南极细菌胞外多糖溶液冻结特性的差示扫描量热研究

采用差示扫描量热法,测定几种南极细菌胞外多糖(简称,EPSs)溶液的结晶、熔融、焓转变以及水合性质等冻结特性,分析了EPSs的浓度和分子量与其抗冻活性的关系.结果表明,在溶液冻结过程中,仅0.25%的Pseudoalteromonas sp.S-15-13 EPSs(分子量,6.2×104Da)可抑制冰核形成,溶液冻结温度较纯水的降低(1.07±0.62)℃;溶液的冻结焓降低说明冰核生长变缓,冰晶形成细小,0.125%的Shewanella sp.5-1-11-4 EPSs(分子量,1.2×103Da)和Moritella sp.2-5-10-1 EPSs(分子量,3.0×103Da)冻结焓分别较纯水的降低17.15%和29.13%,S-15-13 EPSs在0.125%～0.5%的范围内可降低冻结焓,0.125%时冻结焓较纯水的低30%,其不冻水含量为(0.292 ±0.05) g/g.在冰晶熔化过程中,几种EPSs均可降低溶液熔融温度和熔融焓,促进冰晶熔化,使冰晶细小;4.0%的5-1-11-4 EPSs、2-5-10-1 EPSs和0.5% S-15-13 EPSs的熔融温度较纯水的分别降低(2.70±0.15)℃、(2.30±0.39)℃和(4.66±0.42)℃.研究结果阐明EPSs可以通过改变菌体周围水的冻结特性,以抵御冰晶对微生物的损伤,大分子量EPSs对冰晶的抑制作用强于低分子量的.     

溶剂极性对碳酸酐酶热变性的影响

以差示扫描量热技术为手段研究了具不同碳氢链长度及不同浓度的醇－水溶剂对碳酸酐酶热变性温度及热变性焓的影响,以探讨两性分子对蛋白质构象及热稳定性的影响。结果表明,随着甲醇、乙醇及丙醇各自浓度的增加碳酸酐酶的变性温度降低;在相同的醇浓度下随着醇的碳氢链的加长,碳酸酐酶的变性温度明显下降;当醇在低浓度,例如１０％时,碳酸酐酶的热变性焓比在纯缓冲液中要高。而在高浓度时其变性焓比在纯缓冲液中要低,且随着碳氢     

脂肪酸对牛血清白蛋白的水合及热变性的影响

本文用差示扫描量热技术和热重分析技术分别测定了结合有不同脂肪酸的牛血清白蛋白在不同相对湿度下的水合值R、热变性温度T_D及变性焓ΔH_D。实验结果表明,在任一实验相对湿度下,脂肪酸含量的增加均导致牛血清白蛋白(简称BSA)的水合值增加,其增加程度因脂肪酸的种类而异;在低相对湿度下,脂肪酸含量的增加对BSA的热变性起敏化作用即使T_D降低;而在高相对湿度(例97%)及水溶液状态下,脂肪酸含量的增加对BSA的热变性起保护作用即使T_D增加,其影响程度为月桂酸>油酸>硬脂酸。月桂酸和油酸使BSA的ΔH_D增加,硬脂酸使其ΔH_D降低。实验还表明脂肪酸与低温吸热峰的出现无关。     

小牛胸腺脱氧核糖核酸内水的量热研究——Ⅱ.非平衡态下水的恒温冻结动力学

用差示扫描量热法研究了DNA内水的冻结行为和在218K下的恒温冻结动力学.实验表明了低温下水-DNA体系及其冻结的非平衡性.冻结是个复杂的一级串联反应,其速率与初始水含量R及实验条件密切相关.在不同R及不同恒温时间t_k下冻结的微观过程不同.不同含水量样品在相同t_k下具有不同的不冻水量R_(nf)然而只要过程的自由能降低,在不同t_k下却可达到相同的R_(nf).“不冻水”是个纯动力学现象,其量与R、冻结温度及t_k等实验条件密切相关.所谓“不冻水”并非由于水同大分子的特殊相互作用所致.     

水合溶菌酶及其热稳定性的NMR研究

本文用90MHz脉冲NMB波谱仪记录了具不同含水量的溶菌酶的质子宽谱线NMR谱图及自由感应衰减曲线,并记录了水合度为0.10及0.19克水/克溶菌酶的溶菌酶样品从室温到热变性温度范围内的谱图.用线宽参量随水合度及温度的变化讨论溶菌酶在水合及热变性过程中溶菌酶分子及水分子运动性的变化.结果表明,溶菌酶分子及水分子的运动性与水合溶菌酶中的水含量密切相关;低水含量的水合溶菌酶在热变性过程中酶分子运动性的变化经历了两个转变,分别对应于酶分子间的解缔合及分子内的解旋.     

