC in the investigation of the intact hyaline cartilage and intervertebral disc (IVD). The aim of this study was to establish the thermograms of hyaline cartilage and intervertebral disc degeneration according to different stages. The calorimetric experiments with osteoarthritic knee joint samples and degenerated discs repeatedly demonstrated that the method is feasible for the investigation of tissues of the musculoskeletal system. The studies clearly demonstrated thermal differences between various stages of osteoarthritis and intervertebral disc degeneration.
B1466 – A differential scanning calorimetric study on the irreversible thermal unfolding of concanavalin A
A differential scanning calorimetry study on the thermal denaturation of concanavalin A at pH 5.2 where it exists in the dimeric form was carried out. The calorimetric transitions were observed to be irreversible and the transition temperature of the protein increased with increasing scan rate, indicating that the thermal denaturation process is under kinetic control. The thermal unfolding, and its scan rate dependence could be explained according to the kinetic scheme with k as first-order kinetic constant whose change with temperature is given by the Arrhenius equation. Using this model, rate constant as a function of temperature and activation energy of the process have been calculated. The average activation energy of the kinetic process using different approaches is 129±10 kJ mol-1. The differential scanning calorimetric results on transition temperatures and calorimetric enthalpies supported by intrinsic fluorescence indicate that the irreversibility in the calorimetric transitions of concanavalin A includes a combination of post-transition aggregation, chain separation and loss of cofactor.
B1465 – Microcalorimetry as a possible tool for phenanthrene toxicity evaluation to eukaryotic cells
Microcalorimetry and measurement of culture turbidity using a Bioscreen C Analyzer System were applied to study the toxic effect of phenanthrene on Cunninghamella elegans IM 1785/21Gp spore germination. The results of C. elegans spore incubation in Bioscreen C microbiology reader showed the inhibition of spore germination by 70% (with 25 mg l-1 of phenanthrene) and total inhibition of the fungus growth with a higher content of the xenobiotic (50-100 mg l-1). The microcalorimetric technique showed to be useful for the estimation of metabolic activity of C. elegans spores in growth medium up to xenobiotic concentrations of 90 mg l-1. These data corresponded with the microscopic observations. The obtained results showed that the microcalorimetry method could be a valuable supplement in the study on the mechanism of PAHs detoxification by fungi.
B1463 – Thermal stability of chemically denatured green fluorescent protein (GFP) A preliminary study
Green fluorescent protein (GFP) is a light emitter in the bioluminescence reaction of the jellyfish Aequorea victoria. The protein consist of 238 amino acids and produces green fluorescent light (?max=508 nm), when irradiated with near ultraviolet light. The fluorescence is due to the presence of chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-, which buried into beta-barrel.
GFP is extremely compact and heat stable molecule. In this work, we present data for the effect of chemical denaturing agent on the thermal stability of GFP. When denaturing agent is applied, global thermal stability and the melting point of the molecule is decreases, that can be monitored with differential scanning calorimetry. The results indicate, that in 1-6 M range of GuHCl the melting temperature is decreasing continuously from 83 to 38°C. Interesting finding, that the calculated calorimetric enthalpy decreases with GuHCl concentration up to 3 M (5.6-0.2 kJ mol-1), but at 4 M it jumps to 8.4 and at greater concentration it is falling down to 1.1 kJ mol-1. First phenomena, i.e. the decrease of melting point with increasing GuHCl concentration can be easily explained by the effect of the extended chemical denaturation, when less and less amount of heat required to diminish the remaining hydrogen bonds in beta-barrel. The surprising increase of calorimetric enthalpy at 4 M concentration of GuHCl could be the consequence of a dimerization or a formation of stable complex between GFP and denaturing agent as well as a precipitation at an extreme GuHCl concentration. We are planning further experiments to elucidate fluorescent consequence of these processes.
B1441 – Differential scanning calorimetric examination of the osteoarthritic hyaline cartilage in rabbits
Osteoarthritis of the knee is one of the most common musculoskeletal disorders with major pathological changes occurring in the structure of hyaline cartilage. Differential scanning calorimetric (DSC) examination is a well-established method for the demonstration of thermal consequences of local and global conformational changes in biological systems. With foregoing studies authors have demonstrated the feasibility of DSC in the investigation of the hyaline cartilage. The aim of this study was to establish the thermograms of hyaline cartilage degeneration in the knee joint, experimentally induced in rabbits. The calorimetric experiment of osteoarthritic samples in rabbits resulted in scans similar to those observed earlier in human samples. Measurements were reproducible both in terms of changes in total enthalpy and heat capacity and in the shape of DSC scans themselves.
B1440 – Calorimetric and spectroscopic properties of small globular proteins (bovine serum albumin, hemoglobin) after free radical generation
Mild oxidation of ---SH-containing proteins (serum albumin, hemoglobin) by Ce(IV)-ions in the presence of the spin trap phenyl-tert-butylnitrone (PBN) resulted in the appearance of strongly immobilized nitroxide free radicals which evidences the formation of thiyl radicals on the thiol site of the proteins. In hydroxyl free radical generating system a fraction of strongly immobilized nitroxide radicals was also detected in these proteins, which implies that the oxidation of a fraction of the thiol groups was also involved in the free radical reaction. According to the differential scanning calorimetry (DSC) experiments the melting processes of the proteins were calorimetrically irreversible, therefore the two-state kinetic model was used to evaluate the experiments. The results support the view that site-specific interaction of SH-containing proteins with hydroxyl and thiyl free radicals is able to modify the internal dynamics of proteins and affect the conformation of large molecules.
B1439 – Differential scanning calorimetric examination of the human intervertebral disc: establishment of calorimetric standards of different stages of degeneration
Intervertebral disc (IVD) degeneration is a common orthopaedic disorder with significant social and economic impact. The major pathological changes occur in the structure of anulus fibrosus (AF) and nucleus pulposus (NP). According to previous reports differential sacnning calorimetric (DSC) proved to be a suitable method for the demonstration of thermal consequences of local and global conformational changes in the structure of the human intervertebral discs. According to the present study, the DSC results clearly proved that definitive differences are present between the stages of disc degeneration in calorimetric measures. The structural differences between the stages could be also demonstrated by histology.
B1428 – Calorimetric and Fourier transform infrared spectroscopic study of solid proteins immersed in low water organic solvents
Calorimetric heat effects and structural rearrangements assessed by means of Fourier transform infrared (FTIR) amide I
spectra were followed by immersing dry human serum albumin and bovine pancreatic K-chymotrypsin in low water organic
solvents and in pure water at 298 K. Enthalpy changes upon immersion of the proteins in different media are in a good linear
correlation with the corresponding IR absorbance changes. Based on calorimetric and FTIR data the solvents were divided
into two groups. The first group includes carbon tetrachloride, benzene, nitromethane, acetonitrile, 1,4-dioxane, n-butanol,
n-propanol and pyridine where no significant heat evolution and structural changes were found during protein immersion.
Due to kinetic reasons no significant protein^solvent interactions are expected in such systems. The second group of solvents
includes dimethyl sulfoxide, methanol, ethanol, and water. Immersion of proteins in these media results in protein swelling
and involves significant exothermic heat evolution and structural changes in the protein. Dividing of different media in the
two groups is in a qualitative correlation with the solvent hydrophilicity defined as partial excess molar Gibbs free energy of
water at infinite dilution in a given solvent. The first group includes the solvents with hydrophilicity exceeding 2.7 kJ/mol.
More hydrophilic second group solvents have this energy values less than 2.3 kJ/mol. The hydrogen bond donating ability of
the solvents also assists in protein swelling. Hydrogen bonding between protein and solvent is assumed to be a main factor
controlling the swelling of dry solid proteins in the studied solvents.
B1426 – DSC analysis of the abnormalities of human leg skeletal muscles. A preliminary study
The standard calorimetric properties of the healthy human skeletal muscle are presented and compared to the same of human skeletal muscles in primary peripheral leg deformities (congenital clubfeet) and secondary deformities caused by the malfunction of the central nervous system (cerebral palsy). To our best knowledge no calorimetric analysis of either the healthy or pathologic human skeletal muscles have been reported previously. Eleven muscle samples from the three groups of patients were analyzed and compared. We hope to add to the understanding of the primary and consecutive functional behavioral changes of the human skeletal muscle due to different etiologic reasons. It has been found that the human muscles have different DSC scans than the rabbit skeletal ones and they are very characteristic for the actual functional and structural state, they behave as a fingerprint. To find the precise biochemical and structural explanation of these alterations there is need for further detailed examinations.
B1424 – Vanadate(Vi) and ADP induced domain motions in myosin head by DSC and EPR
Thermal stability and internal dynamics of myosin head in psoas muscle fibres of rabbit in the intermediate state AM.ADP.Pi - mimicked by AM.ADP.Vi - of the ATP hydrolysis cycle was studied by differential scanning calorimetry and spin label electron paramagnetic resonance spectroscopy.
Three overlapping endotherms were detected in rigor, in strongly binding ADP and weakly binding AM.ADP.Vi state of myosin to actin. The transition at 54.0°C can be assigned to the 50 k actin-binding domain. The transition at highest temperature (67.3°C) represents the unfolding of actin and the contributions arising from the nucleotide-myosin head interaction. The transition at 58.4°C reflects the melting of the large rod part of myosin. Nucleotide binding (ADP, ATP plus orthovanadate) induced shifts of the melting temperatures and produced changes in the calorimetric enthalpies. The changes of the EPR parameters indicated local rearrangements of the internal structure in myosin heads in agreement with DSC findings.
B1423 – DSC and EPR study on AMP.PNP, BEFx and AlF4 containing myosin nucleotide complexes
Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated skeletal muscle fibres to study the effect of the binding of nucleotides and nucleotide analogues to myosin. The thermal unfolding of muscle fibres in rigor showed three discrete domain regions with thermal stability of 52.2, 58.8 and 67.8°C. AMP.PNP and ATP plus AlF3 or BeF2 affected markedly the transitions, which implies the strong interaction between AMP.PNP or nucleotide analogues and catalytic domain of myosin, and a partial dissociation of heads from actin. ADP.BeFx and ADP.AIF 4 - states model the transition states of the ATP hydrolysis cycle which precede the powerstroke of the muscle fibres.
Spectrum deconvolution on isothiocyanate-labelled fibres in AMP.PNP-state resulted in two populations; 50% of labels was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The myosin heads which showed high degree of order were in the strongly binding ADP-state. The spectra in 4 - and ADP.BeFx state reflected random orientation of labels with increased rotational mobility in comparison with rigor. The results suggest that myosin in muscle fibres in ADP.BeFx state exists in two forms.
B1405 – Effects of some metal ions on the denaturational heat capacity increments in dilute solutions of ds-DNA
The native DNA duplex may be viewed as a cooperatively-ordered H bonded structure, including well localized Watson-Crick base pairs and H-bounded networks of hydration parts of the DNA-solvent interface in the grooves of the helix. In this paper, we present the effect of different metal ions (Li+, Mg2+ and Cu2+) on the denaturational heat capacity increment (?Cp) for calf thymus DNA.
Since the contribution from the ordered hydration water fraction disruption energy to the total enthalpy and heat capacity increment values of double helix melting is significant, it must be possible to detect the effect of metal ions on the structural ordering/disordering of the H-bounded network in the hydration shell of the DNA duplex.
