【作者】 邓斌；

【导师】 臧树良；

【作者基本信息】 辽宁大学 ， 分析化学， 2008， 硕士

【摘要】 血清白蛋白（SA）是生物体内含量最丰富的运输蛋白质。SA有许多重要的生理学和药理学功能,能与许多内源和外源性物质如脂肪酸、氨基酸、荷尔蒙、阴阳离子和药物等结合,起到存储和转运作用,因此,SA己成为研究最广泛的目标蛋白之一。研究毒性物质、药物等小分子配体与SA的相互作用机理和作用过程,在毒理学、药代动力学上具有重要意义。该领域己成为从事生命科学、化学和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）,从以下几方面进行了创新性研究:1.研究了模拟生理条件下杂多酸盐K₇[PTi₂W₁₀O₄₀]·6H₂O（PM-19）、4-硝基苯胺、氟铃脲与牛血清白蛋白（BSA）和人血清白蛋白（HSA）的相互作用。根据Stern-Volmer方程和双对数方程研究了上述小分子与BSA及HSA结合的作用机理（包括荧光碎灭机理、结合常数和结合位点数）,讨论了主要作用力类型。2.研究了实验结果偏离Stern-Volmer曲线的原因。3.利用紫外光谱技术考察了小分子物质对SA构象的影响。4.利用FTIR技术考察了小分子对SA二级结构的影响。论文共分为五部分引言:简要介绍了蛋白质的结构、功能和性质;综述了小分子物质与蛋白质相互作用的研究方法、研究现状以及不同小分子物质与蛋白质相互作用研究。第一章:研究了PM-19同血清白蛋白的作用。利用荧光光谱法研究不同温度时,在模拟人生理条件下PM-19与血清白蛋白之间发生了的相互作用,发现PM-19对血清白蛋白内源荧光（色氨酸和酪氨酸）产生了荧光猝灭作用,探讨了这种荧光猝灭为静态猝灭机制,计算了其结合常数和结合点位,从而得出其主要的作用力类型;并用同步荧光表明PM-19对血清白蛋白的构象并没有明显的影响,这个结果与紫外吸收光谱一致。第二章:讨论了4-硝基苯胺与血清白蛋白的作用。根据荧光光谱的研究,发现4-硝基苯胺对血清白蛋白有强烈的猝灭作用。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。4-硝基苯胺与BSA作用时,当4-硝基苯胺浓度很大时数据处理结果偏离Stern-Volmer方程,表明在高浓度时动态猝灭和静态猝灭同时存在。同步荧光和三维荧光表明4-硝基苯胺的加入强烈的改变了血清白蛋白的构象,这种改变使血清白蛋白的双螺旋结构趋于折叠态。探讨了4-硝基苯胺与血清白蛋白中色氨酸残基和酪氨酸残基的作用方式、作用点及作用距离。第三章:研究了氟铃脲与牛血清白蛋白（BSA）的相互作用。根据荧光光谱的研究研究表明:氟铃脲对牛血清白蛋白的猝灭方式为静态猝灭。同步荧光表明氟铃脲使BSA的双螺旋结构趋于伸展态。紫外光谱和红外光谱都证实了这一结果。第四章:总结论。更多还原

【Abstract】 Serum Albumin (SA) is the most abundant carrier protein in blood circulation. It has many important physiological and pharmacological functions, which can bind many exogenous and endogenous ligands in blood, and realize transport and distribution of many molecules and metabolites, such as fatty acids, amino acids, bilirubin, hormones and many diverse drugs. Therefore, it has been one of the most extensively studied of all proteins. Investigating the binding mechanism of toxic materials and drugs with SA has many importances in toxicology and pharmacokinetics. Thus, 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 combined with UV-visible absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and computational modeling were used to investigate the interaction of several small molecule substances with SA. The following major innovative works were carried out:1 .Several methods were associated to study the interaction of diphacinone and three components of small molecules (heteropoly salt (PM-19), 4-nitroaniline and hexaflumuron) with bovine serum albumin (BSA) and human serum albumin(HAS). The binding properties including the fluorescence quenching mechanisms, binding constants and the number of binding sites were investigated in detail, thermodynamic parameters were calculated, and the main interaction force between drugs and proteins was discussed.2. The cause of departure from the Stem-Volmer plots at different conditions was analyzed.3. The effects of drugs on SA second structure were investigated with FT-IR techniques.4. The computational modeling method was used to study the drug-SA interaction and the study results were in consistent with the experimental results.This dissertation consists of five chapters:Chapter1:The structures, functions and of proteins were briefly introduced. The contents and methods of interaction of different kind of small ligands with protein were reviewed.Chapter2: Under the imitated physiological condition of animal body, the interactions of heteropoly salt (PM-19) with serum albumin (SA) were investigated by fluorescence spectrum and absorption spectroscopy. It was shown that this compound had a quite strong ability to quench the fluorescence launching from SA. we found that SA had reacted with PM-19 and formed a certain new compound. The quenching belonged to static fluorescence quenching. Based on F?rster’s non-radiation energy transfer mechanism, the binding locality (r = 4.14nm) was calculated between donor and accepter. The effect of PM-19 on the conformation of BSA was analyzed by synchronous fluorescence spectroscopy.Chapter 3: the interaction between 4-nitroaniline and serum albumin (SA) has been studied by fluorescence spectroscopy, absorption spectra, synchronous fluorescence and three dimensional fluorescence spectra. 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. 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.Chapter 4: the interaction of hexaflumuron with Bovine serum albumin was investigated by fluorescence spectroscopy at different temperature respectively. It was found that the intrinsic fluorescence can be quenched by hexaflumuron and it is Static quenching. Furthermore synchronous fluorescence spectra showed that hexaflumuron destroyed ordered structure of BSA, resulting in formation of unfolding of BSA. Infrared spectral results revealed that the binding of hexaflumuron induced conformational changes in BSA, resulting in the decrease of a-helical.Total conclusions were given in the fifth chapter.更多还原