【作者】 刘敏；

【导师】 林瑞森； 孙德志；

【作者基本信息】 浙江大学 ， 物理化学， 2007， 博士

【摘要】 蛋白质是生物体中必不可少的基本物质,在生命活动中起着重要的作用。血清白蛋白是血浆中具有广泛结合能力的蛋白质,能结合内源性和外源性物质,并能将这些物质输送到身体的各个部位。研究有机小分子与血清白蛋白的相互作用,可帮助人们更深入地了解亲水-憎水等弱相互作用在生物大分子溶液中的重要性,揭示相关生命过程的奥秘。氨基酸、肽、酰胺及其衍生物被认为是理论研究中重要的生物模型物质。糖类和多羟基化合物能够稳定球形蛋白质分子的天然构象。通过研究水溶液中氨基酸或酰胺与羟基化合物的热力学性质以及pH值对溶液热力学性质的影响,既可以获得水溶液中溶剂化的溶质分子间的相互作用方面的信息,又有助于了解多羟基化合物对蛋白质的稳定机理及氨基酸在蛋白质中的构象稳定性和解折叠过程中所担当的角色。微量热方法是现代热力学和热化学研究中的重要方法,对含有多种组分的生命体系溶液的热力学性质研究具有其它方法不可比拟的优势。因此,大量开展这方面的研究,对开拓物理化学的研究及应用领域、解决生命科学中许多复杂问题有着重要的理论意义和广阔的应用前景。二十一世纪是生命科学的世纪,应用微量热法结合波谱学方法研究拟生命体液中生物大分子与小分子之间的相互作用以及生物模型化合物的热力学性质,对于人类探究生命的本质和揭示生命之奥秘具有非常重要的意义。本论文作为国家自然科学基金资助项目（No.20273061）的部分工作,主要由以下五部分组成:第一部分:血清白蛋白（人血清白蛋白Human Serum Albumin,HSA;牛血清白蛋白Bovine Serum Albumin,BSA）与季铵盐双子表面活性剂的相互作用本部分应用等温滴定量热法研究298.15 K时血清白蛋白与两种季铵盐双子表面活性剂（（C_nN）₂Cl₂,n=12,14）在缓冲溶液（pH=7.0）中的相互作用,由实验数据拟合出结合常数、结合位点数以及热力学函数变化。并利用圆二色谱技术研究了这两种表面活性剂对蛋白二级结构的影响。结果表明:（1）HSA和BSA对这两种表面活性剂均有两类结合位点,一类是表面活性剂的极性基团和蛋白质分子表面的氨基酸残基之间的静电作用造成的强结合位点（有较高的结合常数）,此类结合为吸热过程;另一类是表面活性剂的憎水基团与蛋白质分子疏水空腔的疏水相互作用造成的弱结合位点（有较低的结合常数）,该过程放出热量。（2）HSA和BSA对这两种表面活性剂结合的熵效应均为正值。表面活性剂分子中疏水链的增长可加强它们向HSA或BSA分子上的结合,且同一种表面活性剂对HSA分子的结合强度大于BSA。对于高结合位点,两种表面活性剂分子对应的结合位点数差别不大。但对于弱结合位点,由于(C₁₄N)₂Cl₂疏水链过长,只有部分进入疏水空腔内,因此相应的结合位点数和放热量减小,而熵变增加。（3）这两种表面活性剂与HSA或BSA的结合作用会使蛋白质的二级结构发生变化,主要是α-螺旋向β-折叠的转变,这样会使构成α-螺旋的肽段伸展开来,转化为较为松散的二级结构。第二部分:血清白蛋白与丹皮酚及其两种同分异构体的相互作用本部分应用等温滴定量热法研究298.15 K时血清白蛋白与丹皮酚（2′-羟基-4′-甲氧基苯乙酮,Pae）及其两种同分异构体（2′-羟基-5′-甲氧基苯乙酮,Hma;4′-羟基-3′-甲氧基苯乙酮,Ace）在缓冲溶液（pH=7.0）中的相互作用。由实验数据拟合出结合常数、焓变、熵变及吉布斯自由能变等热力学数据。并利用圆二色谱技术研究了这三种药物分子对蛋白质分子二级结构的影响。结果表明:（1）血清白蛋白（Serum Albumin,SA）对这三种药物分子有两类结合位点,一类是通过药物分子苯环上的取代基与SA分子表面的氨基酸残基的静电及氢键作用形成,另一类则通过药物分子的苯环与SA分子疏水空腔的疏水相互作用达到彼此结合。（2）根据吉布斯自由能的变化特征,这两类结合主要以焓驱动为主。同一种药物分子对HSA结合过程的放热量及结合常数比其对BSA的结合均有所减少,并且在同一类结合位点上,Pae,Hma以及Ace与SA结合过程的焓变绝对值依次减小,这些热力学数据的差异主要是由于蛋白分子结构的不同以及客体分子苯环上取代基的相对位置不同引起的。（3）由于这三种同分异构体与SA的结合,使蛋白的二级结构发生变化,这表明Pae,Hma以及Ace与SA的相互作用既包含结合反应也包含其诱导蛋白质分子结构部分改变的过程。（4）Pae-SA体系的第二类结合位点数远远大于其它体系,所以其它条件相同时,Pae分子结合到蛋白分子上的几率最大,这可能是Pae比Hma及Ace具有更多的治疗功效的主要原因。第三部分:血清白蛋白与三种咪唑基离子液体的相互作用本部分应用等温滴定量热法研究298.15 K时血清白蛋白与三种咪唑基离子液体（[bmim]BF₄,[bmim]PF₆,[omim]BF₄）在缓沖溶液（pH=7.0）中的相互作用。通过对实验数据的非线性拟合得到结合常数、焓变、熵变及吉布斯自由能变等热力学数据。并利用圆二色谱技术研究了这三种离子液体对蛋白质分子二级结构的影响。结果表明:（1）SA对这三种离子液有两类结合位点,一类主要是通过离子液中阴离子与蛋白分子表面的氨基酸残基的静电及氢键作用等形成,另一类则主要通过离子液中的咪唑环及与之键合的烷基链与蛋白分子疏水空腔的疏水相互作用形成。（2）根据吉布斯自由能的变化特征,这两类结合均为熵驱动过程。同一种离子液对HSA结合过程的放热量及结合常数比其对BSA的结合均有所增加。与[bmim]BF₄相比,[omim]BF₄与蛋白的疏水作用结合常数明显增大,这表明[omim]BF₄烷基链长度与蛋白疏水空腔尺寸更匹配。另一方面,与[bmim]BF₄相比,蛋白对[bmim]PF₆结合的第一类结合位点数显著增加,这主要是由于阴离子PF6^-在蛋白表面上的结合机率增大造成的。（3）由于这三种离子液与SA的结合,使蛋白质分子的二级结构发生变化,主要是α-螺旋相对含量的减少,进一步表明了这三种离子液与SA之间存在相互作用。第四部分:甲酰胺和N,N—二甲基甲酰胺与肌醇在氯化钠水溶液中的焓相互作用本部分应用等温流动微量热法测定298.15 K时甲酰胺和N,N—二甲基甲酰胺（DMF）与肌醇分子在不同浓度氯化钠水溶液中的混合过程焓变及这些溶质分子的稀释焓,根据McMillan-Mayer理论关联得到各级异系焓相互作用系数(h_xy,h_xxy及h_xyy)。从溶质—溶质相互作用和溶质—溶剂相互作用角度讨论这两种酰胺与肌醇分子之间的相互作用机制。结果表明:（1）无论是在纯水还是在不同浓度的氯化钠水溶液中,甲酰胺与肌醇分子的异系焓对相互作用系数h_xy均为负值,表明在所研究体系中,h_xy的大小主要决定于这两种溶质分子之间的偶极-偶极作用。（2）DMF与肌醇分子的异系焓对相互作用系数h_xy在所研究的所有氯化钠浓度范围内均为正值,这是由于DMF分子的甲基与肌醇分子的羟基之间的疏水—亲水作用以及这两种溶质分子的去水化超过了DMF分子的极性基团与肌醇分子羟基之间的偶极—偶极作用。（3）在不同浓度的氯化钠水溶液中,随着盐浓度的增大,甲酰胺与肌醇的焓对作用系数h_xy的绝对值逐渐减小,而DMF与肌醇的焓对作用系数逐渐增大,这主要是由于氯化钠浓度的增大导致对被稀释组分水化结构的部分破坏增强（一种溶剂效应）所致。