三种蛋白质与小分子物质相互作用的分子光谱法研究
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摘要
研究蛋白质与小分子物质之间的相互作用,不仅可以揭示二者相互作用的配位本质,而且对改进和发展分析蛋白质的新方法有促进作用,是超分子化学、蛋白质组学等研究领域的重要课题。本论文采用荧光、共振光散射、化学发光等分子光谱法,以溶菌酶,肌红蛋白和牛血清白蛋白为模型蛋白,研究这三种蛋白质与小分子物质的相互作用机理。本论文包括八个章节内容。
     第一章绪论。概述了研究蛋白质与小分子物质相互作用的意义;总结了五年来采用荧光和共振光散射法,对蛋白质与有机小分子、无机离子等作用机理的研究现状;列出了本论文的研究内容。本部分引用了198篇文献。
     第二章溶菌酶与溴酚蓝相互作用的荧光和共振光散射法研究。采用荧光等分子光谱分析手段,详细讨论了溶菌酶与溴酚蓝的相互作用机理。本次实验发现,溴酚蓝对溶菌酶荧光的猝灭是由静态猝灭模式诱发的,二者形成了非辐射基态复合物。通过结合常数、结合位点数、作用类型等参数来表征溶菌酶与溴酚蓝的作用方式;结合荧光共振能量转移理论,计算了溴酚蓝与溶菌酶的结合位点到溶菌酶分子中的氨基酸残基的作用距离。并将溶菌酶-溴酚蓝共振光散射体系成功应用于分析人体唾液和泪液样品中溶菌酶的含量。
     第三章溶菌酶与4-(2-吡啶)-间苯二酚相互作用的共振光散射法研究。在该章节中,采用荧光、共振光散射和紫外吸收等分子光谱手段,详细讨论了溶菌酶与4-(2-吡啶)-间苯二酚的相互作用。研究发现,4-(2-吡啶)-间苯二酚首先通过疏水作用发生自聚集,再与溶菌酶以静电引力等作用相互结合,形成以摩尔浓度比为12∶1的多分子复合物。利用溶菌酶-4-(2-吡啶)-间苯二酚共振光散射体系,分别对鸡蛋清、人体唾液和泪液中的溶菌酶进行了定量分析,获得了令人满意的结果。
     第四章溶菌酶与头孢菌素相互作用的荧光法研究。用荧光法结合荧光共振能量转移理论,研究了溶菌酶与头孢菌素包括:头孢羟氨苄、头孢拉定、头孢呋辛、头孢噻肟和头孢曲松的相互作用机制。实验发现,头孢菌素的存在导致溶菌酶的内源荧光猝灭,猝灭机制主要是由形成非辐射基态复合物的静态猝灭模式引发,推测头孢菌素可能结合溶菌酶分子中Trp62附近的活性基团。头孢菌素与溶菌酶的结合能力的强弱为:头孢曲松>头孢呋辛>头孢噻肟>头孢拉定>头孢羟氨苄,与头孢菌素的代次基本一致。
     第五章牛血清白蛋白与头孢菌素相互作用的荧光法研究。为了对照溶菌酶和头孢菌素的结合情况,还研究了牛血清白蛋白与头孢菌素,包括头孢拉定、头孢呋辛、头孢噻肟和头孢曲松的作用机理。同样发现,头孢菌素与牛血清白蛋白的结合能力的强弱与开发药物的距今由近到远的时间顺序基本一致。
     第六章肌红蛋白与NO_2~-离子相互作用的化学发光法研究。应用本研究组首次提出的Luminol-肌红蛋白化学发光体系,对人体尿液和自来水样品中的NO_2~-离子进行定量分析。肌红蛋白分子中的卟啉铁有六个配位键,其中一个是空缺的,可以结合O_2等小分子或离子,NO_2~-离子就是通过结合铁的这个空缺键与肌红蛋白发生作用形成复合物的。该复合物中的铁没有因键合NO_2~-离子而改变价态,仍然处于高价状态,且不稳定,可以加速Luminol与肌红蛋白之间的电子转移,产生增强的化学发光信号。基于增强的化学发光强度与NO_2~-离子浓度之间的线性关系,将该方法成功用于测定人体尿液和自来水样品中NO_2~-离子的含量。
     第七章肌红蛋白与苏丹红相互作用的化学发光法研究。本章采用流动注射-化学发光法,应用Luminol-肌红蛋白新型发光体系对污染的酱制品中的苏丹红Ⅰ进行定量分析。肌红蛋白分别与苏丹红Ⅰ、Ⅱ、Ⅲ和Ⅳ结合形成复合物,可以加速Luminol与肌红蛋白之间的电子转移,催化二者的发光反应,产生增强的化学发光信号。基于增强的化学发光强度与苏丹红浓度之间的线性关系,将该方法成功用于测定被污染的酱制品中苏丹红Ⅰ的含量。
     第八章总结
The study on the interaction between protein and small molecules is the important subject in the field of Supermolecular Chemistry, Proteomics and Life Science. It can be helpful to understand the binding nature of the small ligands to protein molecules and to develop new methods for analyzing protein. In this paper, lysozyme, myoglobin and bovine serum albumin were employed as the model proteins to study their interaction mechanism with small molecules such as dyes, drugs and inorganic ion adopting fluorescence, resonance light scattering and chemiluminescence methods. The dissertation includes eight chapters.
