Thermodynamic analysis of partially folded states of myoglobin in presence of 2,2,2-trifluoroethanol

The thermal denaturation of myoglobin was studied in the presence of 2,2,2-trifluoroethanol (TFE) at various pH values using differential scanning calorimetry and UV–visible spectroscopy. The most obvious effect of TFE was lowering the transition temperature up to 1.5 mol · kg⁻¹, beyond which no thermal transitions were observed. The protein conformation was analysed by fluorescence and circular dichroism measurements. Quantitative binding of ANS to the TFE induced molten globule state of myoglobin was studied by using isothermal titration calorimetry. The results enable quantitative estimation of the binding strength of ANS with the molten globule state of myoglobin along with the enthalpic and entropic contributions to the binding process. The results suggest occurrence of common structural features of the molten globule states of proteins offering two types of binding sites to ANS molecules which are a widely used fluorescence probe to characterise partially folded states of proteins.     

DMSO对鸡蛋白溶菌酶溶液变性的影响

利用差示扫描量热(DSC)和温度调制差示扫描量热(MDSC)研究了鸡蛋白溶菌酶在纯水及二甲基亚砜(DMSO)/水混合溶剂中的热变性过程, 探讨了酶的浓度、扫描速率和共溶剂的含量对热变性行为的影响规律. 在纯水溶液中, 溶菌酶的变性焓(△Hm)随酶浓度的增大而增大. 而在DMSO/水混合溶剂中, 变性温度(Tm)随DMSO体积分数的增大向低温方向移动, 变性峰变低变宽; 当DMSO体积分数达到70%后, 热变性曲线变成了一条光滑的直线. 另外, 在纯水溶液中溶菌酶的MDSC图除了出现DSC中可观察到的主吸热峰(I)外, 在峰(I)的前面还出现一个小而对称的吸热峰(II), 并且当体系中有DMSO存在时也未能观察到此峰. 当溶菌酶浓度增大时, Tm(II)移向低温, △Hm(II)减小, Tm(I)与Tm(II)之间的距离变长. 吸热峰(II)的出现被认为是由于水溶液中溶菌酶二聚体的可逆离解造成的.     

On the cooperativity of the thermal denaturation of mini-proteins

It is well established that the reversible thermal denaturation of small globular proteins is a cooperative two-state transition, analogous to a first-order phase transition in a finite-size system. Finite-size effects on the cooperativity of the reversible thermal denaturation become more important when the polypeptide chain is very small, as in the case of some synthesized mini-proteins. The analysis of two specific examples of mini-proteins, by means of a statistical mechanical approach, leads to the conclusion that their thermal denaturation, in view of its broadness and energetics, cannot be considered a cooperative first-order phase transition.     

Studies on gelation of soybean globulin solutions

Thermal denaturation of soybean globulin fraction (SBGF) in diluted solution (protein concentration 0.15–0.63%) has been studied by the method of differential adiabatic scanning calorimetry. SBGF thermograms have two maxima. The low temperature maximum is consistent with denaturation of 7S component, while the high temperature maximum with denaturation of 11S components of this fraction. In the investigated range of protein concentrations the thermodynamic parameters (temperature and enthalpy) of denaturation of SBGF and its main components are constant. This fact suggests that differential adiabatic scanning calorimetry gives information purporting a change in the protein state at molecular level. The temperatures and enthalpies of denaturation of the main SBGF components linearly rise with increase of NaCl concentration. The slope of dependences of denaturation temperature on salt concentration,K _s, is extremely large (nearly 20 K · l/mole). The elementary thermodynamic theory of lyotropic effects in thermal denaturation of proteins has been developed based on the two-state model and linear approximation of protein-salt interactions by means of the corresponding second virial coefficient. It shows that the dependences of thermodynamic parameters of thermal denaturation on salt concentration should be linear in the initial section. This conclusion is consistent with the experiment. The differences of enthalpies and entropies of transferring denatured and native forms of the main SBGF components from water into NaCl solution have been determined. They are positive and their quantity increases linearly with salt concentration. This fact is consistent with the concept to the effect that the main factor of salt influence on thermal denaturation of SBGF is confined to a decrease of protein hydration. The effect of protein nature on the quantity of lyotropic effect in thermal denaturation has been considered. Using simple considerations as a basis, the dependence of the ratio betweenK _s and the denaturation temperature in water has been obtained, which characterizes the lyotropic effect, on the molar fraction of hydrophobic residues in the protein molecule. This dependence is linear and the lyotropic effect rises with increase in the content of hydrophobic residues. It is satisfactorily consistent with the experimental data on NaCl effect on thermal denaturation temperature for ichthyocol gelatin, ribonuclease, lysozyme, 7S and 11S SBGF components. An extraordinary strong influence of NaCl on thermal denaturation temperatures for the main SBGF components can be accounted for by a relatively high content of hydrophobic residues.     