第五部分:D-（-）-对羟基苯甘氨酸在不同pH缓沖溶液中的稀释焓本部分应用等温滴定微量热法测定298.15 K时D-（-）-对羟基苯甘氨酸在不同pH磷酸缓冲溶液中的稀释焓,根据McMillan-Mayer理论关联得到各级同系焓相互作用系数。根据溶质—溶质相互作用和溶质—溶剂相互作用对结果进行了讨论。结果表明:（1）D-（-）-对羟基苯甘氨酸在不同pH磷酸缓冲溶液中的稀释焓均为正值,而相应的焓对作用系数h₂均为负值,这表明在所研究体系中,分子之间的氢键、离子—偶极作用以及静电作用在焓对作用中占主导地位。（2）D-（-）-对羟基苯甘氨酸的稀释焓在pH 7.0时具有最小值,导致其焓对作用系数在该pH下具有最大值。这是由于该pH值接近D-（-）-对羟基苯甘氨酸的等电点（6.6）,电解质与D-（-）-对羟基苯甘氨酸之间的静电作用最小。而当pH值高于等电点时,D-（-）-对羟基苯甘氨酸所带电荷随pH的增大而增加,静电作用增强,从而导致稀释焓增大。（3）当pH=6,7,8时,-(（?）Δ_dilH_m/（?）m_f)_mf→0值接近零,而当pH:9,10时该值大大增加。当pH=11时,-(（?）Δ_dilH_m/（?）m_f)_mf→0值再次减小。这种差别可归因于不同pH下溶质之间相互作用的差异。更多还原

【Abstract】 Protein is an indispensable material in living body,which plays an important role in life process.Serum albumin（SA）is a kind of protein which can bind with intrinsic and extrinsic materials,and transfers them to every parts of the body.Study on interaction between small organic molecules and serum albumin is helpful to understand the importance of such weak interaction as hydrophilic-hydrophobic interaction in biomacromolecule solution more deeply,thereby disclose the mystery of relevant life process.Amino acids,small peptides,acylamides and their derivatives have been used extensively as the most important biological model compounds.Sugars and polyols help in stabilizing the native conformation of globular proteins.The principle reasons for studying the thermodynamics of amino acids or acylamides with polyhydric compounds and the influence of pH value on such systems are to obtain（ⅰ）the information that contributes to the growing body of knowledge about solute solvation and solute -solute interactions in aqueous media,and（ⅱ）a better understanding of their role played in the conformational stability and unfolding behavior of proteins.Thermometry is an important method in the modern thermodynamic and thermochemical research,which has incomparable advantages than other methods in the thermodynamic research of life system containing many components.21^th Century is the century of life science.Study on interaction between biology macromolecule and small molecule in life body solutions by utilizing microcalorimetry combined with spectroscopy methods is of great significance for human being to explore the life essences and to disclose the life mystery.As a part of the project supported by National Natural Science Foundation （No.20273061）,the present work consists of five parts.The first part:interactions of human and bovine serum albumin（HSA and BSA） with bis-quatemary ammonium surfactants. In this part,the interactions of serum albumin with two bis-quatemary ammonium surfactants,（C_nN）₂Cl₂（n = 12,14）,in buffer solutions（pH=7.0）has been investigated by isothermal titration calorimetry at 298.15 K.The binding site number, binding constant and thermodynamic function change were obtained by fitting the experimental data.In addition,the influence of the two surfactants on the secondary structure of protein has been studied by Circular dichroism（CD）spectra.The results show that:（1）There are two classes of binding sites on HSA and BSA molecules for the two surfactants.One is high affinity binding（corresponding to larger K value）caused by electrostatic interaction of surfactant head groups with ionic sites on the protein surfaces,the other is low affinity binding（corresponding to smaller K value）due to the binding of alkyl chains of the surfactant to hydrophobic cavities of the protein molecules.The two classes of binding processes respectfvely cause positive and negative thermal effect.