【作者】 梁彦秋；

【导师】 臧树良；

【作者基本信息】 华东师范大学 ， 分析化学， 2007， 博士

【摘要】 蛋白质是细胞内的功能分子，在所有的生命过程中起着关键的作用，具有运载和储存功能，其特定结构与其生物功能密切相关。血清白蛋白是血浆中最丰富的蛋白质，它由一条单独的氨基酸多肽链构成，其三级结构主要为α-螺旋结构，其中包含3个结构域，即domainⅠ、domainⅡ和domainⅢ。每个结构域包含A、B两个亚区，以槽口相对的方式形成一种圆筒状结构。其中位于domainⅡ和domainⅢ的SiteⅠ和SiteⅡ两个结合位点与许多小分子化合物具有较强结合能力，能够与许多内源性物质和外源性物质结合，起到存储和转运的作用。血清白蛋白具有许多重要的生理功能，可与许多化学物质（如金属离子、脂肪酸、氨基酸、激素、药物及污染毒物等）以不同方式结合，并携带着这些物质通过血液在体内进行转运、运输、分配和代谢。通过研究化学物质与血清白蛋白相互作用，可获取化学物质在体内被转运、分配和代谢过程的重要信息。在金属离子、药物药理和毒理研究、污染毒物的危害机理及基因变异研究等方面具有重要意义。该领域已经成为从事生命科学、化学、临床医学科研工作者共同关注的课题之一。本论文基于开展小分子与蛋白质相互作用的重要意义及国内外研究趋势，在前人工作的基础上，综合利用荧光光谱、紫外一可见吸收光谱、傅里叶变换红外光谱(（FTIR）、同步荧光光谱和三维荧光光谱，从以下几方面进行了研究：在第一章的绪论中介绍了五个方面的内容：1．概述；2．小分子与蛋白质相互作用的研究方法；3．小分子与蛋白质相互作用获取的信息；4．小分子与蛋白质相互作用的研究现状；5．立题依据和研究内容。重点介绍了小分子与蛋白质相互作用的研究方法和手段及能够获取的信息。阐述了本论文的目的和意义，并对该方面未来的发展进行了预测和展望。在第二章中在模拟生理条件下利用紫外光谱法和荧光光谱法研究了Cu（Ⅱ）与牛血清白蛋白的相互作用。利用荧光光谱法研究了不同温度下Cu（Ⅱ）对牛血清白蛋白的猝灭情况，采用Stern-Volmer方程和Lineweaver-Burk双倒数方程对数据进行处理，并利用热力学公式推导了两者之间的相互作用力。研究中发现，Cu（Ⅱ）对牛血清白蛋白BSA的内源荧光猝灭以静态猝灭为主，二者之间生成了不发荧光的复合物是导致荧光猝灭的主要原因。Cu（Ⅱ）—牛血清蛋白相互作用过程是一个熵增加、吉布斯自由能降低的自发超分子作用过程，二者之间的作用力以疏水作用力为主。Cu（Ⅱ）会导致血清蛋白色氨酸残基和酪氨酸残基附近微环境极性增加，疏水性略有降低，使得牛血清白蛋白的肽链伸展，α-螺旋含量减少。紫外光谱法进一步验证了Cu（Ⅱ）与牛血清白蛋白之间的相互作用。通过改变激发波长，采用荧光光谱法研究了Cu（Ⅱ）和Cd（Ⅱ）对人血清白蛋白和牛血清白蛋白上的2种荧光基团的猝灭情况及结合位点，紫外光谱法研究了二者共存时与血清白蛋白共有的结合位点的竞争。研究中发现，Cu（Ⅱ）和Cd（Ⅱ）对血清白蛋白的色氨酸残基和酪氨酸残基均具有猝灭作用，Cu（Ⅱ）的猝灭程度远远强于Cd（Ⅱ）。Cu（Ⅱ）只与血清蛋白中214位色氨酸残基发生作用，而Cd（Ⅱ）除了与血清白蛋白的214位发生作用外，还和牛血清蛋白独有的135位色氨酸残基发生作用。Cu（Ⅱ）与Cd（Ⅱ）同时存在时Cu（Ⅱ）与牛血清白蛋白的结合占主导作用。在第三章中在模拟生理条件下利用紫外光谱法和荧光光谱法研究了杂多酸盐K₇[PTi₂W₁₀O₄₀]·6H₂O（PM-19）与牛血清白蛋白和人血清白蛋白的相互作用。利用荧光光谱法研究了不同温度下PM-19对两种血清蛋白的猝灭情况，采用Stern-Volmer方程和Lineweaver-Burk双倒数方程和双对数方程对数据进行处理，获取了结合常数和结合位点数。并利用热力学公式研究了PM-19与蛋白作用的热力学参数及主要作用力类型。采用F（?）rster非辐射能量转移理论计算了PM-19与牛血清白蛋白和人血清白蛋白结合时结合位置与蛋白质分子中荧光发射基团之间的距离。采用同步荧光光谱法探讨了PM-19对血清蛋白构象的影响。研究中发现PM-19对血清蛋白的内源荧光具有强烈的猝灭作用，静态猝灭和非辐射能量转移是导致血清蛋白的内源荧光猝灭的主要原因。PM-19和血清蛋白形成的复合物的结合常数在10⁴-10⁶L·mol^-1，表明两者的结合较稳定，说明PM-19可被血清蛋白存储和运输。与牛血清白蛋白和人血清白蛋白的结合位点数分别为0.89和0.85。PM-19与牛血清白蛋白之间的主要作用力为静电作用力；PM-19与人血清白蛋白之间的主要作用力为氢键和范德华力。PM-19与牛血清蛋白的结合距离为为4.14nm；PM-19与人血清白蛋白中色氨酸残基结合位置距离为4.21nm。PM-19与血清蛋白的作用点位靠近色氨酸残基，二者结合并不影响血清蛋白中荧光基团色氨酸残基和酪氨酸残基附近的微环境，并不改变血清蛋白的构象。在第四章中，采用紫外、荧光、同步荧光和三维荧光光谱法研究了4-硝基苯胺与血清白蛋白的相互作用。利用荧光光谱法研究了不同温度下4-硝基苯胺对牛血清白蛋白和人血清白蛋白的猝灭情况，采用Stern-Volmer方程和双对数方程对数据进行处理，并利用热力学公式研究了4-硝基苯胺与蛋白作用的热力学参数及主要作用力的类型。采用F（?）rster非辐射能量转移理论计算了4硝基苯胺与牛血清白蛋白和人血清白蛋白结合时结合位置与蛋白质分子中荧光发射基团之间的距离。采用紫外光谱法探讨了4-硝基苯胺对血清蛋白构象的影响。采用同步荧光光谱法和三维荧光光谱法探讨了4-硝基苯胺对血清蛋白构象的影响。研究中发现4-硝基苯胺与血清蛋白发生作用生成了新的复合物。其猝灭机理为静态猝灭，同时伴随非辐射能量转移。确定4-硝基苯胺与血清蛋白有一个结合位点，其结合常数数量级为10⁴L·mol^-1。4-硝基苯胺与血清蛋白相互作用过程是一个熵增加、吉布斯自由能降低的自发超分子作用过程，二者之间主要靠静电作用力和疏水作用力结合。4-硝基苯胺与牛血清蛋白的结合距离为3.87nm；4-硝基苯胺与人血清白蛋白中色氨酸残基结合位置距离为3.76nm。4-硝基苯胺对血清蛋白中的色氨酸和酪氨酸残基均具有猝灭作用，并使得色氨酸残基附近极性降低，疏水环境增强。4-硝基苯胺与血清蛋白的结合位点更接近于色氨酸残基。三维荧光光谱进一步证明了4-硝基苯胺的加入使血清白蛋白的色氨酸残基所处的微环境极性减弱，破坏了血清白蛋白的有序结构，使得整个大分子更趋向于折叠态。