Experimental results suggest that Li+, which is preferentially adsorbed in the minor groove of B-DNA and should contribute significantly to the stabilization of B-form, has a pronounced influence on the value of ?Cp. For the system Mg2+-DNA, the values of ?Cp are also significant, which can be explained by the formation of inner hydration sphere complexes and immobilization of structural water by the grooves of the duplex, stabilizing the helix. The effect of Cu2+ ions is much more pronounced. The satellite peaks of calf thymus DNA become lower as the concentration of Cu2+ increases and for concentrations of Cu2+ higher than 0.010 M, they disappear and ?Cp=0. This is indicative of the known preference of Cu2+ for purins and GC rich sites of DNA, binding to the N7 of guanine. As a result, disruption of the base stacking and hydrogen bounded water networks in the grooves of the double helix takes place.
B1404 – Low-temperature DSC study of the hydration of ss-DNA and ds-DNA and the role of hydrogen-bonded network to the duplex transition thermodynamics
The study of thermodynamic parameters of ice-water phase transition in aqueous solutions of ss-DNA, at different concentration of polynucleotide single chains is reported and the critical concentration for which the enthalpy of transition is zero has been determined. The thermodynamic parameters of ice-water phase transitions, obtained for native ds-DNA and for ss-DNA in the whole concentration region (0.2-2.0 g H2O/g DNA) and the hydration values (bound water quantity) for native (helix), denatured (single strand coils) and unfolded ss-DNA are compared. The critical values of the hydration at which all the water in the DNA-H2O system exists in the bound (unfrozen) state are established, for these conformations, with great precision: Nds-DNA?=(0.55±0.01) g H2O/g DNA, Nss-DNA?=(0.40±0.01) g H2O/g DNA. According to this calorimetric data the transformation "double stranded helix"=>"single stranded chains" is accompanied by dehydration of ds-DNA: ?N=Nds?-Nss?=(0.15±0.01) g H2O/g DNA. We suggest that the formation of B-DNA duplex from mixing of their complementary single strands is mostly accompanied by the uptake of structural water molecules and so, the overall hydration of a duplex is mainly determined by its conformation and stability.
B1361 – Nucleotides induced changes in skeletal muscle myosin by DSC, TMDSC and EPR
Electron paramagnetic resonance (EPR, ST-EPR) and differential scanning calorimetry (DSC) were used in conventional and temperature modulated mode to study internal motions and energetics of myosin in skeletal muscle fibres in different states of the actomyosin ATPase cycle. Psoas muscle fibres from rabbit were spin-labelled with anisothiocyanate-based probe molecule at the reactive sulfhydryl site (Cys-707) of the catalytic domain of myosin. In the presence of nucleotides (ATP, ADP, AMPPNP) and ATP or ADP plus orthovanadate, the conventional EPR spectra showed changes in the ordering of the probe molecules in fibres. In MgADP state a new distribution appeared; ATP plus orthovanadate increased the orientational disorder of myosin heads, a random population of spin labels was superimposed on the ADP-like spectrum.
In the complex DSC pattern, higher transition referred to the head region of myosin. The enthalpy of the thermal unfolding depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding to myosin. This was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head region The monoton decreasing TMDSC heat capacities show that Cp of biological samples should not be temperature independent.
B1351 – Differential scanning calorimetric and circular dichroistic studies on plant antifreeze proteins
Antifreeze protein (AFP) can lower the freezing point by inhibiting the growth of ice crystals. In this article, the thermal hysteresis activity (THA) of a plant AFP was measured with differential scanning calorimetry (DSC). As is shown, when the amount of ice in the sample was less than 5% THA of this AFP reached as high as 0.35°C. The secondary structure of this AFP was studied with circular dichroism (CD). The CD spectrum from 195 to 240 nm indicated a well-defined secondary structure consisting 11% -helix, 34% antiparallel -sheet and 55% random coil.
B1342 – Investigation of adsorption-induced structural changes of proteins at solid/liquid interfaces by differential scanning calorimetry
Adsorption of proteins on solid surfaces is widely studied because of its importance in various biotechnological, medical, and technical applications, e.g. biosensors, cardiovascular implants and chromatography.
One of the main problems is to what extend adsorption-induced conformational changes occur since they often modify the biological activity of the protein. Thus, the investigation of structural rearrangement due to interaction with the solid surface is of great interest. As protein molecules are in many cases continuously exchanged between the adsorbed and dissolved states, the question arises whether protein molecules re-adopt their original native structure after release from the surface.
In this study, two model proteins with well-characterized properties (human serum albumin (HSA) and alpha-chymotrypsin) were adsorbed from aqueous buffered solution onto finely dispersed hydrophilic silica particles. Adsorption isotherms were determined from the depletion of the supernatant, protein concentrations were analyzed by photometric methods. Reversibility of the processes was tested.
The structural rearrangements in the protein molecules induced from the adsorption process were probed by highly sensitive differential scanning calorimetry (micro-DSC). Transition temperature and enthalpy measurements for thermal unfolding were compared between given amounts of protein in free (native) and adsorbed/desorbed form.
From these measurements it appears that under the chosen adsorption conditions, HSA may lose essentially all of its cooperatively folded structure, while alpha-chymotrypsin retains full structure. The relation between the native protein structural stability on the one hand, and structural rearrangement on the other is discussed. Finally, the importance of conformational changes for spontaneous adsorption to occur is considered.
B1325 – Cerium-mediated free radicals in contractile proteins – EPR and DSC study
The effect of free radicals obtained in hydroxyl and cerium(IV)-nitrilotriacetic acid free radical generating systems on contractile proteins (actin, myosin and their complexes in glycerinated muscle fibres) was studied using differential scanning calorimetry and spin trapping electron paramagnetic resonance technique. The analysis of spectra showed that selective attack of thiol groups - Cys-257 and Cys-374 residues of actin, and among others Cys-707 residue of myosin - and random attack of sidechains of the main proteins of muscle tissue produced structural and functional changes, which affected the ATP hydrolysis cycle and very likely the dynamics of actin. The melting curves obtained on protein systems support the view that global conformational changes accompany the local damage of free radicals.
B1324 – Binding of nucleotides at the active site modulates the local and global conformation of myosin in muscle fibres
Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated muscle fibres to study the effect of the binding of nucleotides (ADP and AMPPNP) to myosin. The thermal unfolding of muscle fibres showed three discrete domain regions with thermal stabilities of 52.2, 58.8 and 67.8°C. AMPPNP markedly affected the transitions, implying the strong interaction between AMPPNP and catalytic domain, and partial dissociation of heads from actin. ADP produced only small changes in transition temperatures.
Spectrum deconvolution performed on isothiocyanate-labelled fibres in AMPPNP-state resulted in two populations; 50% of labels was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The conformation of the myosin heads which showed high degree of order were in the strongly binding ADP-state, the heads being attached to actin differ from those of heads in rigor. The results support the suggestion that the attached heads in strongly binding state to actin might have different local conformations.
B1323 – Differential scanning calorimetric examination of the human intervertebral disc: a preliminary study
Intervertebral disc degeneration is a common orthopaedic disorder with significant social and economic impact. The major pathological changes occur in the structure of anulus fibrosus and nucleus pulposus. Differential scanning calorimetry (DSC) is a well-established method for the demonstration of thermal consequences of local and global conformational changes in biological systems. It has never been applied for the investigation of intervertebral disc. According to the present study, the thermograms may prove and follow the changes in the structure of degenerated intervertebral discs. Differences were clearly demonstrated between the two major parts of the intervertebral discs as well as healthy and degenerated samples with the changes in total enthalpy and heat capacity.
B1322 – The effect of nucleotides (ADP ans ADP+Vi) on the thermal stability of rat uterus
Thermal unfolding of stripes prepared from rat uterus has been studied in the presence of nucleotides by differential scanning calorimetry (DSC). Using ADP, ATP and inorganic phosphate analogue orthovanadate, three intermediate states of the ATP hydrolysis cycle were simulated in the uterus stripes. In the main transition of the DSC pattern at least four overlapping endotherms were detected in rigor (AM), in strongly binding (AM.ADP) and weakly binding state (AM.ADP.Vi) of myosin to actin. It was found that nucleotide binding induced a shift of the main melting temperatures (from 60.7 to 61.1°C) and produced changes in the total calorimetric enthalpies (0.45 J/g for rigor, 0.4 J/g for strong binding, and 0.6 J/g for weak binding state). In the Krebs-Ringer bicarbonate buffer containing 100 nM estrogen (Oe) the main transition temperature shifted to 62.4°C and the total enthalpy change was 0.56 J/g. It seems to be an intermediate phase between the strong and weak binding state. The changes of the parameters of the peak functions suggest global rearrangements of the internal structure in myosin heads in the intermediate states.
B1317 – Nucleotide-induced changes in muscle fibres studied by DSC and TMDSC
Differential scanning calorimetry (DSC) was used in conventional and temperature-modulated mode to study the energetics of myosin in skeletal muscle fibres in different states of the actomyosin ATPase cycle. Psoas muscle fibres from rabbit were used in the experiments with and without the presence of nucleotides (ATP, ADP, AMP-PNP) and ATP or ADP+orthovanadate.
In the complex DSC pattern, the higher transition referred to the head region of myosin. The enthalpy of the thermal unfolding depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding to myosin. This was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head region. The monoton decreasing TMDSC heat capacities show that the Cp of biological samples should not be temperature independent.
B1316 – The role of bound water on the energetics of DNA duplex melting
A combination of common and low-temperature differential scanning calorimetry (DSC) techniques was used to detect the thermodynamic parameters of heat denaturation and of ice-water phase transitions for native and denaturated DNA, at different low water contents. We suggest that the main contribution to the enthalpy of the process of the heat denaturation of DNA duplex (35±5 kJ/mol bp) is the enthalpy of disruption of the ordered water structure in the hydration shell of the double helix (26±1 kJ/mol bp). It ispossible that this part of the energy composes the non-specific general contribution (70%) of the enthalpy of transition of all type of duplexes. For DNA in the condensed state the ratio=C p/S 2 is smaller than for DNA in diluted aqueous solutions (2-4). This means that there are other sources for the large heat capacity change in diluted solutions of DNA - for example the hydrophobic effects and unstacking (unfolding) of single polynucleotide chains.
B1296 – Thermodynamics and conformational properties of DNA triplexes containing 3′, 3′-phosphodiester bond
The thermodynamic characterization of the stability of two DNA triple helices of alternate sequence was obtained by differential scanning calorimetry (DSC). The conformational properties of these triple helices were investigated by circular dichroism (CD) and molecular mechanics. The triplexes under investigation form by way of major groove Hoogsteen association of a Watson-Crick 16-mer duplex with an all pyrimidine 16-mer or 15-mer third strand. The target duplex is composed of two adjacent oligopurine-oligopyrimidine domains where oligopurine sequences alternate on the two duplex strands. Both the third strands contain a 3',3'-phosphodiester junction, which introduces the appropriate inversion of polarity and let the switch from one oligopurine strand of the duplex to the other. The two-third strands differ for the lack of a cytidine monophosphate in the junction region. Thermal denaturation profiles indicate the initial loss of the third strand followed by the dissociation of the target duplex with increasing temperature. Transition enthalpies, entropies and free energies were derived from DSC measurements. The comparison of Gibbs energies reveals that a more stable triplex is obtained when in the third strand there is the lack of one nucleotide in the junction region. The thermodynamic data were discussed in relation to structural models.
