【作者】 刘春丽；

【导师】 林瑞森；

【作者基本信息】 浙江大学 ， 物理化学， 2007， 博士

【摘要】 氨基酸作为蛋白质的基本结构单元，是研究蛋白质结构和性质的良好模型化合物。通过对氨基酸在水及混合溶剂中的热力学性质的研究，既可以获得溶质-溶剂相互作用的信息，又有助于认识蛋白质的稳定性机理。盐溶液对蛋白质的结构和功能有着重要的影响，酰胺衍生物具有与蛋白质的骨架结构相同的肽键，是多肽结构的一种很好的模型化合物，研究氨基酸在盐及酰胺衍生物水溶液中的热力学性质，有助于揭示蛋白质与盐之间、蛋白质的氨基酸残基之间以及蛋白质与周围介质之间的相互作用。本论文是国家自然科学基金资助项目（No．20273061）的部分工作，主要由以下五个部分组成：第一章：简要介绍了蛋白质模型分子溶液热力学的研究背景、理论和研究现状。第二章：用迁移焓方法研究了甘氨酸、L-丙氨酸和L-丝氨酸三种典型的天然氨基酸与含氧酸盐：LiNO₃、NaNO₃、KNO₃、Na₂SO₄和NaClO₄之间的相互作用。用RD496-Ⅱ型微量热量计分别测定了298.15 K三种氨基酸在含氧酸盐水溶液中的溶解焓，以迁移焓为探针，系统研究了氨基酸的结构和离子的性质对迁移焓的影响，探讨了离子对蛋白质稳定性的影响及含氧酸盐水溶液的结构特征。研究表明：1)除L-丙氨酸在Na₂SO₄水溶液外，氨基酸在五种含氧酸盐中的迁移焓均为负值。不同的氨基酸从水到同浓度的含氧酸盐水溶液中的迁移焓的差异取决于氨基酸侧链的性质。三种氨基酸在NaNO₃，KNO₃，Na₂SO₄及低浓度的NaClO₄和LiNO₃溶液中迁移焓的大小均表现为：L-丙氨酸＞甘氨酸＞L-丝氨酸。在NaClO₄和LiNO₃溶液中，当盐的浓度大于1.5 mol·kg^-1时，迁移焓的大小为：L-丙氨酸＞L-丝氨酸＞甘氨酸，体现了高浓度时溶剂结构的变化。2)离子的水化性质对迁移焓有明显的影响，阴离子的影响要大于阳离子。离子越容易去水化，对迁移焓的负贡献越大。对于结构相似的阳离子或阴离子，半径越大，与氨基酸之间的结构相互作用越大，结构相互作用在大多数情况下超过了静电作用而成为影响氨基酸与盐之间迁移焓规律的主要因素。3)阴离子使蛋白质变性的能力与阴离子-甘氨酸之间相互作用的放热效应有近似平行的关系。第三章：用体积方法研究甘氨酸、L-丙氨酸和L-丝氨酸与LiNO₃、NaNO₃、KNO₃、Na₂SO₄和NaClO₄之间的相互作用。用DMA55型精密密度测定仪分别测定了298.15 K三种氨基酸在含氧酸盐水溶液中的密度。计算了氨基酸的迁移偏摩尔体积，讨论了氨基酸侧链对迁移偏摩尔体积和理论水化数的的影响。探讨了离子的性质对迁移体积和蛋白质稳定性的影响。研究发现：1)三种氨基酸在五种含氧酸盐水溶液中的迁移体积均为正值，并且随着盐浓度的增大而增大。在盐浓度相同时，三种氨基酸的迁移偏摩尔体积的大小顺序为：L-丝氨酸＞甘氨酸＞L-丙氨酸。2)氨基酸两性头部与盐之间的静电作用对迁移偏摩尔体积的贡献是主要的，静电作用的大小取决于离子的半径和电荷。同一种氨基酸在含氧酸盐中的迁移体积表现为：Na₂SO₄＞NaNO₃＞KNO₃＞NaClO₄＞LiNO₃。3)离子对蛋白质结构的破坏能力与离子对甘氨酸迁移体积影响减小的顺序基本一致。第四章：用溶解焓方法研究298.15 K甘氨酸、L-丙氨酸和L-丝氨酸与N-甲基甲酰胺（NMF），N，N-二甲基甲酰胺（DMF），N-甲基乙酰胺（NMA）和N，N-二甲基乙酰胺（DMA）之间的相互作用，并与氨基酸在甲酰胺（FA）和乙酰胺（AD）中的迁移焓进行比较，得到以下结论：1)除L-丝氨酸在低浓度NMF溶液外，三种氨基酸从水到四种酰胺衍生物水溶液中的迁移焓都为正值。三种氨基酸在同一种酰胺衍生物水溶液中的迁移焓表现为：L-丙氨酸＞甘氨酸＞L-丝氨酸，这种差异反映了侧链与溶剂之间的结构相互作用的不同。2)氨基酸两性离子头部与酰胺衍生物之间的结构相互作用对迁移焓的贡献是主要的，氨基酸与酰胺衍生物之间主要表现为两分子相互作用。3)同一种氨基酸在酰胺及酰胺衍生物溶液中的迁移焓大小为：DMA＞NMA＞DMF＞NMF＞AD＞FA，这一顺序与酰胺的疏水性大小是一致的。第五章：用与第三章中相同的方法研究298.15 K时甘氨酸、L-丙氨酸和L-丝氨酸分别在NMF、NMA、DMF和DMA水溶液中的体积性质。讨论了氨基酸的结构和酰胺衍生物的结构对迁移体积的影响，探讨了温度对三种氨基酸在DMA水溶液中迁移体积的影响。结果表明：1)三种氨基酸从水到酰胺衍生物水溶液中的迁移体积与氨基酸和酰胺的相对疏水性有关。甘氨酸和L-丝氨酸与四种酰胺间的亲水-亲水相互作用对迁移体积的贡献是主要的，迁移体积均为正值，并且随溶剂浓度的增大而增大，水化数随浓度的增大而减小。L-丙氨酸与NMA、DMF和DMA之间的疏水-疏水作用略占优势，迁移体积为负值，水化数较水溶液中稍大，但随溶剂浓度变化迁移体积和水化数变化很小。L-丙氨酸在低浓度NMF溶液中疏水-疏水作用对迁移体积的贡献较大，溶剂浓度增大，亲水-亲水作用逐渐明显，并且随浓度的增大而增大，水化数逐渐减小。2)在同一溶剂浓度下(除在m＞2.5 mol·kg^-1DMA溶液中)，三种氨基酸迁移偏摩尔体积的大小均表现为：L-丙氨酸＜L-丝氨酸＜甘氨酸，在m＞2.5 mol·kg^-1DMA溶液中为：L-丙氨酸＜甘氨酸＜L-丝氨酸，这反映了不同结构的氨基酸与酰胺衍生物在水溶液中结构相互作用的特征。3)对甘氨酸和L-丝氨酸，在整个研究的溶剂浓度范围内NMF、NMA和DMF结构的不同对迁移体积的影响很小，在较高浓度的DMA水溶液中的迁移体积明显大于在其它三种酰胺溶液中的迁移体积。对L-丙氨酸来讲，NMA、DMF和DMA的结构和浓度对迁移体积的影响不明显，而在NMF水溶液中随着浓度的增大，对迁移体积的正贡献逐渐增大并明显高于其它三种酰胺。4)温度对氨基酸在DMA溶液中的体积性质有着重要的影响，温度升高，氨基酸的极限偏摩尔体积增大，迁移体积减小。更多还原