第五章中采用紫外、荧光、同步荧光和三维荧光光谱法研究了2，4-二硝基苯胺与牛血清白蛋白的相互作用。采用荧光方法中常用的Stern-Volmer方程和双对数方程对数据进行处理。采用F（?）rster非辐射能量转移理论计算了2，4-二硝基苯胺与牛血清白蛋白结合时结合位置与蛋白质分子中荧光发射基团之间的距离。采用紫外光谱法研究了不同浓度的2，4-二硝基苯胺对血清蛋白紫外吸收光谱的影响。采用同步荧光光谱法和三维荧光光谱法探讨了2，4-二硝基苯胺对血清蛋白构象的影响。研究中发现2，4-二硝基苯胺与血清蛋白发生作用生成了新的复合物。其猝灭机理为静态猝灭，同时伴随非辐射能量转移。确定2，4-二硝基苯胺与BSA的结合位点数为1.17，2，4-二硝基苯胺与牛血清蛋白的结合距离为为3.13nm。2，4-二硝基苯胺对牛血清蛋白中的色氨酸和酪氨酸残基均具有猝灭作用，并使得酪氨酸残基附近极性降低，疏水环境增强。三维荧光光谱进一步证明了4-硝基苯胺的加入破坏了血清白蛋白的有序结构，使得整个大分子更趋向于折叠态。第六章中，采用荧光光谱法分别研究了不同温度下十六烷基三甲基溴化铵CTAB及十二烷基苯磺酸钠SDBS与牛血清白蛋白的作用。采用红外光谱法研究了十六烷基三甲基溴化铵对牛血清白蛋白构象的影响。研究中发现CTAB和SDBS均对BSA内源荧光具有荧光猝灭作用。但两者对蛋白的猝灭程度有所不同，SDBS对蛋白的内源荧光猝灭作用较强。两者均与蛋白有两类结合部位。基于荧光猝灭理论得出第一类结合部位的荧光猝灭为静态猝灭。表面活性剂和牛血清蛋白相互作用以氢键和范德华作用力为主，其作用过程一个熵减、吉布斯自由能降低的自发超分子作用过程。并使得色氨酸残基附近极性降低，疏水环境增强。采用红外光谱法进一步证实了加入表面活性剂后引起了蛋白质二级结构的变化，导致了牛血清白蛋白中α-螺旋含量的降低。第七章为总结论。更多还原

【Abstract】 Protein as the essential biological material in the cells has been playing many vital roles for all kinds of biological phenomena. It has many important functions such as transportation and distribution, whose biological function is dependent on its special structure. Serum Albumin （SA） is the most abundant carrier protein in blood. Serum Albumin （SA） is a small protein with a single polypeptide chain, which is largely α-helical. It consists of three structurally homologous domains: domain I 、 domain II and domain III. Each domain is composed of two sub-domains （A and B）, which forms a columnar cavity with grooves towards each other. Site I and Site II located in domain II and domain III are known as locations of high affinity binding ability for many small molecules such as endogenous and exogenous ligands. Serum albumin has many important physiological functions. For example, by binding to chemicals such as metallic ions, fatty acids, steroids, amino acids, drugs, serum albumin realize their transportation, distribution and metabolism in the body. As a result, investigation of the interaction of serum albumin with chemicals noted previously can offer us very important information for understanding the absorption, transportation, distribution and metabolism of the chemicals in vitro.It is important to study the interaction of small molecules and metallic ion with the protein because protein-drug binding plays an important role in pharmacology, pharmacodynamics, toxicology and gene mutation. Therefore, it has been an interesting research field of life sciences, chemistry and clinical medicine. In this dissertation, on the basis of the previous research, the fluorescence spectroscopy including synchronous fluorescence and three-dimensional fluorescence combined with U-visible absorption spectroscopy, Fourier transform infrared （FT-IR） spectroscopy were used to investigate the interaction of Cu（II）, Cd（II） and several small molecules