B1293 – Interaction between lipopolysaccharide and detergents detected by differential scanning calorimetry
The Gram-negative bacteria can cause very serious diseases. One of their pathogenetic factors is the endotoxic lipopolysaccharide (LPS). This molecule is a component of outer membrane of Gram-negative bacteria. The LPS is released from the living bacteria in a modest measure, but after killing off bacteria by antibiotics a larger quantity of endotoxin is released into the surrounding. This molecule can induce different biological effects including pyogenicity and toxic shock. The LPS is an amphiphilic molecule: it has a hydrophobic and a hydrophilic part of molecule. In water, the LPS can aggregate. This aggregation process has been followed in our experiment by calorimetry. When we mixed the LPS extracted from Shigella sonnei Re 4350 with different detergents (polymyxin B (PmB), deoxicholat (DOC)) in different concentration, we could diminish the measure of aggregation. The heat treatment process was reversible within the temperature range and detergent concentration used by us.
B1292 – Metabolic depression and investigation of glucose/ethanol conversion in the european eel (Anguilla anguilla Linnaeus 1758) during anaerobiosis
The European eel (Anguilla anguilla Linnaeus 1758) is an important commercial fish species for aquaculture and fisheries. In aquaculture, one of the main hazards for the farmer is low oxygen condition due to technical disturbances or calamities. Therefore, in this study, we investigated the tolerance and metabolic response of European eel to anoxic conditions. In a 1 l flow-through Sétaram microcalorimeter we measured a 70% reduction of the Standard Metabolic Rate (SMR) during a period of 1 h anoxia, a process called metabolic depression. This strategy has the advantage that the survival time during anaerobioses can be extended because of a reduction of energy consumption and reduction of end product accumulation. Correcting for the time constant of the calorimeter by deconvolution techniques (time lag correction), we could describe the dynamics of the process of metabolic depression in European eel. From the deconvoluted signal it can be concluded that the 70% metabolic depression of European eel under anoxia takes place within 30 min. In parallel asphyxia experiments with European eel, no increased levels of ethanol were observed in blood plasma or ambient water. Ethanol concentrations in blood plasma were even significantly lower in the asphyxia group, probably indicating a decreased microbial activity. The 18-fold increase of plasma lactic acid is indicative for activation of Embden-Meyerhof glycolysis during anaerobioses.
B1287 – Nucleotide analogue induces global and local changes in muscle fibres
The effect of AMP.PNP on the thermal stability and dynamics of myosin head were investigated by using DSC and different spin label technique for chemically skinned muscle fibres prepared from rabbit. The thermal unfolding of the fibres in rigor, strong as well as weak-binding state showed a complex process characterizing at least three discrete domain regions with different stability (T m=54, 58.4 and 62.3°C). The unfolding at 54°C refers to the catalytic domain of myosin, whereas transition at T m=58.4°C represents the rod-like region. In the presence of AMP.PNP only the parameters of the last transition changed significantly (T m=70.4°C) showing an increased interaction between actin and myosin heads being attached to actin. Measurements on MSL-fibres (labelled at Cys-707 of myosin) in the presence of AMP.PNP showed that about half of the cross-bridges dissociated from actin. This fraction had a dynamic disorder, the other population had the same spectral feature as in rigor. In contrast, on TCSL-fibres AMP.PNP increased the orientational disorder of myosin heads, a random population of spin labels was superimposed on the ADP-like spectrum showing conformational and motional changes in the internal structure of myosin heads. ST EPR measurements reported increased rotational mobility of spin labels in the presence of AMP.PNP. The DSC and EPR results suggest that in the presence of AMP.PNP the attached heads have the same global orientation as in rigor, but the internal structure undergoes a local conformational change.
B1266 – Calorimetric study of the glass transition process in humid proteins and DNA
By method of differential scanning calorimetry the absolute values of heat capacity for the systemwater-biopolymer (globular and fibrillar proteins and DNA) were measured in a wide range of temperatures (from -30 up to 130°C) and concentrations of proteins both in native and denatured states. Thermal properties of humid denatured biopolymers demonstrate a characteristic anomaly in the form of the heat capacity jump at temperature depending on the bound water content. It has been shown that in the systems studied a glass transition, where water serves as a native plasticizer, is observed. It has been established that the S-shaped character of all heat capacity curves obtained on dehydration for native and denatured biopolymers is due to the gradual transition to the glassy state of both native and denatured samples. It was found that thermally denatured humid small globular proteins at subsequent dissolving in water at room temperature are able to restore their native structure.
B1265 – DSC study of the postdenaturated structures in biopolymer-water systems
The temperature dependences of heat capacity for water-denaturated biopolymer (globular proteins, collagen and DNA) were measured in a wide range of temperatures (0-140°C) and water content of the systems. It has been shown that thermally denaturated globular proteins (lysozyme, myoglobin and catalase) are able to form the thermoreversible heat-set structures under the certain conditions studied. The additional endothermal maximum observed is the calorimetric manifestation of the phase transition related to the melting of these thermotropic non-native structures. The melting gels are completely formed just after denaturation during relatively short time and only their prolonged state at T>T d leads to their transformation to thermoirreversible non-melting ones. The post denaturated structures from water-denaturated protein (Mb, Lys and RN-ase) systems with a different amount of free water were also studied. The thermoreversible cold-set gels are formed from both water-denaturated DNA and water-denaturated collagen systems. These thermotropic structures are metastable. A spatial gel network of both collagen and DNA is formed from the native-like renaturated structures.
B1260 – Effect of oxygen free radicals on myosin in muscle fibres
Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR, both conventional and saturation transfer EPR) were used to study the motional dynamics and segmental flexibility of myosin in muscle fibres in the presence of free radical generating system.
Muscle fibre bundles isolated from psoas muscle of rabbit were spin-labelled with maleimide- and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe molecules decreased with time following a single exponential curve. MgADP and MgATP plus orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system. The analysis of the EPR spectra of spin-labelled and oriented fibres showed that the narrow distribution of spin labels changed in the presence of hydroxyl free radicals. Spectrum analysis by computer subtraction showed that short irradiation by UV light resulted in the enhancement of the ordered population at the expense of the disordered population. This suggests a transition of myosin heads from weak- binding state into strong-binding state.
DSC measurements performed on calf cardiac myosin resulted in two main transitions at 49.4 and 54.1°C, respectively. Addition of MgADP produced a decrease of the 49.4°C transition, whereas a shift towards higher temperature was detected at the 54.1°C transition. It shows that there is an inter-site communication between the domains of the myosin. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves.
B1259 – Thermodynamic stability of ribonuclease B
The thermodynamic stability of pancreatic ribonuclease B (RNase B), which possesses identical protein structure of pancreatic ribonuclease A (RNase A), but differs by the presence of a carbohydrate chain attached to Asn 34, was studied by means of differential scanning calorimetry (DSC) at different pH conditions. The comparison between the two proteins has shown a little but significant stabilization of RNase B with respect to the unglycosylated one at pH values higher than 7.0. The thermodynamic analysis reveals the carbohydrate moiety to have a small stabilization effect of 3 kJ mol-1 at pH 8.0 and 63°C on the protein.
B1216 – Differential scanning calorimetric examination of the human hyaline cartilage. A preliminary study.
Arthritis of major joints especially osteoarthritis of the knee is a very frequent disease of human beings mainly in the developed countries, the major pathological changes occur in the structure of hyaline cartilage. Differential scanning calorimetry (DSC) is a well established method for the demonstration of thermal consequences of local and global conformational changes in biological systems. It has never been applied for the investigation of cartilage. According to the present study the thermograms may prove and follow the changes in the structure of cartilage in different stages of osteoarthritis. The differences were clearly demonstrated between the various anatomical origins of the cartilage as well as intact and osteoarthritic samples with the changes in total enthalpy and heat capacity, as well as by the shape of DSC scans themselves.
B1208 – UV generated oxygen free radicals in cardiac myosin. DSC and EPR study.
Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) were used to study the motional dynamics and segmental flexibility of cardiac myosin in the presence of free radical generating system.
Cardiac myosin isolated from calf heart muscle were spin-labelled with maleimide- and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe molecules attached to cardiac myosin decreased with time following a single exponential curve. MgADP and MgADP plus orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system.
DSC measurements resulted in two main transitions at 49.4°C and 54.1°C, respectively. Addition of MgADP produced a decrease of 49.4°C transition, whereas a shift towards higher temperature was detected at 54.1°C transition. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves. The total enthalpy of the UV irradiated myosin decreased significantly in different intermediate states of the ATP cycle.
B1207 – Functional and structural differences in skeletal and cardiac myosins. A molecular dynamic approach.
conventional and saturation transfer electron paramagnetic resonance spectroscopy and differential scanning calorimetry were used to study the internal dynamics and stability of cardiac myosin.
Intact and LC 2-deficient myosin isolated from bovine heart were spin-labelled with maleimide and iodoacetamide probe molecules at the SH1 sites. It was found that the probe molecules rotate with an effective rotational correlation time of 42 ns, which is at least six times shorter than the rotational correlation time of the same label on skeletal myosin. Addition of MgADP induces intrinsic changes in the multisubunit structure of myosin, but it does not lead to changes of the overall rotational properties of the myosin head.
Temperature dependence of the EPR spectra of maleimide-labelled myosin shows continuous decrease of the spectral parameters (intensity ratio of the peak heights, hyperfine splitting) at increasing temperature. However, marked changes were obtained at about 16°C in LC 2-deficient myosin. DSC measurements also support the view that the removal of the LC 2 light chain produces change in the internal structure of cardiac myosin.
B1203 – DSC studies of the effects of cisplatin and transplatin on G-actin.
The effects of cisplatin and its trans isomer transplatin on the thermal denaturation of G-actin were studied with a Micro DSC-III differential scanning calorimeter. The denaturation enthalpy of G-actin was found to be 12 J g-1, and the denaturation temperature was 328 K. The thermal denaturation curve showed that increasing cisplatin concentration decreased the enthalpy change. However, after the ratio of cisplatin to G-actin attained 8:1 (mol:mol), the denaturation enthalpy no longer decreased. Transplatin decreased the enthalpy change more rapidly. In contrast with cisplatin, the denaturation peak at 328 K disappeared, and a strong exothermic peak appeared at 341 K when the ratio of transplatin to G-actin was 8:1 (mol:mol). The enthalpy change was 75 J g-1, which is far in excess of the range of weak interactions. This strong exothermic phenomenon probably reflects the agglutination of protein. The effects of cisplatin and transplatin on the number of the free thiol groups of G-actin are discussed.
B1200 – Thermal denaturation of bacterial cells examined by differential scanning calorimetry.
Thermal stability of vegetative cells of Listeria monocytogenes, Escherichia coli and Lactobacillus plantarum was studied by counting viable fractions and determining DSC curves of their suspensions. DSC curves in the 5-99°C range showed a series of endothermic transitions between 50 and 60°C, where the heat destruction of cells occurred. Heat denaturation of DNA required a higher temperature than cell killing. Thermal death was strongly influenced by the pH, composition and NaCl content of the suspending buffer. A mathematical model developed by us enabled comparison of DSC peak temperatures and temperatures required for loss of viability.