溶液热历史对蛋白质晶体生长影响的内在原因研究

从溶液中聚集体的角度研究了溶液的热历史改变生长出的蛋白质晶体的数目和尺寸的内在原因. 将在281 和309 K下保存1 d的两组溶菌酶溶液按不同比例混合, 加入沉淀剂生长晶体. 随着高温溶液的比例增加, 生长出的晶体数目减少, 同时溶液中生长基元的尺寸增大. 在5周内, 采用动态光散射对281, 293和309 K三种温度下保存的溶菌酶溶液中聚集体的变化情况进行监测, 发现溶液中均存在大小不同的两部分聚集体, 称之为小聚集体与多聚体. 前者的尺寸基本不随保存时间而变化, 而后者尺寸随保存时间增加而减小, 减小的速度与保存温度有关. 多聚体的尺寸经过5周后和小聚集体基本相同. 研究结果表明, 处于无序聚集阶段的溶液的均一化程度和成核阶段生长基元的尺寸受到了溶液热历史的影响, 并最终对晶体的数目产生影响.     

On the application of CZE to the study of protein denaturation

Experimental mobilities obtained from CZE are used to study protein denaturation through a model based on known physicochemical theories. This model is able to provide additional information concerning the folded and unfolded protein states from mobility data. Its use comprises first the evaluation of relevant parameters of the protein microstates like the electrostatic free energy, apart from the classical conformational free energy, and second the expression of raw experimental data concerning the folding-unfolding transition into more specific physicochemical parameters like protein hydrodynamic radius, net charge number, and hydration. Spurious effects that are intrinsic to the experimental evaluation of the mobility of protein states, like BGE viscosity, pH, and ionic strength variations accompanying the changes of the denaturant agent intensity are eliminated. In order to illustrate the proposal of this work, two case studies are considered here. The first one concerns thermal and urea denaturations of horse heart ferricytochrome c and the second one involves thermal denaturation of hen egg-white lysozyme. Thus, relevant theoretical thermodynamic considerations of the folded-unfolded protein transition are presented, where the electrostatic free energy is included explicitly in the effective free energy. It is found that this transition involves sharp increases of hydrodynamic radius and protein hydration.     

Conformational relaxation dynamics of a poly(N-isopropylacrylamide) aqueous solution measured using the laser temperature jump transient grating method

We observed phase transition and phase relaxation processes of a poly(N-isopropylacrylamide) (PNIPAM) aqueous solution using the heterodyne transient grating (HD-TG) method combined with the laser temperature jump technique. The sample temperature was instantaneously raised by about 1.0 K after irradiation of a pump pulse to crystal violet (CV) molecules for heating, and the phase transition was induced for the sample with an initial temperature just below the lower critical solution temperature (LCST); the following phase relaxation dynamics was observed. Turbidity relaxation was observed in both the turbidity and HD-TG responses, while another relaxation process was observed only in the HD-TG response, namely via the refractive index change. It is suggested that this response is due to formation of globule molecules or their assemblies since they would have nothing to do with turbidity change but would affect the refractive index, which is dependent on the molar volume of a chemical species. Furthermore, the grating spacing dependence of the HD-TG responses suggests that the response was caused by the counter propagating diffusion of the coil molecules as a reactant species and the globule molecules as a product species and the lifetime of the globule molecules ranged from 1.5 to 5 seconds. Thus, we conclude that the turbidity reflects the dynamics of aggregate conditions, not molecular conditions. The coil and globule sizes were estimated from the obtained diffusion coefficient. The sizes of the coil molecules did not change at the initial temperatures below the LCST but increased sharply as it approaches LCST. We propose that the coil-state molecules associate due to hydrophobic interaction when the initial temperature was higher than LCST minus 0.5 K and that the globule-state molecules generated from the coil-state molecules showed a similar trend in temperature. The phase transition was also induced by heating under a microscope, and the relaxation process was followed using the fluorescence peak shift of a fluorescent molecule-labeled PNIPAM. The result also supports the existence of a globule molecule or its assembly remains for several seconds in the phase relaxation.     