（2）The entropy effects for the two surfactants binding sites are all positive. Elongation of the alkyl chains in the surfactant molecules can strengthen their binding to HSA or BSA,and the binding force of the both surfactants to HSA is stronger than that to BSA.For high affinity sites,the difference of the binding site number between the two surfactants is small.However,for low affinity sites,the binding site number for(C₁₄N)₂Cl₂ is much smaller than that for(C₁₂N)₂Cl₂,this is because the alkyl chains of(C₁₄N)₂Cl₂ molecules are too long to be completely enclosed in the molecular cavity of a single binding site,which would lead to the reduction of evolved heat and the increasing of entropy.（3）The binding of the two surfactants to HSA or BSA can change the secondary molecular structure of the two kinds of proteins.The change of the secondary structure is mainly the conversion fromα-helix toβ- sheet.This is because the adsorption of surfactant cations on the protein macromolecule surface would lead to a swelling of the macromolecule and exposing of the hydrophobic residues.Thus the originalα-helices are partly broken,which gives a more open disordered structure.The second part:interactions of serum albumin with paeonol and two of its isomers.In this part,the interaction of serum albumin with paeonol （2’-hydroxyl-4’-methoxyacetophone,Pae）as well as two of its isomers （2’-hydroxyl-5’-methoxyacetophone,Hma and 4’-hydroxyl-3’-methoxyacetophone, Ace）in buffer solutions（pH=7.0）has been determined by isothermal titration calorimetry at 298.15 K.The binding constants,changes of enthalpy,entropy and Gibbs free energy are obtained by fitting experimental data.Moreover,the influence of the three drug molecules on the secondary structure of protein has been studied by CD spectra.The results show that:（1）There are two classes of binding sites on SA molecules for the three drugs. The first-class binding is caused by electrostatic interaction and hydrogen bonding of substitute groups on benzene ring of drug molecules with amino acid residues on SA surface,and the second one is due to the binding of benzene ring of drug to hydrophobic cavities of SA.（2）The binding process is predominantly driven by enthalpy according to the characteristics of standard Gibbs energy change.For the binding of the same drug molecule,the evolved heat and binding constant to HSA are smaller than those to BSA.On the same class of binding site,the negative value ofΔH°decreases in the order of Pae,Hma and Ace.The difference of thermodynamic data is caused by the different protein structure and locations of substitute groups on aromatic benzene ring of guest molecules.