B1199 – Thermodynamic behavior of mixed biopolymers in solution and in gel phase.
The thermodynamic properties of mixtures of two biopolymers, namely maltodextrin and gelatin, have been studied from the gelation as well as from the solution properties points of view. Differential scanning calorimetry has been used to monitor the changes in enthalpy due to the melting of the gel and to evaluate the cooperativity parameter of the gelatin in dilute and semi-dilute concentration. Heats of dilution of the single biopolymers and heats of dilution of the mixed biopolymer solutions have been used to evaluate the Flory interaction parameters within the framework of a new experimental procedure. These data are useful in the description of the thermodynamics of mixed biopolymers and complement other data in progress on this or similar systems.
B1195 – Vanadate and nucleotide induced domain motions in myosin head
Differential scanning calorimetry (DSC) and conventional and saturation transfer electron paramagnetic resonance spectroscopy were used to study the rotational dynamics and structural stability of myosin in different states of the actomyosin ATPase cycle.
B1194 – A calorimetric study of methylcellulose gelation.
Thermograms of methylcellulose solutions have been obtained using samples with different distributions of methyl substituents. It is
concluded that hydrophobic interactions between zones of highly substituted units are at the origin of the gelation. A minimal value of the
degree of substitution (equal to 1.2) is demonstrated to be necessary for the gelation to be observed either from enthalpy calculations or by
B1191 – Thermodynamics of a 24-mer triple helix formation and stability
In this work we report a thermodynamic characterization of stability and melting behaviour of two 24-mer DNA triplexes. The third strand, that binds the Watson-Crick double helix with Hoogsteen hydrogen bonds, contains 3-3 phosphodiester junction that determines the polarity inversion. The target double helix is composed of adjacent and alternate fragments of oligopurine-oligopyrimidine tracts. The two helices differ from the substitution of the cytosine, involved in the junction, with the thymine. Calorimetric data reported here provide a quantitative measure of the influence of pH and base modification on the stability of a DNA triplex.
B1166 – Structural stability of actin filaments as studied by DSC and EPR.
Thermal stability of actin, isolated from skeletal muscle, was studied in monomeric and polymerized form using DSC and EPR spectroscopy. The analysis of the DSC profiles in both forms enabled the assignment of the components: two endothermic components in the G-form (47.3±1.4°C and 53.4±1.8°C with enthalpies of 184.4±10.9 and 120.0±7.5 kJ/mol), and three endothermic components in the F-form (Tm1=59.7±1.4°C, H1=171.3±1.7 kJ/mol; Tm2=60.6±1.6°C, H2=231.2±9.6 kJ/mol; Tm3=61.3±1.4°C, H3=148.3±4.6 kJ/mol).
B1146 – Thermal stability of alcohol dehydrogenase enzyme determined by activity assay and calorimetry.
The thermostability of pure yeast alcohol dehydrogenase was investigated at various temperatures, in the presence and absence of sucrose, by both activity assay and differential scanning calorimetry. The thermal inactivation exhibited nonlinear biphasic behavior. The thermal inactivation rate constants and the magnitude of the heat-stable and heat-labile fractions of the enzyme were quantified. The values of the denaturation temperature were experimentally measured by calorimetry. It was found that although activity assay and calorimetry are based on different principles, they yield results that agree well with each other. However, each technique provides unique data (e.g. enzyme activity vis-a-vis basic thermodynamic properties, such as the denaturation enthalpy) and the two techniques may be considered complementary to each other.
B1141 – Synergetic effect of polyols with tetrabutylammonium bromide and urea on the thermal stability of lysozyme.
The thermal denaturation of lysozyme was studied in 2.0 molal aqueous solutions of polyols at pH 2.50 and varying concentration of glycerol at pH 2.50 and 6.00 using differential scanning calorimetry (DSC). The transition temperature, heat capacity, enthalpy, entropy and free energy of stabilization have been determined by a least square fit of the excess heat capacity data to the two-state model. Polyols are found to stabilize lysozyme and the stabilization increases with an increase in the number of hydroxyl groups. The stabilization increases with an increasing concentration of glycerol. The stabilization has been explained in terms of preferential hydration or due to the strengthening of the water structure which in turn intensify the hydrophobic interactions of the protein.
B1129 – Comparative study of myosins in solutions and supramolecular complexes. Effect of nucleotides.
Electron paramagnetic resonance (EPR) and differential scanning calorimetric (DSC) measurements were performed to
study the motional dynamics and structural stability of myosins prepared from skeletal muscle of rabbit and cardiac myosin of
bovine heart in different intermediate states of the ATP pathway in solution and supramolecular complexes. ADP, the
nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate (AMP.PNP) and ADP plus orthovanadate (Vi) were used in
different muscle model systems, as chemically skinned muscle fibres, myofibrils prepared from chemically skinned fibres and
myosin solutions to simulate the pre-power (AMP.PNP-state, ADP.Vi-state) and post-power (rigor state, ADP-state) stroke
conformations of myosin heads.
Both DSC and EPR measurements support the view that the myosin heads following attachment to actin undergo a sequence
of conformational states that differ both dynamically and energetically from each other. The differences, in the melting
temperatures and rotational mobility at the different intermediates clearly indicate significant alterations in the internal
microstructure of myosin head region induced by binding of nucleotides.
B1085 – Internal flexibility of cardiac myosins.
Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) and differential scanning calorimetry (DSC) were used to study the motional dynamics and segmental flexibility of cardiac myosins.
Cardiac myosins isolated from bovine and human heart muscle were spin-labelled with isothiocyanate- or maleimide-based probe molecules at the reactive sulfhydryl sites (Cys-697 and Cys-707) of the motor domain. The maleimide probe molecules attached to human cardiac myosin rotated with an effective rotational correlation time of 33 ns which was at least eight times shorter than the rotational correlation time of the same label on skeletal myosin (260 ns). In the presence of MgADP and MgADP plus orthovanadate, flexibility changes in the multisubunit structure of myosins were detected, but this did not lead to changes of the overall rotational property of the myosin heads. Significant difference in the internal flexibility was detected on myosin samples isolated from ischemic tissue, the rotational correlation time decreased to 25 ns.
DSC measurements supported the view that addition of nucleotides produced additional loosening in the multisubunit structure of cardiac myosin. It is postulated that there is an intersite communication between the nucleotide binding domain and the 20 kDa subunit where the reactive thiol sites are located.
B1055 – Thermal denaturation of bovine serum albumin and its oligomers and derivatives pH dependence.
In a previous paper, we report a preliminary DSC study on bovine (BSA) and human (HSA)
serum albumins. However, at accurate HPLC analysis the commercial proteins show three peaks:
Fraction V-I, probably globulins (as declared by the producers), Fraction V-II (about 15-18%
of the product) and Fraction V-Ill that represents pure BSA or HSA. A hypothesis is that the
Fraction II is a covalent dimer, or trimer or a mixture of both, generated during the scalf-life of
the commercial product.
Denaturation enthalpies of the purified Fraction V-Ilk and Fraction V-II of BSA, have been determined
calorimetrically, at changing the pH, and the results of both compared with those obtained
on the untreated protein. Few calorimetric experiments have been also carried on a BSA
monomer derivative with sulphidril group protected. Computer program have been developed for
the deconvolution of exo- and endothermic effects and for the analysis of thermal denaturation
B1039 – Kinetic and calorimetric investigations on micelle formation of block copolymers of the poloxamer type
Block copolymers of the Poloxamer type EOxPOyEOx (where EO = ethylene oxide and PO = propylene oxide) form micelles in aqueous solution that have a hydrophobic core of PO blocks and a strongly hydrated shell of EO blocks. The influence of electrolyte and surfactant on the aggregation behavior of three Poloxamers (F127, F88 and P123) was investigated by differential scanning calorimetry (DSC) and T-jump measurements. The micelle formation is influenced both by addition of electrolyte and of surfactant. The critical micellization temperature of the Poloxamer decreases linearly with increasing weight fraction of electrolyte and the decrease is independent of the cation size (Li+, Na+, K+). The DSC peak that is due to the micelle formation of the Poloxamer disappears with addition of surfactant. The adsorption of the anionic surfactant sodium dodecyl sulfate (SDS) starts at a lower concentration than the adsorption of the cationic surfactant N-dodecyl-N, N, N-trimethyl ammonium bromide (DTAB), whereas the latter is adsorbed to a larger extent.
For all investigated Poloxamers one relaxation time ? in the millisecond range is obtained. For constant temperature the relaxation time decreases with the total concentration of block copolymer. For constant concentration of block copolymer the relaxation time decreases with increasing temperature and increasing surfactant concentration. The relaxation process can be described by the Aniansson-Wall mechanism and values for the association rate constant for the poloxamers were determined. The evaluated rate constants are in the range of 0.2 x 10^(7) - 2 x 10^(7) l mol-1 s-1 and are not diffusion controlled. The micellization kinetics depends on the molecular structure of the Poloxamer, which determines the structure of the micelles. The thicker the shell of the micelle (i.e. the EO block length), the smaller the association rate constant k+. Keeping the EO block length constant and decreasing the PO block size again increases k+. Now the hydrophobic part of monomeric Poloxamer controls the penetration through the EO shell.
B1038 – Interaction of ABA block copolymers with ionic surfactants: influence on micellization and gelation
Block copolymers of the poloxamer type EO,PO,EO, (with EO = ethylene oxide and PO = propylene oxide)
from micelles in aqueous solution that have a hydrophobic core of PO blocks and a strongly hydrated shell
of EO blocks. The critical micellization temperature (cmt) of the poloxamers is strongly influenced by cosolutes
such as surfactsnts. The interaction of F127 (E0#06&097) with the anionic surfactant SDS was investigated
by small-angle neutron scattering (SANS), static light scattering, and differential scanning calorimetry (DSC).
It is found that addition of SDS can suppress the micellization of F127 completely. A simple model is
proposed which describes the suppression of polymer micelles by ionic surfactant. The surfactant binds
cooperatively on the block copolymer molecules, and the hydrophobic block is thereby made hydrophilic.
At saturation conditions four to five SDS molecules bind to one F127 molecule. The bound amount of SDS
increases somewhat with increasing polymer concentration. At higher concentration (w I 20 wt %), pure
F127 forms a cubic gel with increasing temperature. The gel region increases with increasing F127
concentration. Addition of SDS to a fixed F127 concentration decreases the gel region, until the gel completely
disappears. The "melting" of the gel is a result of the suppression of the poloxamer micelles. With increasing
surfactant concentration the hard sphere volume fraction 0 decreases below 0.53, the critical value for hard
B1036 – L3 phase in a binary block copolymer/water system.
Block copolymers of the Poloxamer type EO,PO,EO, (with EO = ethylene oxide and PO =
propylene oxide) can form lyotropic liquid crystalline phases in water, as is known for small hydrocarbon
surfactants. The phase behavior of PE6200 (E06-P036-E06) is investigated in more detail for
concentrations up to 50 wt % and temperatures from 0 to 100 "C. The technical product contains about
10 wt % hydrophobic contaminations, which induce clouding at ambient temperatures. These 'Tmpurities"
can be removed hy filtration, hut they do not affect the subsequent phase behavior. A lamellar phase
(La) and a spongelike phase (Ld are found and charaeterized by DSC, SANS, electric birefringence, dynamic
light scattering, and specific conductivity. The temperatures for the phase transitions decrease linearly
with increasing NaCl concentration. In the L3 phase the orientational relaxation time from electric
birefringence decreases with the third power of the volume fraction and the reduced diffision coefficient
derived from dynamic light scattering increases linearly with Poloxamer concentration. The conductivity
of added electrolyte clearly shows the hicontinuous structure of the Poloxamer L3 phase. Comparison of
the systems PEBZOO/water and ClnEOdwater shows analogies for the phase behavior and the properties
in the different phases.