【Abstract】 Amino acids have been useful models for understanding the thermodynamic behavior of proteins. Investigations on the thermodynamic behavior of amino acids in mixed aqueous solutions would not only provide information about interaction between solute and solvent but also the mechanism of action of proteins. As a part of the project supported by National Natural Science Foundation （No. 20273061）, this paper consists of the following five parts.The fast part （chapter 1） summarizes the studies on the thermodynamic theories and properties of protein model molecules.In the second part （chapter 2） the enthalpies of solution of glycine, L-alanine and L-serine in aqueous solutions of LiNO₃, NaNO₃, KNO₃, Na₂SO₄ and NaClO₄ were determined by RD496-Ⅱmicrocalorimeter at 298. 15 K. Enthalpy of transfer of amino acids were derived and interpreted in terms of electrostatic interaction and structural interaction. The results show that: 1) the transfer enthalpies (Δ_trH) of amino acids in the five oxacid salts solutions are positive except L-alanine in aqueous Na₂SO₄ solutions. The transfer enthalpies of amino acids from water to NaNO₃, KNO₃, Na₂SO₄ and low concentration NaClO₄ and LiNO₃ solutions vary in the sequence L-serine＜glycine＜L-alanine while glycine＜L-serine＜L-alanine in NaClO₄ and LiNO₃ solutions at the concentration above 1.5 mol·kg^-1. The difference may be due to the effect of anions and cations association at high concentration. 2) Anions exert much marked influences on the transfer enthalpy than cations. In most salts solutions, structure interactions influenceΔ_trH more strongly than electrostatic interactions. 3) There is an approximate parallel between the potency of the anions in inducing protein denaturation and the exothermicity of the anion-glycine ineractions. In the third part （chapter 3）, the densities of glycine, L-alanine and L-serine in aqueous solutions of LiNO₃, NaNO₃, KNO₃, Na₂SO₄ and NaClO₄ have been measured at 298.15 K with an Anton Paar Model 55 densimeter. Apparent molar volumes, limiting partial molar volumes, transfer volumes (Δ_trV) and hydration numbers have been calculated. The results show that: 1) all the transfer volumes of amino acids are positive and increase with increasing concentration of salts. At the same concentration of salts, the relative order ofΔ_trV is L-serine＞glycine＞L-alanine. 2) The dominating contribution to transfer volume comes from the electrostatic interaction between the head groups of amino acids and ions. The order of transfer volume of the same amino acid in oxyacid salts solutions is: Na₂SO₄＞NaNO₃＞KNO₃＞NaClO₄＞LiNO₃. 