（3）The binding of the three isomers to HSA or BSA can change the secondary structure of the two protein molecules.These results indicate that the interaction includes contributions of the binding and the partial change of molecular structure of SA induced by the three isomers.（4）The second binding site number of Pae- SA system is much larger than that of other systems.So the chance for the binding of Pae to SA is largest under the same conditions,which may be helpful to understand that Pae has more therapeutic efficacy than its two isomers. The third part:interactions of serum albumin with three imidazole-based ionic liquids.In this part,the interactions of serum albumin with three imidazole-based ionic liquids（[bmim]BF₄,[bmim]PF₆,[omim]BF₄）in buffer solutions（pH=7.0）have been determined by isothermal titration calorimetry at 298.15 K.The binding constants, changes of enthalpy,entropy and Gibbs free energy are obtained by the non-linear fitting of experimental data.In addition,the influence of the three ionic liquids on the secondary structure of protein has been studied by CD spectra.The results show that:（1）There are two classes of binding sites on SA molecules for the three ionic liquids.The first-class binding is caused by electrostatic interaction and hydrogen bonding of anions of ionic liquids with amino acid residues on SA surface,and the second one is due to the binding of imidazole ring of ionic liquid and the alkyl chains thereon to hydrophobic cavities of SA.（2）The binding processes are both entropy driven according to the characteristics of standard Gibbs energy change.For the binding of the same ionic liquid,the evolved heat and binding constant to HSA are larger than those to BSA.The binding constant for the hydrophobic interaction of[omim]BF₄ with SA is much larger than that for[bmim]BF₄,which shows that the match degree between alkyl chains and hydrophobic cavity of protein for[omim]BF₄ is better than that for[bmim]BF₄.In addition,the first-class binding site number for the binding of[bmim]PF₆ to SA is much larger than that for the binding of[bmim]BF₄,which may be caused by the increasing of binding chance of anion PF₆^- to the surface of SA molecule.（3）The binding of the three inoic liquids to HSA or BSA can change the secondary structure of the two protein molecules,mainly the decrease ofα-helix content.These results proved the interactions of SA with the three ionic liquids.The fourth part:Enthalpic interactions of formamide and N, N-dimethylformamide with myo-inositol in aqueous sodium chloride solutions.In this part,the enthalpies of mixing of formamide and N,N-dimethylformamide with myo-inositol along with those of their dilution in aqueous sodium chloride solution of different concentration have been determined by using flow-mix-isothermal microcalorimetry at 298.15 K.These results were used to determine the heterotactic enthalpic interaction coefficients(h_xy,h_xxyand h_xyy) according to the McMillan-Mayer theory.The pairwise interactions between the two acylamide and myo-inositol have been discussed by solute-solute interactions and solute-solvent interactions.The results show that:（1）The heterotactic enthalpic pairwise interaction coefficients h_xybetween formamide and myo-inositol in water and aqueous sodium chloride solution of different concentration are all negative,which shows that the value of h_xyis mainly dependent on the dipole-dipole interaction between solute molecules.