B1031 – Relaxational transitions and ergodicity breaking within the fluid state: the sugars fructose and galactose
We describe calorimetric and other studies of time-dependent proceses in some molecular liquids in which there is a higher level of complexity than normally encountered in viscous liquid studies. Best characterized is the sugar -fructose, which melts to give a liquid which is not in an equilibrium state, and in which slow relaxation processes occur in the liquid far above Tg. These have time scales up to 10 orders of magnitude longer than the shear relaxation time, yet are shown to have an important effect on the liquid viscosity and hence on the value of Tg. Three different heat capacity anomalies are identified, and are assigned to three possible tautomerization equilibria, one of which provides a major contribution to Cp and becomes slow not far below the melting point. The effects of this slow degree of freedom on liquid behaviour are discussed.
B1028 – The effects of scan rate and protein concentration on DSC thermograms of bovine superoxide dismutase
DSC thermograms of bovine Cu---Zn superoxide dismutase (BSOD) have been recorded at different scan rates and protein concentrations in order to clarify the process of its unfolding. The lack of calorimetric reversibility made direct thermodynamic analysis of the thermograms impossible. The study of the effect of the scan rate on the shape of the heat capacity (Cp) profiles of BSOD has allowed the calculation of the apparent activation energy (Eapp) for the whole irreversible process. Extrapolation of the excess heat capacity curves (Cpexc) to infinite scan rate has provided the scan-rate-independent part of the thermograms. The protein concentration effect is explained by a change in molecularity that takes place before the kinetically-controlled step. The data collected suggest that, on thermal denaturation, BSOD dissociates during the thermally-induced transitions of the protein; this is followed by a kinetically controlled, exothermic step which is responsible for the global irreversibility of the entire process.
B1022 – Thermogelation of methylcellulose. Part I : molecular structures and processes
Thermogelation of methylcellulose (A4M from Dow) shows two distinct 'waves' of increase in G', preceded by an initial reduction at lower temperature. The reduction and first wave of increase are accompanied by a sigmoidal change in optical rotation (indicating a co-operative conformational transition). Light transmission and detectable 1H-NMR reach their maximum values at the end of the first structuring process, but drop to almost zero over the temperature range of the second. Both structuring processes are reversible on cooling, but offset, to lower temperature, and are accompanied by enthalpy changes in DSC (endothermic on heating; exothermic on cooling). About 40% of the high-resolution NMR signal remains undetectable in the solution state at low temperature, and the shear rate dependence of viscosity is appreciably different from that of disordered polysaccharide coils.
The proposed interpretation of these findings is that methylcellulose chains exist in solution as aggregated 'bundles', held together by packing of unsubstituted or sparingly substituted regions of cellulosic structure, and by hydrophobic clustering of methyl groups in regions of denser substitution. As the temperature is raised, the ends of the bundles come apart, exposing methyl groups to the aqueous environment and causing a large increase in volume (with consequent increase in G' ). At higher temperature the methyl substituents shed structured water, and form a hydrophobically crosslinked network (giving the second 'wave' of increase in G' ). As in other polysaccharide systems, the thermal hysteresis is attributed to aggregation stabilising the cellulosic 'bundles' to temperatures higher than those at which they will re-form on cooling.
B1008 – Scanning calorimetric and EPR studies on the thermal stability of actin
The thermal stability of actin isolated from skeletal muscle was studied in monomer and polymerized forms using DSC and EPR spectroscopy. Actin was labelled with the paramagnetic derivative of maleimide at the reactive thiol site Cys-374 in the C-terminal subdomain-1. The unfolding of actin induced by heating in the range 10-70°C resulted in a single transition: the transition temperature was 51 ± 2°C for G-actin, whereas 63 ± 2°C was obtained for the transition temperature of filamentous actin. Assuming a simple two-state transition for F-actin, the van't Hoff enthalpy was calculated to be 343.6 ± 25.1 kJ mol-1. The spectral changes were reversible in the temperature interval 10-65°C for F-actin, and actin did not lose its ability to polymerize.The analysis of the DSC profiles for actin in monomer and polymerized forms enabled the assignment of three endothermic components in the F-form (Tm1 = 59.7 ± 1.4°C, H1 = 60.3 ± 1.7 kJ mol-1; Tm2 = 60.6 ± 1.6°C, H2 = 348.2 ± 9.6 kJ mol-1; Tm3 = 61.4 ± 1.4°C, H3 = 104.3 ± 4.6 kJ mol-1), and two components in the G-form (52.5 ± 1.4 and 56 ± 1.8°C with enthalpies 184.4 ± 10.9 and 110.2 ± 7.5 kJ mol-1).Comparison of the spectroscopic and calorimetric behavior of actin showed remarkable differences in transition temperature and enthaply in both forms of actin, suggesting intramolecular and intermolecular interactions between subdomains and monomers that stabilize actin, especially in filament form.
B1006 – Study of pH and temperature-induced transitions in urate oxidase (Uox-EC22.214.171.124) by microcalorimetry (DSC), size exclusion chromatography (SEC) and enzymatic activity experiments
Purified recombinant urate oxidase (urate oxygen oxidoreductase EC 126.96.36.199. re-Uox) has been studied by means of differential scanning calorimetry (DSC) in correlation with enzymatic activity measurements and size exclusion chromatography. Differential scanning calorimetry curves versus pH show two endothermal effects in the pH range 6-10. The first endotherm reveals a maximum stability between pH 7.25 and pH 9.5 corresponding to a temperature of transition Tm1 of 49.0°C and an enthalpy of transition of 326 kJ mol-1. This value dramatically decreases below pH 7.25. The behavior of the second endotherm is more complex but the temperature of transition Tm2 is constant between pH 9 and 7.25 and a maximum for the corresponding enthalpy is obtained near pH 8 with ?H2=272 kJ mol-1. An optimal pH of 8.0 for the stability of the enzymatic activity at elevated temperature was also found which was in good agreement with calorimetric results. Reversibility of the first endotherm is obtained from 20 to 51.5°C. The calorimetric result is correlated to enzymatic activity, purity by size exclusion chromatography (SEC) and protein concentration measurements. In contrast, for the second endotherm, after heating up to 68.9°C, no reversibility was found. Interaction with structural analogues of urate has been studied by DSC. 8-Azahyooxanthine has only a small effect and caffeine has no effect at all. With 8-azaxanthine, a rapid increase of the Tm1 function of the concentration is obtained. At high concentration Tm1 reached the Tm2 value which remained unaffected.
B1003 – Changement de conformation de l’ovalbumine : étude par analyse enthalpique différentielle
B1000 – Effect of D-Glucose on yeast hexokinase denaturation
B0999 – Surfactant binding to bovine serum albumine : a DSC study
B0998 – Deconvolution of DSC denaturation profiles of DNAs from some phages
B0997 – Heat effects and water sorption by human serum albumin on its suspension in water-dimethyl sulphoxide mixtures
The heat effects on suspending solid human serum albumin (HSA) in various water-dimethyl sulphoxide (DMSO) mixtures were measured calorimetrically at 298 K. The isotherm of the water sorption for HSA suspended in the water-DMSO mixtures was also measured. The recording of the calorimetric heat effects exhibits endothermic and exothermic peaks. The endothermic heat effects were estimated graphically from the calorimetric curves. These values are shown to obey the Langmuir isotherm of the water sorption. The quasi-thermodynamic constant of water adsorption (1.2 ± 0.3 M-1) and the monolayer formation energy (-20.1 ± 1.0 J g-1) were estimated from the calorimetric data with the Langmuir model. The adsorption constant (0.16 ± 0.08 M-1) was evaluated from fitting the water sorption isotherm by the Langmuir model also. There is a divergence between the latter constant and the adsorption constant obtained from the calorimetric data. It appears that the processes accompanying the exothermic heat evolution influence the HSA's ability to bind water. The surface area of the water monolayer was also calculated from the fitting of the water sorption isotherm. It essentially exceeds the recognised values for proteins estimated from the data for water vapour sorption. The aqueous solubility of the protein after the exposure of the HSA preparation in the water-DMSO mixtures is also essentially decreased. Hence, changes in the protein-protein interactions of a diverse nature might accompany the exothermic heat evolution on suspending HSA in water-DMSO mixtures.
B0988 – Thermodynamics of water binding by human serum albumin suspended in acetonitrile
Heat effects resulting from the introduction of solid human serum albumin (HSA) into various water-acetonitrile mixtures were measured calorimetrically at 298 K. The amount of water bound to the suspended HSA as a function of the water content of the solvent was also determined. Introducing HSA into water-acetonitrile mixtures involves water binding according to the Langmuir isotherm with an adsorption constant Kc = 1.0 ± 0.1 M-1, enthalpy ?h = -9.0 ± 1.5 kJ mol-1 and entropy ?S = -30 ± 6 J mol-1 K-1. Placing HSA in the solvent has an additional heat effect of 46 ± 19 J g-1, which is attributed to an unknown transformation of the protein preparation.
B0984 – Microcalorimetric study on the interaction of F-actin with myosin and its proteolytic fragments
The heat effect produced in the interaction of rabbit muscle F-actin with myosin and its proteolytic fragments (HMM, S1) was measured with an MS-80 Calvet microcalorimeter (Setaram, France). An improved reaction cell of the microcalorimeter was used. There were three thermokinetic peaks in the measured curve of the interaction of myosin with F-actin in the absence of ATP, which were exothermic, endothermic and exothermic in order of appearance. When ATP was added to F-actin before mixing with myosin, the curve remained similar in feature to that in the absence of ATP. But the first peak (exothermic) became smaller and the second and third peaks became larger. There was only one exothermic peak in the measured curve of the interaction between S1 and F-action. But ATP caused big changes in the curve of their interaction. With addition of ATP to F-action solution before it was mixed with S1, there were four thermokinetic peaks in the curve which were endothermic, exothermic, endothermic, and exothermic in order of appearance. The measured curves of interaction between HMM and F-actin in the presence and absence of ATP were quite similar to that of interaction between S1 and F-action.