牛胰RNaseA的热转变的差示扫描量热研究

用差示扫描量热法对含水量为0.05-3.15克/克的牛胰核糖核酸酶A的热转变进行了研究.当R<0.40克/克时,在315-345和400-450K左右,分别观察到峰Ⅰ和峰Ⅲ.文中对峰Ⅰ的“恢复”进行了讨论.在R<1.1克/克时,通常被认为热变性峰的峰Ⅱ的峰温,随R的增加而降低,变性热随R的增加而减少,但在 R≥1.1克/克时,二者均取稳定值,Ttr=335.5K和Qtr=7.38CaⅠ/go峰Ⅱ的半宽在R=0.40克/克处取极小值,在R≥1.65时取稳定值,△T1/2=7.34K,文中首次给出了水合状态下热变性峰的转变热和峰温的关系曲水线.对水合球状蛋白的热变性的一种可能解释为,变性焓是温度的函数,而转变温度直接受含水量影响.     

牛胰RNaseA的热转变的差示扫描量热研究

用差示扫描量热法对含水量为0.05-3.15克/克的牛胰核糖核酸酶A的热转变进行了研究.当R<0.40克/克时,在315-345和400-450K左右,分别观察到峰Ⅰ和峰Ⅲ.文中对峰Ⅰ的“恢复”进行了讨论.在R<1.1克/克时,通常被认为热变性峰的峰Ⅱ的峰温,随R的增加而降低,变性热随R的增加而减少,但在 R≥1.1克/克时,二者均取稳定值,Ttr=335.5K和Qtr=7.38CaⅠ/go峰Ⅱ的半宽在R=0.40克/克处取极小值,在R≥1.65时取稳定值,△T1/2=7.34K,文中首次给出了水合状态下热变性峰的转变热和峰温的关系曲水线.对水合球状蛋白的热变性的一种可能解释为,变性焓是温度的函数,而转变温度直接受含水量影响.     

水合牛血清白蛋白热稳定性的热力学研究

蛋白质的初级结合水对于蛋白质分子的构象及热稳定性有着重要影响。将不同吸附水量的牛血清白蛋白样品密封入铝制挥发型样品盘中,用P/E DSC_(-2)型差示扫描量热计对蛋白样品的变性温度、变性焓及变性前后的比热变化等热力学参数进行测量。实验证明,随水含量增多变性温度T_D下降,当含水量R(克H_2O/克BSA)>0.24时T_D的下降渐微,当R=0.5时T_D通过最小值后又略有增大。变性焓ΔH_D也与水含量密切相关,当R<0.5时ΔH_D渐趋恒值,为200千卡/克分子。本实验还观察到在低含水量范围内0.02相似文献   

小牛胸腺脱氧核糖核酸内水的量热研究——Ⅰ.水的热力学状态

在0.40-4.35g(water)/g(DNA)范围内用差示扫描量热法(DSC)研究了DNA内水的热力学状态与水合度R的关系.实验中观察到突出的冻结/熔融滞后.在R≤0.70g/g时无冻结放热峰可见,然而在218K下恒温15min于0.49g/g时即测到吸热峰.在R≥0.95和0.60-0.95g/g范围,积分熔融热Q_f-R分别呈线性和接近线性关系,表观微分熔融热分别为72.29和47.73cal/g.R≤0.60g/g时Q_f-R呈非线性关系.在0.49g/g时,Q_f随恒温时间t_k指数增加.实验表明,水的状态参数是依赖于t_k等动力学因素而随R连续变化的热力学量.由此我们提出了有关大分子内不冻水的两个概念.     