with serum albumin. The following major works were carried out:In the first chapter, there are five parts are introduced. First of all, a brief introduction of knowledge of protein and characteristics of serum albumin is offered. Secondly, common methods and instruments are elaborately introduced in this research field. Thirdly, information obtained by spectroscopy was exhibited, including binding constants K, binding sites n and major force etc. In the fourth part, current research situation was discussed. Eventually, our research contents, its innovation, purpose and theoretical significance were stated. Among them, emphasis was placed upon research methods and obtained information. In addition, future was predicted and prospected.In the second chapter, the binding of Cu（II） to bovine serum albumin （BSA） was investigated by spectroscopic （fluorescence spectroscopy, synchronous fluorescence and ultraviolet spectrum） techniques under simulative physiological conditions. Quenching for intrinsic fluorescence of serum albumin by Cu（II） was investigated in different temperatures by fluorescence spectroscopy. Data were handled by using Stern-Volmer and Lineweaver-Burk double reciprocal equations. The major force between them was obtained by thermodynamic parameters. It was found that non-fluorescence complex formed between Cu（II） and bovine serum albumin resulted in the decrease of intrinsic fluorescence of BSA. Thus static quenching predominated in the binding process. Their interaction is a spontaneous super-molecule formation （ΔS >0, ΔG <0）. The hydrophobic force played a major role in the binding BSA to Cu（II）. The spectral results observed showed that the binding of Cu（II） to BSA induced conformational changes in BSA. That is to say, polarity around tryptophan residues was increased and the hydrophobicity was decreased. Moreover, the interaction of Cu（II） and Cd（II） with serum albumin was investigated by fluorescence spectroscopy and the competition between Cu（II） and Cd（II） was analyzed by absorption spectra. Results showed that fluorescence emitted by tyrosyl and tryptophanyl residue could be quenched by Cu（II） and Cd（II）.The quenching produced by Cu（II）was much stronger than that produced by Cd（II）. Cu（II） only bound the Trp 214 in II A subdomain, but Cd（II） interacted with both Trp 214 in IIA subdomain and Trp 135 in I B subdomain of bovine serum albumin. The binding of Cu（II） with bovine serum albumin predominated in the presence of Cu（II） and Cd（II） together. In the third chapter, under the imitated physiological condition of animal body, the interactions of heteropoly salt （PM-19） with bovine serum albumin （BSA） were investigated by fluorescence spectrum and absorption spectroscopy. Quenching for intrinsic