B0980 – Thermal denaturation of bacterial cells examined by differential scanning calorimetry
Differential scanning calorimetry (DSC) with a microcalorimeter was used to detect thermal transitions in cells of Listeria monocytogenes, Escherichia coli and Lactobacillus plantarum, suspended in pH 6.8 physiological phosphate buffer. DSC-scanning at 0.5°C min[-1] showed multi-peaked thermograms. Thermograms taken from 10 to 99°C temperature range showed an exothermic heat flow below the maximum temperature of growth and a series of endothermic transitions began to appear between 50 to 60°C, in the temperature range, where the heat destruction of vegetative cells occurred. Our observations strengthen the premise that the process responsible for thermal death is the irreversible thermal denaturation of a crucial proteinaceous component of the cells and it is strongy influenced by the pH of the suspending citric/phosphate buffer
B0954 – O-Glycosylation and stability. Unfolding of glucoamylase induced by heat and guanidine hydrochloride
B0952 – Studies by UV spectroscopy of thermal denaturation of B-lactoglobulin in urea and alkylurea solutions
The thermal denaturation of beta-lactoglobulin in aqueous solutions at three different pH's and in aqueous solutions of urea, methyl-, N,N'-dimethyl- and ethylurea was studied by UV spectroscopy. The UV-melting curves were analyzed on the basis of two-state approximation to obtain the apparent equilibrium constant, Kapp, and the apparent standard enthalpy of transition, ?Happ0, for protein unfolding as a function of temperature. From Kapp, calculations of ?Gapp0, as functions of temperature around transition temperature, T1/2, in urea and alkylurea solutions and different buffer solutions have been carried out. An increase in the observed transition temperature, T, and the corresponding transition enthalpies, ?Happ0, with decreasing pH or denaturant concentration indicate increased stability of protein in these conditions. However, comparison of ?Happ0 with ?Hcal shows that the thermal transition of beta-lactoglobulin in aqueous urea and alkylurea solutions is not a two-state process. It has also been observed that urea and all alkylureas cause a red shift in the absorbance spectrum of beta-lactoglobulin which increases with increasing denaturant concentration and decreasing pH. A similar increase in red shift of beta-lactoglobulin absorbance spectrum has also been observed with increasing temperature.
B0950 – Denaturation of ribonucleases from different sources in the presence of denaturing or stabilizing agents
B0949 – High-pressure unfolding and aggregation of B-lactoglobulin and the baroprotective effects of sucrose
The effects of processing at 450 MPa and 25 "C for 15 min on the unfolding and aggregation of an
industrial P-lactoglobulin protein isolate (PLG) have been studied at pH 7.0 (at 0.1 MPa) and two
protein concentrations, at 04% sucrose. Mer 2.5 or 5% protein solutions (no sucrose) were pressure
processed, PLG remained soluble, but the residual enthalpy of denaturation (AH, as determined by
DSC 20-27 h after pressurization) was decreased by 44 or 54%, respectively, indicating significant
unfolding. Solubility in 2 M ammonium sulfate was similarly decreased, evidencing pressure-induced
protein aggregation. Some soluble aggregates (36-103 kDa) were observed by gel permeation
chromatography. Pressure-induced unfolding or aggregation (at 2.5% protein) was found to be
partially reversible with storage time after pressurization (up to 26 or 33%, respectively, of the
initial changes, after 7 days at 4 "C). The presence of 5% sucrose during pressurization at 2.5%
protein reduced PLG unfolding (AH was decreased by 27% instead of 44%) and slightly increased
the rate of recovery of protein solubility in 2 M ammonium sulfate.
B0942 – Ligand-induced biphasic thermal denaturation of RNAase A
DSC measurements have been accomplished in aqueous solutions of bovine pancreatic ribonuclease
A (RNAase A) in the presence of subsaturating amounts of 3' cytidine monophosphate
(3'CMP) and 2' cytidine monophosphate (2'CMP) at pH 5.0 and 5.5. In these conditions the experimental
profiles do not conform to a one-step unfolding process. It can be emphasized, as a
general phenomenon, that a strong linkage between the temperature-induced protein unfolding
and the ligand binding, when the ligand is less than the saturation level, causes marked distortions
from a two-state transition. A purely equilibrium thermodynamic analysis gives a correct
account of this behaviour and allows to simulate calorimetric curves. It is thus possible to obtain,
in an indirect manner, information about the thermodynamic parameters concerning the binding
process, namely the association constant and the binding enthalpy. The values of Kb and ?bH for
3'CMP and 2'CMP, so determined, are consistent with the literature data.
B0941 – Comparison of the results of thermal denaturation of B-lactoglobulin obtained by DSC and UV-spectroscopy
The thermal denaturation of beta-lactoglobulin in the presence of urea and alkylurea solutions
were measured. In the presence of a high concentration of urea this protein shows not only heat
but also cold denaturation. For studying the effect of temperature two methods were used, differential
scanning calorimetry (DSC) and UV-speetroseopy. DSC provides direct model-independent
determination of the transition enthalpy in comparison with UV-speetroscopy, which
gives only apparent or van't Hoff enthalpy of transition. The UV-melting curves were analyzed
on the basis of a two-state approximation. The apparent standard enthalpies of thermal denaturation,
?Happ°, were compared with calorimetric ones.
B0940 – Thermodynamic characterisation of RNAase A in the presence of urea and GuHCl
It is presented a study concerning the influence of guanidinium chloride (GuHCl) and urea
on thermal stability of Bovine Pancreatic Ribonuclease A (RNAase A) at different pH values. As
expected, at increasing the denaturant concentration, the protein thermostability decreases. This
is shown by a decrease of both the thermodynamic parameters, temperature and heat effect, characterising
the denaturation process. In order to analyse the calorimetric curves we adopt a statistical
thermodynamic approach. The individual one-dimensional DSC profiles have been
expanded into another dimension by varying the GuHCl concentration, so that a heat capacity
surface is defined for each pH. By means of the ICARUS program, developed in our laboratory,
we accomplish a two dimensional deconvolution of the experimental data linking the binding
equilibrium to the denaturation process. This analysis provides a well founded and complete statistical
thermodynamic characterisation of denaturation process of RNAase A in the presence of
GuHCl and allows to calculate the thermodynamic parameters associated to the binding of denaturant
B0933 – Differential scanning calorimetry of the irreversible denaturation of bovine superoxide dismutase
The thermal denaturation of bovine superoxide dismutase has been studied by means of differential scanning calorimetry (DSC). Analysis of the calorimetric profile by means of Freire and Biltonen's algorithm to determine the accessible states in the transition path did not succeed. At least three factors influenced this analysis negatively: (a) under the experimental conditions used, the denaturation of the enzyme was completely irreversible, as judged by the absence of any endotherm on re-scanning; (b) the denaturation process was followed by a change in molecularity, as the dimeric nature of the enzyme suggested; and (c) there was distortion at the high temperature side of the DSC profile, caused by the existence of an exothermic aggregation process.Moreover, the difference between the van't Hoff and the calorimetric enthalpy on one hand, and the dependence of the specific heat Cp on scan rate on the other, allowed us to exclude a simple two-state reversible or irreversible transition for the denaturation of the enzyme. In the light of these results, we attempted to fit the experimental Cpexc curve using the minimization algorithm by taking in account only three processes: two endothermic and one exothermic. The deconvolution of the calorimetric profile agreed very well with the experimental data and allowed us to suggest a tentative mechanism for the thermal denaturation of this dimeric enzyme.
B0922 – A combined scanning dilatometric and differential scanning calorimetric study of the thermal unfolding of bovin serum albumin
Here we report the first results of combined scanning calorimetric and dilatometric investigations carried out on bovine serum albumin in aqueous solution at 30-97°C. Comparison of experimental data obtained by the two techniques suggests a "steps" model for the thermal denaturation of the protein.Moreover, the results in the high-temperature region, after aggregation of the unfolded chain, show a subsequent, previously unreported, spatial rearrangement of the polypeptidic chain network, during which the order of the system increases with an increase in temperature.The results indicate that scanning dilatometry is a very useful method of detecting phenomena which are not seen by calorimetry. The results also demonstrate the determinant role of water in the unfolding process.
B0909 – Enthalpies of mixing in PrCl3-CaCl2 and NdCl3-CaCl2 liquid systems
The molar enthalpies of mixing ?mixHm in the PrCl3---CaCl2 and NdCl3---CaCl2 liquid binary systems were measured at 1073 K over the whole composition range under argon at atmospheric pressure. The apparatus used was a Calvet-type high-temperature micro-calorimeter and mixing of the two liquid components was obtained by the break-off ampoule technique. In both systems the plots of enthalpies of mixing versus composition were "S-shaped" curves.The enthalpy of mixing values are positive for NdCl3- and PrCl3-rich compositions, and negative for other compositions. These values are very small (from about -200 to about 180 J mol-1). In the PrCl3---CaCl2 and NdCl3---CaCl2 liquid binary systems,?mixHm˜0 at ?PrCl3 ˜ 0.6 and at ?NdCl3 ˜ 0.4, respectively. These results are discussed in terms of equilibrium between the LnCl3 (where Ln is Pr, Nd) polymers and anionic complexes as a function of the composition of the melts
B0904 – Characterization of actin by DSC and EPR
B0894 – A calorimetric study of cooperative phase equilibria in microbial polysaccharides
Some calorimetric data of the order-disorder conformational transitions of natural polysaccharides from microorganisms are presented in the more general thermodynamic framework of the cooperative helix-coil transition of linear biopolymers. A new home-mad
B0892 – The deconvolution of multi-state transition DSC curves of biological macromolecules : bovine serum albumin and bovine seminal ribonuclease
The denaturation of the ordered, specific structures of biological macromolecules is a highly cooperative process which many researchers have likened to the melting or solution of a crystal. However, with increasing numbers of studied systems using differ
B0876 – Heat capacity of hydrated and dehydrated globular proteins. Denaturation increment of heat capacity
B0861 – Thermodynamic characterization of RNase A in the precence of GdnHCl
B0839 – Thermal denaturation of ribonuclease T1: a DSC study
The thermal denaturation of microbial Ribonuclease TI (RNAase T1) as a function ofpH,
was studied by means of DSC microcalorimetry. The midpoint denaturation temperatures, enthalpy
changes and heat capacity changes of Ribonuelease T1 were compared with those obtained
for pancreatic Ribonuelease A (RNAase A). It was found that the microbial T1 protein
undergoes a more complex eonformational transition than the simple two-state transition
shown by Ribonuelease A. The hypothesis of the presence of a 'molten globule' form is discussed.
The conformational stability of RNAase T1 is lower than that of RNAase A at high pH
values. Indeed, the maximum stability of RNAase T1 occurs atpH ~ 5, whereas that of RNAase
A occurs at pH ~ 8. AtpH = 3.7 an irreversible aggregation phenomenon was indicated by the
existence of a reproducible exothermie peak. The conformational transition of RNAase T1 is
reversible in the range of pH 4.5-7.0, whereas it becomes irreversible at pH >_ 8.0 as for
B0838 – Theseus : a new software package for the handling and analysis of thermal denaturation data of biological
A new software package (THESEUS) has been assembled for the analysis of the DSC data,
concerning the thermal denaturation of biological macromolecules. The system is useful to obtain
accurate physico-chemical information, bypassing the casual and systematic errors, very
common in these experiments. It can also be used for handling data from other instruments and
methodologies giving thermodynamic, spectroscopic or other kind of data as a function of
temperature. Because many of the researches in this field are of exploratory nature and continuously
new unfolding mechanisms are described or hypothesized in the current literature, we
have written and assembled this powerful and flexible program of general applicability, in
order to put the operator in a position to control each step of the calculation procedure and use
his own experience for choosing the better way to solve unexpected problems.