蛋白质热变性前新峰形成机制探讨

蛋白质热变性前新峰是蛋白质热变性过程的共性。通过对蛋白质三级结构特征的理论分析及实验验证的方法,研究了蛋白质热变性前新峰的变化规律,从而揭示了该峰产生的机理。采用ＤＳＣ方法对以不同结构水和十二烷基硫酸钠溶液水合溶菌酶样本进行了研究, 结果表明蛋白质的这种热变性前新峰的存在是由于维持其三级结构的疏水相互作用所造成, 新峰出现的峰温及其焓变与水的结构改变及由此而造成的蛋白质中结合水的含量和结构功能的变化有着直接的关系。     

Calorimetry of rat tail tendon collagen before and after denaturation: the heat of fusion of its absorbed water

The specific heat, of rat tail tendon at various water contents was measured as a function of temperature. The resulting graphs showed peaks arising from the melting, near 50°C, of helical material in the collagen, and from the melting of absorbed water in the range -40°C to 0°C. The heat of melting of helical material was 11.7 cal per gram of dry tendon. Determination of the heat and temperature of fusion of the absorbed water allowed resolution of the water into four states in the case of tendon before denaturation, and three states after denaturation. The four states are (1) water not freezable on cooling to - 70°C, (2) freezable water with-both heat and temperature of fusion different from the values for ordinary water, (3) freezable water with the heat of fusion of ordinary water, but a different temperature of fusion, and (4) water not distinguished from ordinary water. The fourth state was absent in denatured tendon. The results are discussed in terms of increasing size of clusters of absorbed water molecules.     

胶原纤维内吸附水熔融热的差示扫描量热法的研究

用DSC法研究了天然与变性牛蹄跟腱胶原蛋白内吸附水的熔融热.在含水量(R)为0.33克(水)/克(蛋白)时观察到冰的熔融峰.在0.57克/克以下,水的积分熔融热(Q_f)与水含量呈非线性关系,由此得到出现熔融峰的临界水含量为0.26克/克.在0.57—1.05克/克范围内Q_f同R接近线性关系,其表观微分熔融热dQ/dR=69.5卡/克(水).R>1.05克/克时,dQ/dR=79.2卡/克.     

Thermal conformational transformation of tropocollagen. I. Calorimetric study

Effects of heat in heated solution of tropocollagens of different origins were calorimetrically studied. It was found that denaturation enthalpy and entropy of different tropocollagens increase with increasing imino acid content and thermostability. It is shown that the value and dependence of denaturational enthalpy and entropy on the denaturation temperature for tropocollagens with different imino acid contents are inconsistent with the assumption that the native structure of tropocollagen is stabilized only by intramolecular hydrogen bonds. A supposition is made that the regular water structure near the macromolecule plays an essential role in stabilizing the structure. From the character of tropocollagen melting curves in salt-free solution it is found that the tropocollagen macromolecule is linearly heterogeneous. It is shown that the complex pattern of thermal absorption observed in tropocollagen salt, solution is connected with pre-denaturational conformational transformation when approaching conditions close to the physiological.     

Characterizing the secondary hydration shell on hydrated myoglobin, hemoglobin, and lysozyme powders by its vitrification behavior on cooling and its calorimetric glass-->liquid transition and crystallization behavior on reheating.

For hydrated metmyoglobin, methemoglobin, and lysozyme powders, the freezable water fraction of between approximately 0.3-0.4 g water/g protein up to approximately 0.7-0.8 g water/g protein has been fully vitrified by cooling at rates up to approximately 1500 K min-1 and the influence of cooling rate characterized by x-ray diffractograms. This vitreous but freezable water fraction started to crystallize at approximately 210 K to cubic ice and at approximately 240 K to hexagonal ice. Measurements by differential scanning calorimetry have shown that this vitreous but freezable water fraction undergoes, on reheating at a rate of 30 K min-1, a glass-->liquid transition with an onset temperature of between approximately 164 and approximately 174 K, with a width of between approximately 9 and approximately 16 degrees and an increase in heat capacity of between approximately 20 and approximately 40 J K-1 (mol of freezable water)-1 but that the glass transition disappears upon crystallization of the freezable water. These calorimetric features are similar to those of water imbibed in the pores of a synthetic hydrogel but very different from those of glassy bulk water. The difference to glassy bulk water's properties is attributed to hydrophilic interaction and H-bonding of the macromolecules' segments with the freezable water fraction, which thereby becomes dynamically modified. Abrupt increase in minimal or critical cooling rate necessary for complete vitrification is observed at approximately 0.7-0.8 g water/g protein, which is attributed to an abrupt increase of water's mobility, and it is remarkably close to the threshold value of water's mobility on a hydrated protein reported by Kimmich et al. (1990, Biophys. J. 58:1183). The hydration level of approximately 0.7-0.8 g water/g protein is approximately that necessary for completing the secondary hydration shell.     