fluorescence of serum albumin by PM-19 was investigated in various temperatures by fluorescence spectroscopy. Data were handled by some equations. Thermodynamic parameters ΔH, ΔG, ΔS were calculated and their interaction forces were revealed. The distance r between donor （serum albumin SA） and acceptor （PM-19） was obtained according to fluorescence resonance energy transfer （FRET）. The effect of PM-19 on the conformation of serum albumin has been analyzed by synchronous fluorescence spectroscopy. It was proved that the fluorescence quenching of SA by PM-19 is mainly a result of static quenching and non-radiation energy transfer. The binding constants of the complex formed by PM-19 and SA is 10⁴-10⁶L·mol^-1, which illustrated that the complex is more stable. It indicated that PM-19 can be stored and transported by SA. Electrostatic force played an important role in the binding reaction; hydrogen bonds and van der waals interactions play a major role in stabilizing the complex. In addition, the binding instance r is 4.14nm （BSA-PM-19） and 4.21nm （HSA-PM-19） respectively. Furthermore synchronous fluorescence spectra showed that the binding sites for PM-19 is close to tryptophan residue （Trp 212） of BSA and Trp 214 of HSA. The microenvironment of the tyrosine and tryptophan residue has not obvious changed, which indicated that the interaction of PM-19 with SA does not affect the conformation of serum albumin.In the fourth chapter, the interaction between 4-nitroaniline and serum albumin （SA） has been studied by fluorescence spectroscopy, absorption spectra, synchronous fluorescence and three dimensional fluorescence spectra. Quenching of intrinsic fluorescence of SA was investigated at different temperature. Stern-Volmer and double logarithm equations were used to study various binding parameters. The thermodynamic parameters had also been calculated. The binding average distance, r between the donor （SA） and acceptor （4-nitroaniline） was determined based on the F（o|¨）rster’s theory. The effect of 4-nitroaniline on the conformation of serum albumin has been analyzed by means of synchronous and three dimensional fluorescence spectroscopy. A strong fluorescence quenching reaction of 4-nitroaniline to SA was observed and the quenching mechanism was suggested as static quenching and non-radiation energy transfer. In addition, for BSA, at higher ligand concentration, the Stern-Volmer plot exhibits an upward curvature, concave towards the y-axis. It illustrated both dynamic and static quenching was involved. There is about one class binding sites for binding of 4-nitroaniline to SA. Thermodynamic analysis showed that electrostatic and hydrophobic interactions were the mainly binding force Negative gibbs free energy （ΔG）, positive entropy （ΔS） and negative enthalpy （ΔH） values indicated the interaction between 4-nitroaniline and SA is spontaneous. The binding instance r is 3.87nm for BSA and 3.76nm for HSA respectively. Furthermore synchronous fluorescence spectra showed that the binding sites for 4-nitroaniline is close to tryptophan residue of SA. It is also indicated that the polarity around the tryptophan residues were decreased and the hydrophobicity was increased. The three dimensional fluorescence spectra proved that 4-Nitroaniline destroyed ordered structure of SA, resulting in formation of more folding of SA.In the fifth chapter, absorption, fluorescence, synchronous fluorescence and three dimensional fluorescence spectra were used to investigate the interaction of 2,4-dinitroaniline with BSA. Data was treated by several equations. The distance r between acceptor and donor was calculated using F（o|¨）rster’s theory. In addition, the impact of ligand on conformation of BSA was studied. Results revealed that static quenching occurs together with non-radiation energy transfer. As a result, 2,4-dinitroaniline caused the fluorescence quenching of BSA by the formation of complex. The number of binding sites is 1.17 and the distance r is 3.13 nm. Fluorescence originating from tryptophan residues and tyrosine residues can be quenched. It indicated that the polarity around the tyrosine residues was decreased and the hydrophobicity was increased. The three dimensional fluorescence spectra proved that 2,4-dinitroaniline destroyed ordered structure of SA, resulting in formation of more folding in SA.In the sixth chapter, the interaction of hexadecyl trimethyl ammonium bromide （CTAB） and sodium dodecyl benzene sulfonate （SDBS） with Bovine serum albumin was investigated by fluorescence spectroscopy at different temperature respectively. The effect of SDBS and CTAB on conformation of protein was discussed. It was found that the intrinsic fluorescence can be quenched by either CTAB or SDBS. Quenching by SDBS is stronger than that by CTAB. There are two types of sites in the binding of surfactant to BSA. The fluorescence quenching for the first type binding site was proved that Static quenching is predominant. Hydrogen bonds and van der waals interactions play a major role in stabilizing the complex. Negative entropy （ΔS） and negative enthalpy （ΔH） indicated that the interaction of CTAB or SDBS is spontaneous process. Polarity around tryptophan residue was decreased and hydrophobicity was increased by addition to CTAB or SDBS. Infrared spectral results revealed that the binding of CTAB or SDBS induced conformational changes in BSA, resulting in the decrease of a-helical.Total conclusions were given in the seventh chapter.更多还原