B0785 – Conformational stability of proteins and peptide-peptide interactions in the presence of carbohydrates
The denaturation temperatures and enthalpies of bovine pancreatic RNAase A in the presence of different amounts of D-glucose or its oligomers have been determined from DSC measurements and compared with literature results for other globular proteins in the presence of oligosaccharides or polyhydroxylated compounds. Both parameters increase almost proportionally for RNAase A at increasing sugar concentration (the denaturation appearing as a reversible, one-step process) and the evaluated Gibbs energy-temperature plots show an expansion of the stability range and an increase in relative stability. Isothermal measurements were also obtained by dilution-flow calorimetry to determine the virial coefficients of the excess enthalpies for aqueous solutions of some model peptides (N-acetylamides of simple amino acids) in the presence of l M D-glucose. These results provide an insight into the role of sugars in preventing peptide-peptide interactions
B0784 – DSC studies on the denaturation and aggregation of serum albumins
Denaturation temperatures and enthalpies of bovine serum albumin (BSA) and human serum albumin (HSA) measured by DSC at different pH values and protein concentrations are compared with the literature data on BSA, or SH-blocked BSA. Both parameters display a trend in the region of pH 6.0-8.5 with a maximum at pH 7.1-7.2, in agreement with the literature data. An exothermic peak related to protein aggregation and precipitation was found at higher temperatures. For BSA, at lower pH values, this peak moves towards lower temperatures and overlaps the denaturation endothermic peak at pH < 5.5. The maximum temperature of aggregation, however, depends markedly on the protein concentration; therefore a separation of the two peaks was attempted. At pH 4.0, a shoulder appears at a temperature lower than that of denaturation. A gelification phenomenon also occurs and partially interferes with the other thermal processes.
B0783 – Microcalorimetric measurements of thermal denaturation and renaturation processes of salmon sperm DNA in gel and liquid crystalline phases
Thermally induced denaturation and subsequent renaturation of salmon sperm DNA in gel (G) and liquid crystalline (LC) phases have been studied by a microcalorimetric approach, using differential scanning calorimetry. An unusual very high renaturation percentage of the denatured LC-DNA and a greater thermodynamic stability of the LC phase were observed.
B0695 – Effect of collagen crosslinking on collagen-water interactions (A DSC investigation)
The effect of collagen cross-linking state on the collagen-water interaction was studied, using Differential Scanning Calorimetry (DSC) which allows the determination of unfreezable water, the variation of enthalpy (delta H) and temperatures of denaturation of collagen to gelatin transition. DSC was performed on intramuscular connective tissue purified with trypsin (control C), depolymerized with penicillamin (P), and reduced with borohydride (B); samples were adjusted with different water contents. For the three tissues, unfreezable water (Wu) and denaturation enthalpy change (delta H) increased with increasing moisture level (Wt); whereas, maximum denaturation temperature (phi M) decreased. The ability of this calorimetric method of investigation to characterize the collagen crosslink state is discussed: maximum limit values of delta H and of Wu decreased significantly with increasing collagen cross-linking degree. Minimum Wt necessary to reach the maximum delta H decreased with crosslinking degree. Likewise significantly different limit values of unfreezable water Wu were reached for smaller Wt the greater the crosslinking of collagen. These results show that the less connective tissues were cross-linked, the more they could bind water. They also demonstrated that the water of collagen hydration can be classified into four states, whose limits vary according to the degree of crosslinking.
B0692 – Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin
The thermal denaturation of beta-lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes accompanying denaturation have been obtained. This allowed calculation of the enthalpy, entropy, and Gibbs free energy changes for thermal denaturation in the temperature range scanned, i.e., 20-100°C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity ([?]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80°C, conformational changes are to a large extent reversible.
B0676 – Microcalorimetric measurement of the specific heat of thynnus thunnus
The specific heat of Thynnus Thunnus from 295 to 413 K was measured using sealed samples of ca. 10 mg in a Calvet high temperature microcalorimeter. A drop method was used, the equipment being calibrated using the specific heat of copper, which gave values from 3.16±0.03 J K-1 g-1 at 295 K to 3.75 ± 0.05 J K-1 g-1 at 413 K.
B0648 – Thermal stability of herring DNA in the presence of clupeine fractions
The denaturation temperatures and enthalpies of the complexes formed between herring DNA and each of the three clupeines (protamine fractions replacing histones in fish seminal fluid and vescicles) have been determined. An increase of about 20 K was found for the melting temperature and of about 100% for the denaturation enthalpy (compared with free DNA). The large increase in the stabilisation of DNA against denaturation after formation of its complexes with clupeines is expressed by the temperature dependence of the unfolding free energy
B0605 – Studies on the thermal behaviour of pea (pisum sativum) vicilin
B0543 – Conformational transition of kappa-carrageenan
The calorimetry of kappa carrageenan using differential scanning calorimetry and
calorimetry at constant temperature is presented. Both sets of experimental results confirmed the
formation of a helical dimer in the absence of gel formation. When gel is formed, the formation of
a helical dimer is the major effect, the contribution of gelation represents only 20% of the
B0532 – Calorimetric investigation in the fields of biology and biochemistry
A new calorimeter based on the Calvet heat flux principle is presented. It has been optimized for investigation in the fields of Biology and Biochemistry. Its main feature is a very high sensitivity associated with removable vessels. It offers many possibilities: upwards and downwards temperature scanning, heat capacity measurements, isothermal batch calorimetry and flow calorimetry.
B0515 – DSC study of reversible and irreversible thermal denaturation of concentrated globular protein solutions
A calorimetric study of the thermal denaturation of bovine serum albumin, RNAase and catalase in concentrated solutions (crystals) has been carried out. The results obtained for RNAase studied within the pH range 2.5-8.5 show that for concentrated solutions there is an interval of pH where, on cooling of the solution which had undergone denaturation, its renaturation is observed. In the case of concentrated and dilute solutions of RNAase these intervals coincide. The study of RNAase under such conditions at various heating rates shows that there is a range of rates in which the process of denaturation of concentrated solutions can be considered as reversible. The dependences of Td and Hd on pH and concentration of solutions have been determined. The denaturation enthalpy of concentrated solutions like in dilute ones, has been found to be independent of the pH of solutions, and the experimentally registered change has been proved to be the result of its dependence on temperature. A new method of determination of protein denaturation enthalpy under the conditions of intensive molecule aggregation is suggested. The forms of irreversibility as appearing in the calorimetric experiment were determined by comparing reversible and irreversible denaturation under continuous and step-heating regimes. It is shown that the decrease in Tmax and the narrowing of the heat absorption peak in the case of decreasing heating rates of protein solutions, observed under certain environmental conditions, results from the irreversibility of the denaturation process.
B0464 – Beitrag zum problem der energiemessung in aquatischen ökosystemen: Mikrokalorimetrische untersuchungen von planktonalgen
The energy content from a batch culture ofPediastrum boryanum was measured in a Differential Scanning Calorimeter as combustion enthalpy. A clear linear relation exists between dry weight and energy content. The energy content in the exponential growth phase is constant, approx. 22.1 kJ/g dry weight (not ashfree).
The proportion of various organic substances-documented by different peaks in the thermograms-change in time, apparently due to increasing growth-limitation by P and N.
B0453 – Calorimetric determination of the conformational transition of kappa carrageenan
This paper deals with measurements of the heat of the helix-coil transition by microcalorimetry for kappa carrageenans; data are given in the absence of gel in various solvents (H2O, Me2SO, and formamide) with two counterions (K+, Rb+). In water, the influence of gel formation on ?H is pointed out and the influence of the time of ageing is demonstrated. The ?H values measured in the absence of gel are interpreted in terms of the electrostatic model proposed by Manning; the agreement is quite good if a double helix is formed in water, but a monochain, ordered conformation is suggested for solutions in Me2SO and formamide.
B0366 – Calorimetry of small animals
A2311 – Novel magnetic chitosan/poly(vinyl alcohol) hydrogel beads: Preparation, characterization and application for adsorption of dye from aqueous solution
Novel magnetic chitosan/poly(vinyl alcohol) hydrogel beads (m-CS/PVA HBs) were prepared by an instantaneous gelation method and characterized by X-ray diffraction (XRD), vibrating sample magnetometry (VSM) and thermogravimetric analysis (TGA). Results of characterization indicated that m-CS/PVA HBs have been prepared successfully without damaging the crystal structure of Fe3O4 and their saturation magnetization were about 21.96 emu g?1. The adsorption capacity of Congo Red on the m-CS/PVA HBs was 470.1 mg g?1. The adsorption was well described by pseudo-second-order kinetics and Langmuir equation. Positive value of enthalpy change (?H?) (13.32 kJ mol?1) showed that the adsorption was endothermic and physical in nature. The values of Gibbs free energy change (?G?) were found to be ?3.321 kJ mol?1 at 298 K for m-CS/PVA HBs, indicating the spontaneity of Congo Red adsorption. Therefore, the m-CS/PVA HBs could be employed as a low-cost alternative to other adsorbents in the removal of dyes from aqueous solution.
A2186 – Solvent composition of one-step self-etch adhesives and dentine wettability
Our aim was to determine the wettability of dentine by four commercial self-etch adhesives and evaluate their spreading rate on the dentine surface. Any correlation with chemical composition was sought, particularly with the amount of solvent or HEMA present in the adhesive. The adhesives used were AdheSE One, Optibond All.In.One, Adper Easy Bond and XenoV.
A2145 – Isotherms and thermodynamics for the sorption of heavy metal ions onto functionalized sporopollenin
In this study, sporopollenin of Lycopodium clavatum spores was used for the sorption experiment. Glutaraldehyde (GA) immobilized sporopollenin (Sp), is employed as a sorbent in sorption of selected heavy metal ions. The sorbent prepared by sequential treatment of sporopollenin by silanazing compound and glutaraldehyde is suggested for sorption of Cu(II), Zn(II) and Co(II) from aqueous solutions. Experimental conditions for effective sorption of heavy metal ions were optimized with respect to different experimental parameters using batch method in detail. Optimum pH range of Cu(II) has occurred at pH?5.5 and Zn(II), Co(II) at pH?5.0, for the batch method. All of the metal ions can be desorbed with 10 cm(3) of 0.5 mol dm(-3) of ethylenediaminetetraacetic acid (EDTA) solution. Langmuir, Freundlich and Dubinin-Radushkevich (D-R) isotherm equations were applied to the experimental data. Thermodynamic parameters such as free energy (?G(o)), entropy (?S(o)) and enthalpy (?H(o)) were also calculated from the sorption results used to explain the mechanism of the sorption. The results indicated that this sorbent is successfully employed in the separation of trace Cu(II), Zn(II) and Co(II) from the aqueous solutions
A2139 – Production and characterization of polyhydroxyalkanoates by recombinant Methylobacterium extorquens: Combining desirable thermal properties with
Polyhydroxyalkanoates (PHAs) are renewable and biodegradable polyesters which can be synthesized either by numerous of microorganisms in vivo or synthase in vitro. The synthesis of PHAs in vitro requires an efficient separation for high yield of purified enzyme. The recombinant Escherichia coli harboring phaC gene derived from Ralstonia eutropha H16 was cultivated in the chemically defined medium for overexpression of synthase in the present work. The purification and characteristics of PHA synthase from clarified feedstock by using aqueous two-phase systems (ATPS) was investigated. The optimized concentration of ATPS for partitioning PHA synthase contained polyethylene glycol 6000 (30%, w/w) and potassium phosphate (8%, w/w) with 3.25 volume ratio in the absence of NaCl at pH 8.7 and 4°C. The results showed that the partition coefficient of enzyme activity and protein content are 6.07 and 0.22, respectively. The specific activity, selectivity, purification fold and recovery of phaCRe achieved 1.76 U mg?1, 29.05, 16.23 and 95.32%, respectively. Several metal ions demonstrated a significant effect on activity of purified enzyme. The purified enzyme displayed maximum relative activity as operating condition at pH value of 7.5 and 37°C. As compared to conventional purification processes, ATPS can be a promising technique applied for rapid recovery of PHA synthase and preparation of large quantity of PHA synthase on synthesis of P(3HB) in vitro.