（2）The h_xyvalues for interaction between DMF and myo-inositol are all positive over the whole concentration of the salt aqueous solutions investigated.This indicates that the cooperative effects of the hydrophobic-hydrophilic interaction between the methyl group of DMF molecule and the hydroxyl group of myo-inositol molecule and the partial dehydration of the two solutes can surpass the dipole-dipole interaction between the polar group of DMF molecule and the hydroxyl group of myo-inositol molecule.（3）In aqueous sodium chloride solution of different concentration,the heterotactic enthalpic pairwise interaction coefficients(h_xy)between formamide and myo-inositol become less negative with increase of the concentration of sodium chloride.In contrast,the values of h_xybetween N,N-dimethylformamide and myo-inositol become more positive with elevated concentration of sodium chloride solutions.This mainly due to the increase of dehydration effects of solute molecules caused by the increase of the concentration of sodium chloride.The fifth part:Enthalpies of dilution of D-p-hydroxyphenylglycine in buffer solutions at different pH.In this part,enthalpies of dilution of D-p-hydroxyphenylglycine in phosphate buffer solutions at different pH have been determined by isothermal titration microcalorimetry at 298.15 K.The corresponding homogeneous enthalpic interaction coefficients have been calculated according to the McMillan-Mayer.The results are discussed according to solute-solute interactions and solute-solvent interactions.The results shows:（1）The enthalpies of dilution of D-pHPG in phosphate buffer solutions at different pH are all positive,while the enthalpic pair interaction coefficients h₂ are all negative.This suggests that the hydrogen bond,ion - dipole interaction and electrostatic interaction dominate the pairwise interactions.（2）There is a minimum for the enthalpies of dilution at pH 7.0,which leads to the enthalpic pair interaction coefficient of D-pHPG pass through a maximum at pH 7.0.This is because the pH value is near to the isoelectric point（pI）of D-pHPG （6.6）,at which D-pHPG becomes electro-neutralized,the electrostatic interactions between the cation and anion of electrolyte with D-pHPG are minimized.When pH>pI,the higher the pH value is,the more electric charges D-pHPG molecule will possess,so the electrostatic interactions reinforces,which results in the increase of dilution enthalpies.（3）At pH = 6,7,8,negative value of partial differential of molar enthalpy of dilution versus final molality of the diluted component（m_f）when m_f→0 (i.e.-(（?）Δ_dilH_m/（?）m_f)_mf→0)is approximately zero,while such values are much larger when pH = 9,10.But when pH = 11,-(（?）Δ_dilH_m/（?）m_f)_mf→0value decreases again. This kind of difference can be attributed to their different interaction between solutes.更多还原