Saccharide‐structure effects on poly N‐isopropylacrylamide phase transition in aqueous media; Reflections on protein stability

Protein stability in aqueous solutions is important in numerous fields, particularly biotechnology and food‐science. To shed new light on the protective effect of carbohydrates on proteins, we studied saccharide‐structure effects in aqueous solutions on the coil‐to‐globule transition occurring at the lower critical solution temperature (LCST) of poly‐N‐isopropylacrylamide (PNIPA), an isomer of polyleucine, as a simple model representing certain key behaviors of proteins (e.g., denaturation/renaturation). We systematically selected sugars and polyols to relate structural and physical characteristics of these carbohydrates to their effect on PNIPA solutions. Using isothermal titration‐microcalorimetry, we showed that no significant binding of saccharides to the polymer occurs. Using micro‐DSC, we studied the decreasing polymer LCST temperature with rising carbohydrate concentration. Beyond the expected observation that steric exclusion is important, we observed previously‐unreported significant differences among the effects of isomeric aldohexoses and also among the effects of isomeric diglucoses on PNIPA LCST. We found good correlation between the sugar hydration number and its effect on LCST. We conclude that the larger and denser the hydrated cluster a carbohydrate forms, the worse a cosolvent is for the polymer, and the stronger it's lowering effect of the coil‐to‐globule transition. Such favoring of the compact globule state provides a protective effect against denaturation of globular proteins. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 2307–2318, 2008     

Denatured Thermodynamics of Proteins in Weak Cation－exchange Chromatography

The thermostability of some proteins in weak cation-exchange chromatography was investigated at 20—80 ℃. The results show that there is a fixed thermal denaturation transition temperature for each protein. The appearance of the thermal transition temperature indicates that the conformations of the proteins are de-stroyed seriously. The thermal behavior of the proteins in weak cation-exchange and hydrophobic interaction chromatographies were compared in a wide temperature range. It was found that the proteins have a higher thermostability in a weak cation-exchange chromatography system. The thermodynamic parameters (△H^0,△S^0) of those proteins were determined by means of Van′t t Hoff re|ationship(lnk′-1/T). According to stan-dard entropy change(△S^0) , the conformational change of the proteins was judged in the chromatographic pro-cess. The linear relationships between △H^0 and △S^0 can be used to evaluate “compensation temperature“ (β) at the protein denaturation and identify the identity of the protein retention mechanism in weak cation-ex-change chromatography.     

不同结晶度的乙二醇及其水溶液玻璃化转变与焓松弛

为了考察晶体成分对无定形成分玻璃化转变和结构松弛行为的影响,利用差示扫描量热法(DSC),结合低温显微技术,研究了乙二醇(EG)及其50％水溶液在不同结晶度时的玻璃化转变和焓松弛行为.采用等温结晶方法控制骤冷的部分结晶玻璃体中的晶体份额.DSC结果表明,对于部分结晶的EG,只有单一的玻璃化转变过程,而对于50％EG,当结晶度不同时,不同程度地表现出两次玻璃化转变（无定形相Ⅰ和无定形相Ⅱ）.相Ⅰ的玻璃化转变温度和完全无定形态的含水EG的玻璃化转变温度相一致;相Ⅱ的玻璃化转变温度要比此温度约高6 ℃.低温显微观察结果印证了DSC实验结果.DSC等温退火的实验和KWW(Kohlrausch-Williams-Watts)衰变函数分析结果表明,EG无定形和50％EG中的两种无定形有不同的焓松弛行为.     