A2061 – Immobilization and characterization of hemoglobin on modified sporopollenin surfaces
Hemoglobin was covalently immobilized onto modified sporopollenin surface with different functional groups by chemical reactions to enhance binding ability of protein. In this study, the influence of various silane linker molecules on the capacity of protein binding was studied. For this purpose, activated sporopollenin was modified by 3-aminopropyltriethoxysilane (APTS), 3-chloropropyltrimethoxysilane (CPTS) and (3-glycidyloxypropyl)trimethoxysilane (GPTS). Hemoglobin (Hb) was immobilized on modified sporopollenin surfaces in phosphate buffer saline solution (PBS, pH 7.4) at 4 °C. Results showed that GPTS modified sporopollenin surfaces resulted in the highest binding capacity for Hb. Micro porosity of samples was observed through scanning electron microscopy (SEM) and thermal behavior of the samples were studied with thermogravimetric analysis (TGA) within a temperature range: 25–900 °C. TGA studies demonstrated the advantages of silane modification for high temperature applications and illustrated differences of the structures due to the different tail groups.
A2053 – Chitosan hydrogel films as a template for mild biosynthesis of CdS quantum dots with highly efficient photocatalytic activity
Cadmium sulfide (CdS) semiconducting quantum dots (QDs) were prepared using in situ synthesizing method in crosslinked chitosan hydrogel films under relative mild experimental conditions and characterized by X-ray diffraction (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The experimental results confirmed that crosslinked chitosan hydrogel films provided a confined matrix for CdS QDs growth in uniform size through chelation and electrostation between cadmium ions and amino groups in chitosan chains. The CdS QDs/chitosan composite films exhibited a highly efficient photocatalytic activity for decolorization of methyl orange (MO) solution under visible light irradiation. The good linearity relationship between ln (C0/Ci) and irradiation time (t) indicated that the decolorization of MO dye under present experimental conditions followed pseudo-first-order kinetics. These results suggested that CdS QDs/chitosan composite films were suitable material for potential application in decolorization of organic dye pollutants under visible light irradiation.
A1902 – Structural characterization and thermal behaviour of biological hydroxyapatite
Thermal study and structural characterization of biological hydroxyapatite (HA) samples were done as well as their comparison with commercial and synthetic samples in this study. The X-ray micro analyser shows that all three samples of human teeth (HT1–HT3) contain two types of HA structures with different crystallite sizes, unlike sample of bovine thigh-bone (BTB). The bone sample was composed only of one HA phase with varied porosity. The molar Ca/P ratio in biological samples was lower compared to theoretical ratio for pure HA; moreover,
in the case of teeth, Ca/P ratio varyies between the centre and the periphery of the cross-sectional samples. Thermogravimetry of the biological samples showed mass decreases—three regions for the bone and four regions for the teeth. In comparison, commercial HA has only two-step weight loss and synthetic HA three-step weight loss. After the calcination up to 1280 °C all the samples of teeth transformed into whitlockite, b-(Ca,Mg)3(PO4)2 (98 wt%) and 2 wt% HA. Besides, HT3 contained further trace amount of hilgenstockite (HIL, Ca4P2O9). The sample BTB partly transited from natural HA into HIL (6 wt%) and lime, CaO (14 wt%). X-ray powder diffraction (XRD) proved occurrence of HIL (9 wt%) beside stability part HA (91 wt%) in the commercial HA after thermal treatment but the synthetic HA composed from Ca3(PO4)2 (74 wt%) and HA (26 wt%).
A1747 – Kinetic studies on the pyrolysis of chitin and chitosan
The thermal degradation of chitin and chitosan have been studied by using simultaneous TG and DSC in nitrogen atmosphere. The model-free iso-conversional method has been employed to evaluate the activation energies as a function of the extent of degradation. The obtained dependencies are interpreted in terms of degradation mechanisms. Under nitrogen, the thermal
degradation of chitin and chitosan follows a random scission pathway, which is initiated at weak links. The activation energies of thermal degradation of chitin are constant and this process follows 1.23 order reaction. As for chitosan, the activation energies of thermal decomposition are obviously dependent on the extent of conversion. The pyrolysis reaction of chitosan is analysed by using a distributed activation energy model (DAEM) because the residual N-acetylated side-chains are randomly distributed on the chitosan main chains. Also, the parameters of the DAEM are evaluated.
A1499 – The investigation of thermal decomposition pathways of phenylalanine and tyrosine by TG–FTIR
An online-coupled TG–FTIR evolved gas analysis (EGA) instruments have been used to identify and monitor the evolution of gaseous products during the thermal decomposition of phenylalanine and tyrosine in flowing N2 atmosphere up to 800 °C. The results indicate that the thermolysis processes of these two compounds are similar. For both of them the main primary decomposition steps are two competing paths: the direct decarboxylation and the concerted rupturing of C–C bonds. And the primary decomposition reactions also include deamination and dehydration. The main secondary reaction is the crack of cyclic dipeptide which also presents two competing pathways. The main gaseous products are NH3, H2O, CO2, CO, HNCO, HCN and some organic compounds. However, compared with tyrosine, the decomposition of phenylalanine is more complete and involves less secondary reaction. That is caused by their different thermal stability and decomposition temperature
A1484 – TGMS analysis of archaeological bone from burials of the late Roman period
The use of thermogravimetric analysis–mass spectrometry (TGMS) to study the state of preservation of archaeological bones has been investigated. As part of a collaborative multi-analytical study, bones exhumed from graves of the late Roman period in France and Italy were examined. A decrease in organic matter for the archaeological bones compared to that for new bone was confirmed, demonstrating that diagenesis of aged bones can be detected using TGMS. Different amounts of collagen were determined for bones from different graves and could, for the
majority of specimens, be correlated with the visually observed preservation states.
A1480 – TG-MS analysis of the thermal decomposition of pig bone for forensic applications
In order to investigate the potential of thermal analysis for the determination of post-mortem age, rib bone specimens were collected from the remains of a number of slaughtered pigs that were allowed to decompose in the Australian bush in a controlled site under a range of conditions for time periods ranging from 1 to 5 years. The bone specimens were cut in cross-section with the compact bone collected for analysis. TG-MS curves were collected by heating bone samples to 1100°C in an argon atmosphere. The TG-MS data showed significant differences for the pig bone specimens derived from the different environments and showed trends in peak size correlating with age. The reported data suggest that TG-MS has significant potential for the identification of origin as well as the ageing of skeletal remains in a forensic context.
Alginic acid, a biopolymer produced by brown seaweed, is incorporated between the sheets of a layered double hydroxide (LDH)
via direct coprecipitation. The growth of the inorganic crystalline seeds over the polymer gives rise to a lamellar structure. The
obtained nanocomposite presents a basal spacingin agreement with the ideal picture of the polymer lyingperpendicularly to the
inorganic sheets. A study using FTIR and 13C CP-MAS spectroscopies suggests that the interaction between the organic guest and
the inorganic framework is weak. However, the polymer has a stabilizing effect in temperature, since ZnO is observed at 350°C,
whereas it appears at 200°C for the chloride LDH pristine material.
A1322 – Biomimetic nucleation and growth of CaCO3 in hydrogels incorporating carboxylate groups
Poly-acrylamide hydrogels were modified by copolymerization with acrylic acid and used as growth medium for CaCO3 in a
double-diffusion arrangement. The carboxylate functionalities in the gel network facilitate the nucleation of a multitude of small
crystallites of vaterite and calcite, which are temporarily stabilized even while supersaturation is increasing within the hydrogel.
After an extended induction period the rapid spherulitic growth of calcite crystals along their c-axis is observed yielding spheres with
diameters exceeding 300 µm. In the center of those aggregates disordered, porous regions can be identified as starting point of this
The results are compared to previous studies on native poly-acrylamide hydrogels and networks modified with -SO3H functional
groups. The mineralization mechanism is significantly altered by specific interactions between the -COOH functionalized network
and the evolving mineral phase.
A1312 – Structural characterization and antimicrobial activity of chitosan/betaine derivative complex
Complex of chitosan acetate (CSAC) with C12-C18 alkyl amido prophyl dimethylamine betaine (AAPDB) was prepared and
characterized by Fourier transform-infrared (FT-IR), thermal analysis (TG, DTG and DSC), X-ray diffractogram. Results showed that
complex formed mainly by the electrostatic interaction between NH3+ of CSAC and COO- of AAPDB. Minimum inhibitory concentrations
(MICs) were evaluated against E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923,
Staphylococcus epidermidis and Candida albicans. Results showed that CS/AAPDB solution could or even more strongly inhibit growth of
all the tested microorganisms, whereas CSAC could not inhibit growth of C. albicans and AAPDB could not inhibit growth of S. epidermidis,
E. coli, P. aeruginosa. Relative inhibition times (RITs) of CS/AAPDB solutions with different concentration and ratio of CSAC and AAPDB
were also investigated against the five microorganisms. Results showed that CS/AAPDB solutions with CCSAC + CAAPDB of
0.05% + 0.015%, 0.05% + 0.0075%, and 0.025% + 0.0075% showed higher antimicrobial activity. The relationship between complex
formation and antimicrobial activity was discussed.
A1294 – Studies on bone marrow stromal cells affinity of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)
The objective of this study is to investigate the biocompatibility of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx)
with bone marrow stromal cells in vitro. The adsorption of fibronectin on the material was studied by enzyme-linked
immunosorbent assay. After bone marrow stromal cells were seeded and cultured on PHBHHx, their proliferation was investigated
by MTT. Differentiation of the cells was assessed by measuring alkaline phosphatase activity and by histochemical assay. The
wettability and thermal property of PHBHHx films were also studied by contact angle goniometer, thermogravimetry and
differential scanning calorimetry, respectively. The results show that bone marrow stromal cells can attach, proliferate and
differentiate into osteoblasts on PHBHHx films. These results suggest that PHBHHx has good affinity with bone marrow stromal
cells and may have potential applications in bone tissue engineering.
A1282 – Synthesis of luminescent bioapatite nanoparticles for utilization as a biological probe
A europium-doped apatitic calcium phosphate was synthesized at low temperature (37°C) in water-ethanol medium. This apatite
was calcium-deficient, rich in hydrogen phosphate ions, and poorly crystallized with nanometric sized crystallites. It is similar to the
mineral part of calcified tissues of living beings and is thus a biomimetic material. The substitution limit of Eu3+ for Ca2+ ions in
this type of bioapatite ranged about 2-3%. The substitution at this temperature was facilitated by vacancies in the calcium-deficient
apatite structure. As the luminescence of europium is photostable, the doped apatite could be employed as a biological probe.
Internalization of these nanoparticles by human pancreatic cells in culture was observed by luminescence confocal microscopy.