3) The destabilizing effect of ions on protein has approximately the same order as the decreasing transfer volumes of glycine.The forth part （chapter 4） have studied the enthalpies of transfer of glycine, L-alanine and L-serine in aqueous solutions of N-methylformamide, N, N-dimethylformamide, N-methylacetamide and N, N-dimethylacetamide with the same method as the second part. The results show that: 1) The transfer enthalpies of amino acids in the four amide solutions are positive except L-serine in low concentration NMF solutions and the relative order is L-alanine＞glycine＞L-serine in the same amide solutions. 2) The structure interaction between head groups of amino acids and amides have a large contribution toΔ_trH. The interaction type between amino acids and amides are dominated by 1:1 from the results of the enthalpic pair interaction coefficients. 3) For the same amino acid the value of transfer enthalpy is consist with the hydrophobicity of amides i. e. DMA＞NMA＞DMF＞NMF＞AD＞FA.In the last part （chapter 5）, the volumetric properties of glycine, L-alanine and L-serine in aqueous solutions of NMF, DMF, NMA and DMA have been studied using the same method as the third part. The volumes, of transfer of amino acids were discussed in terms of solute-solute interaction between amino acids and amides. It has been show that: 1) the transfer volumes of amino acid are related with the relative hydrophobicity of amino acids and amides. Hydrophilic-hydrophilic interactions are predominant in the case of glycine and L-serine in the four amides solutions and L-alanine in higher concentration NMF solutions, and hydrophobic-hydrophobic interactions are predominant in the case of L-alanine in NMA, DMF and DMA solutions and low concentration NMF solutions. Positive transfer volumes are observed for glycine and L-serine in the presence of the four amides and L-alanine in higher concentration NMF solutions. 2) The relative order of the transfer volume in the same concentrations amides solutions is L-alanine＜L-serine＜glycine except in m＞2.5 mol·kg^-1 DMA solutions, while in m＞2.5 mol·kg^-1DMA solutions the order is L-alanine＜glycine＜L-serine. 3) DMA has significant effect but NMA, NMF and DMF have small influence on the transfer volumes of glycine and L-serine. The transfer volumes of glycine and L-serine in higher concentration DMA solutions are much larger than in the other three amides. For L-alanine, NMA, DMF and DMA seem have little influence on the transfer volumes, but NMF has markedly influence and the transfer volumes of L-alanine in higher concentration NMF solutions are larger than that in other amides solutions. 4) An increase in temperature increases the standard partial molar volumes but decreases the transfer volumes of amino acids in DMA solutions.更多还原