     1. Overview of the interaction protein with ligands. It was summarized the significance for studying the interaction mechanism between protein and small molecules, reviewed the research status for the interaction of protein with small organic molecules and inorganic ions using fluorescence and resonance light scattering methods, and given the research content of the thesis.
     2. Study on the interaction between lysozyme and bromophenol blue. It was discussed in detail the mechanism of the interaction between lysozyme and bromophenol blue (BPB) by fluorescence and resonance light scattering (RLS) ways. The quenching process of lysozyme-BPB was induced by the static quenching mode to form the nonradiative ground-state complex. The binding parameters such as the binding constant, the number of binding site and the binding mode were calculated. The binding distance from the binding site of BPB on lysozyme molecule to tryptophan residues was obtained using the fluorescence resonance energy transfer theory. The binding system of lysozyme-BPB was applied successfully to determine lysozyme in human saliva and tear samples using RLS method.
     3. Optical behavior of lysozyme and 4-(2-pyridylazo)-resorcinol. It was described a study on the interaction of lysozyme with 4-(2-pyridylazo)-resorcinol (PAR) by fluorescence and resonance light scattering (RLS) methods. It was proposed that PAR molecules can self-assemble and then form the polymolecular complexes with lysozyme. And it was found that the molecular ratio of lysozyme and PAR arrived to 1:12 the reaction to form the lysozyme-PAR complex got saturated. Based on the formation of lysozyme-PAR polymolecular complex, the RLS singnal of PAR was enhanced obviously and regularly in the presence of lysozyme. The present system was used to deal with lysozyme in hen egg white, human saliva and tear samples, respectively.
     4. Study on the interaction mechenism of lysozyme and cephalosporin analogues. The interaction between lysozyme and cephalosporin including cefadroxyl, cefradine, cefuroxime, cefotaxime and ceftriaxone was studied by fluorescence combined with fluorescence energy transfer theory. It was clearly shown that the intrinsic fluorescence of lysozyme was quenched by cephalosporin through the static quenching ang nonradiative energy transferring procedure. It was suggested that cephalosporin probably bound to the active site near Trp62 in lysozyme. It was observed that the binding capability of cephalosporin to lysozyme was in the order of ceftriaxone > cefuroxime > cefotaxime > cefradine > cefadroxyl and almost consistent with its antibacterial ability.
     5. Investigation of the interaction between bovine serum albumin and cephalosporin. It was depicted the study on the interaction mechanism between bovine serum albumin and cephalosporin analogues adopting fluorenscence spectroscopy. It was also found that the order of the binding ability of cephalosporin to BSA was close to its antibacterial capability.
     6. Chemiluminescence behavior for the interaction of myoglobin with NO_2~- anion. A new chemiluminescence system luminol-myoglobin was introduced to the determination of NO_2~- anion in human urine and tap water samples.
     7. Study on the interaction between myoglobin and sudan dyes by chemiluminescence. The new method of chemiluminescence combined with flow-injection was adopted to determine sudan I in the contaminated hot chilli products using luminol-myoglobin system.
     8. Conclusions
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