Thermal depolarization in ammonium chloride crystals,I

The thermal depolarization of crystals of NH₄Cl polarized above the phase transition temperature (242.5K) by an applied electric field was studied in 13 crystals prepared under a variety of conditions in order to examine the effects of doping with divalent anions or cations and of acid or alkaline conditions during crystal growth. All the features found in crystals grown from solution also occur in a crystal grown from the vapor. We may conclude, therefore, that the depolarization effects observed are properties of crystalline NH₄Cl and are not due to water or urea occluded during crystal growth. Four main depolarizations were observed: one of these is ascribed to space charge and the other three are attributed to dipole relaxation processes. The activation energies for these are 0.58, 0.73, and 0.85 eV, respectively. The various doping experiments lead to the conclusion that one of these is favored by acid conditions and the presence of cation vacancies and the other two by alkaline conditions and the presence of anion vacancies. Specific models for the dipolar defects are formulated and discussed.     

Molecular dynamics simulations for water and ions in protein crystals

The spatial and temporal properties of water and ions in bionanoporous materials-protein crystals-have been investigated using molecular dynamics simulations. Three protein crystals are considered systematically with different morphologies and chemical topologies: tetragonal lysozyme, orthorhombic lysozyme, and tetragonal thermolysin. It is found that the thermal fluctuations of C(alpha) atoms in the secondary structures of protein molecules are relatively weak due to hydrogen bonding. The solvent-accessible surface area per residue is nearly identical in the three protein crystals; the hydrophobic and hydrophilic residues in each crystal possess approximately the same solvent-accessible surface area. Water distributes heterogeneously and has different local structures within the biological nanopores of the three protein crystals. The mobility of water and ions in the crystals is enhanced as the porosity increases and also by the fluctuations of protein atoms particularly in the two lysozyme crystals. Anisotropic diffusion is found preferentially along the pore axis, as experimentally observed. The anisotropy of the three crystals increases in the order: tetragonal thermolysin < tetragonal lysozyme < orthorhombic lysozyme.     

Temperature calibration of an electrical compensation DSC on cooling using thermally stable high purity liquid crystals

Temperature calibration of DSCs is usually carried out on heating. In order to accurately control the temperature during cooling experiments, the calibration has to be carried out on cooling. Therefore, three high-purity, thermally stable liquid crystals were used to perform a temperature calibration of an electrcial compensation DSC on cooling. All three liquid crystals have several liquid crystalline phases, and they all were purified to a 99.9% lovel. Temoperatures of the isotropic to nematic or cholesteric and the mesophase to mesophase transitions were used. It was verified that these liquid crystals have sufficient thermal stability for carrying out the calibration on cooling. The dependence of the real temperature on the indicated temperature has been established for all the combinations of the heating and cooling rates of practical interest. It is also shown that the vant's Hoff equation can only be applied to the crystal to a liquid crystal transition, but not to the liquid crystal to liquid crystal or liquid crystal to isotropic transitions.     

On the Use of the Hypothesis of Statistically Homogeneous Suspensions in the Calculation of Their Conductivity

Electrostatic effects on protein adsorption were investigated using differential scanning calorimetry (DSC) and adsorption isotherms. The thermal denaturation of lysozyme, ribonuclease A (RNase), and alpha-lactalbumin in solution and adsorbed onto silica nanoparticles was examined at three concentrations of cations: 10 and 100 mM of sodium and 100 mM of sodium to which 10 mM of calcium was added. The parameters investigated were the denaturation enthalpy (DeltaH), the temperature at which the denaturation transition was half-completed (T(m)), and the temperature range of the denaturation transition. For lysozyme and RNase, adsorption isotherms depend strongly on the ionic strength. At low ionic strength both proteins have a high affinity for the silica particles and adsorption is accompanied by a 15-25% reduction in DeltaH and a 3-6 degrees C decrease in T(m), indicating that the adsorbed state of the proteins is destabilized. Also, an increase in the width of the denaturation transition is observed, signifying a larger conformational heterogeneity of the surface bound proteins. At higher ionic strengths, both with and without the addition of calcium, no significant adsorption-induced alteration in DeltaH was observed for all three proteins. The addition of calcium, however, decreases the width of the denaturation transition for lysozyme and RNase in the adsorbed state. Copyright 2001 Academic Press.     