X-ray diffraction studies on the structure of hydrated collagen

The temperature dependence of the humidity-sensitive spacing, d, related to the lateral packing of collagen molecules was measured for fully hydrated collagen. In the vicinity of 0°C, a sudden change in d was observed, which was reversible with temperature. In the diffraction profile, below 0°C, a set of diffraction peaks identified with the hexagonal crystalline form of ice was observed. With the reduction in water content, the intensity of the set of diffraction peaks decreased and was found to be zero at a water content of 0.38 g/g collagen. These results were considered to be caused by the frozen water in collagen fibril below 0°C. According to the water content dependence of d, it was considered that up to a certain water content water absorbed would be stowed in the intermolecular space of collagen and above that water content water molecules would aggregate to make pools, i. e., extrafibrillar spaces. The unfreezable bound water was considered to be located in the intermolecular space of collagen. Size of the extrafibrillar space, determined from the intensity analysis of a smallangle x-ray scattering pattern, corroborates the speculation that the water showed in the extrafibrillar space is freezable and free. The formation of the hexagonal crystalline form of ice in the extrafibrillar space was considered to cause the sudden change in d at 0°C.     

Effects of hydrated water on protein unfolding

The conformational stability of a protein in aqueous solution is described in terms of the thermodynamic properties such as unfolding Gibbs free energy, which is the difference in the free energy (Gibbs function) between the native and random conformations in solution. The properties are composed of two contributions, one from enthalpy due to intramolecular interactions among constituent atoms and chain entropy of the backbone and side chains, and the other from the hydrated water around a protein molecule. The hydration free energy and enthalpy at a given temperature for a protein of known three-dimensional structure can be calculated from the accessible surface areas of constituent atoms according to a method developed recently. Since the hydration free energy and enthalpy for random conformations are computed from those for an extended conformation, the thermodynamic properties of unfolding are evaluated quantitatively. The evaluated hydration properties for proteins of known transition temperature (Tm) and unfolding enthalpy (delta Hm) show an approximately linear dependence on the number of constituent heavy atoms. Since the unfolding free energy is zero at Tm, the enthalpy originating from interatomic interactions of a polypeptide chain and the chain entropy are evaluated from an experimental value of delta Hm and computed properties due to the hydrated water around the molecule at Tm. The chain enthalpy and entropy thus estimated are largely compensated by the hydration enthalpy and entropy, respectively, making the unfolding free energy and enthalpy relatively small. The computed temperature dependences of the unfolding free energy and enthalpy for RNase A, T4 lysozyme, and myoglobin showed a good agreement with the experimental ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mobility of molecules and ions solubilized in protein gels: diffusion in the thick fraction of hen egg white

The thick fraction of hen egg white is a protein hydrogel with an immeasurably high viscosity composed of ～90% water that can serve as a model system for mammalian mucous membrane. Measurements of the rate constants of diffusion-controlled reactions occurring within the gel (and corresponding activation energies) and electric conductivity revealed that the thick fraction of egg white can be envisioned as a 3D network comprising hydrated protein molecules (held by intermolecular S-S bridges) surrounded by water pools and channels (of nonuniform diameters) that have a microviscosity that is very similar to that of bulk water. This was corroborated by differential scanning calorimetry measurements that revealed that 16% of water is bound to proteins. The melting kinetics of ice crystallites (produced from the freezable water) indicates nonhomogeneous water pool size.     

Calorimetric study of crystal growth of ice in hydrated methemoglobin and of redistribution of the water clusters formed on melting the ice.

Calorimetric studies of the melting patterns of ice in hydrated methemoglobin powders containing between 0.43 and 0.58 (g water)/(g protein), and of their dependence on annealing at subzero temperatures and on isothermal treatment at ambient temperature are reported. Cooling rates were varied between approximately 1500 and 5 K min-1 and heating rate was 30 K min-1. Recrystallization of ice during annealing is observed at T > 228 K. The melting patterns of annealed samples are characteristically different from those of unannealed samples by the shifting of the melting temperature of the recrystallized ice fraction to higher temperatures toward the value of "bulk" ice. The "large" ice crystals formed during recrystallization melt on heating into "large" clusters of water whose redistribution and apparent equilibration is followed as a function of time and/or temperature by comparison with melting endotherms. We have also studied the effect of cooling rate on the melting pattern of ice with a methemoglobin sample containing 0.50 (g water)/(g protein), and we surmise that for this hydration cooling at rates of > or = approximately 150 K min-1 preserves on the whole the distribution of water molecules present at ambient temperature.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.