Thermal denaturation of some proteins and its effect on their electrospray mass spectrat

The effects of beat on the electrospray mass spectra of eight globular proteins in solution were studied. These ranged from hardly noticeable to a dramatic shift in the mass spectrometric profile and a concomitant increase in ion abundance. This change is believed to be the result of thermal denaturation of the protein species in solution resulting in a transition from a more compact to a less compact conformation. We accounted for this transition by means of a recently proposed model based on aqueous solution acid/base equilibria. For cytochrome c, profiles calculated by means of this model agree well with experimental data. The ΔH of the denaturation reaction of cytochrome c in aqueous solution containing 0.2% acetic acid was calculated from experimental data to be 103.8 ± 9.2 kJ mol^?1, in good agreement with previous measurements.     

Spectral, optical, and thermal studies of pure and Zn(II)-doped l-histidine hydrochloride monohydrate (LHHC) crystals

The influence of doping the transition metal Zn(II) on the growth, spectral, optical, and thermal properties of l-histidine hydrochloride monohydrate (LHHC) crystals grown by slow solvent evaporation method has been investigated. Structural characterizations of the grown crystals were carried out by single crystal X-ray diffraction analysis and it shows slight structural changes as a result of doping. The FT-IR spectral study reveals the presence of various functional groups and confirms the slight distortion of the structure of the crystals due to doping. The energy dispersive X-ray analysis reveals the incorporation of Zn(II) in the crystalline matrix of LHHC crystal. The UV?CVis spectral study was carried out to analyze the optical transmittance of the grown crystals and found that the transmittance is very high in the visible and UV regions for both pure and doped crystals. The second harmonic generation (SHG) for the grown crystals was confirmed by Nd:YAG laser. The scanning electron microscopy reveals the presence of defect centers and crystal voids. The thermal stability and purity of the grown crystals were analyzed by thermogravimetry, differential thermal analysis, and differential scanning calorimetry techniques.     

Conformational motion and disorder in aliphatic nylons The case of nylon 6.6

On the basis of thermal analysis it is suggested that the crystals of aliphatic nylons exhibit conformational disorder above the glass transition. The disorder begins gradually at about room temperature and is evidenced by an increase of the heat capacity to values higher than that of the melt. The specific case of nylon 6.6 is investigated by thermal analysis and x-ray diffraction. The onset of conformational disorder can be clearly separated from premelting. It is shown that the Brill transition, as defined by the merging of the two main peaks in the x-ray diffraction pattern, occurs gradually and is thermal-history-dependent. The transition is not a first-order one, it is only an incidental thermal effect, associated with a packing change in the crystal. In solution-crystallized (sc) samples this change is related to a distinct endothermic peak, while in melt-crystallized (mc) samples it is related to a broad endotherm.Dedicated to Professor Dr. W. Pechhold on the occasion of his 60th birthday     

侧链型丙烯酸酯共聚物相变的正电子湮没谱研究

利用正电子湮没技术对侧链型热致高分子液晶丙烯酸酯共聚物进行了变温相变研究．除实验标识出样品的相变温度点外，根据试样中自由体积随温度的变化关系，对高分子液晶材料内部立链、侧链以及介晶基元的相变行为特点进行了探讨，并就与小分子液晶变化特点的一些不同做了解释．     

Acoustic Emission from Organic Martensites

In salient effects, still crystals of solids that switch between phases acquire a momentum and are autonomously propelled because of rapid release of elastic energy accrued during a latent structural transition induced by heat, light, or mechanical stimulation. When mechanical reconfiguration is induced by change of temperature in thermosalient crystals, bursts of detectable acoustic waves are generated prior to self‐actuation. These observations provide compelling evidence that the thermosalient transitions in organic and organic‐containing crystals are molecular analogues of the martensitic transitions in some metals, and metal alloys such as steel and shape‐memory alloys. Within a broader context, these results reveal that, akin to metallic bonding, the intermolecular interactions in molecular solids are capable of gradual accrual and sudden release of a substantial amount of strain during anisotropic thermal expansion, followed by a rapid transformation of the crystal packing in a diffusionless, non‐displacive transition.