【摘要】：血清白蛋白是血漿中含量最為豐富的蛋白質(zhì),具有貯存轉(zhuǎn)運(yùn)內(nèi)源代謝產(chǎn)物和外源藥物小分子等重要的生理功能,能與多種內(nèi)源以及外源分子進(jìn)行結(jié)合,通過形成復(fù)合物從而完成血漿的各項(xiàng)生理功能。研究降糖藥物與血清白蛋白大分子的相互作用能夠在藥物代謝動(dòng)力學(xué)、制藥學(xué)以及新藥研發(fā)等方提供一定的基礎(chǔ)信息。而藍(lán)銅蛋白(RUS)是一類載銅蛋白,是嗜酸氧化亞鐵硫桿菌中鐵呼吸電子傳遞的重要組分,研究該蛋白的性質(zhì)對(duì)銅蛋白家族中的其他蛋白如生物血漿銅藍(lán)蛋白等性質(zhì)結(jié)構(gòu)提供一定的依據(jù)。本論文采用熒光光譜技術(shù)和紫外-可見光技術(shù)為研究手段,研究了兩種降糖藥物(鹽酸吡格列酮(PIO)和鹽酸二甲雙胍(MET))與牛血清白蛋白(BSA)的相互作用,探究了這兩種降糖藥物與BSA的作用方式,為降糖藥的研發(fā)以及更好的指導(dǎo)用藥提供依據(jù)。同時(shí)采用光譜法和分子結(jié)構(gòu)模擬技術(shù)研究RUS His143在電子傳遞中的作用,并通過對(duì)His143定點(diǎn)突變進(jìn)一步驗(yàn)證。主要工作內(nèi)容如下:1.光譜法研究PIO與BSA的相互作用。采用熒光光譜法和紫外-可見光譜法,發(fā)現(xiàn)PIO對(duì)BSA熒光有明顯猝滅作用,且其猝滅方式為動(dòng)態(tài)猝滅;進(jìn)一步測(cè)定了PIO與BSA的結(jié)合常數(shù)K、結(jié)合位點(diǎn)數(shù)及ΔG、ΔH、ΔS等熱力學(xué)常數(shù);并根據(jù)三個(gè)熱力學(xué)常數(shù)確定PIO與BSA之間的相互作用為疏水作用力。2.光譜法研究MET與BSA的相互作用。采用熒光光譜法等方法初步探究MET對(duì)BSA熒光的猝滅作用,得到其猝滅方式為動(dòng)態(tài)猝滅;根據(jù)測(cè)定的MET與BSA的結(jié)合常數(shù)K和結(jié)合位點(diǎn)數(shù)及熱力學(xué)常數(shù),確定MET與BSA之間的相互作用為氫鍵和范德華力,且通過分子對(duì)接方法進(jìn)一步驗(yàn)證該結(jié)論。3.光譜法和分子模擬技術(shù)研究RUS His143的性質(zhì)。通過對(duì)His143位點(diǎn)的定點(diǎn)突變,聚丙烯酰胺凝膠電泳,光譜學(xué)數(shù)據(jù)初步確定His143位點(diǎn)是RUS與外界電子傳遞的重要位點(diǎn),并結(jié)合分子結(jié)構(gòu)模擬結(jié)果顯示His143位點(diǎn)的咪唑基團(tuán)形成疏水屏障從而使銅離子不會(huì)暴露于溶液環(huán)境,進(jìn)一步說明RUS His143在電子傳遞中的重要性。
[Abstract]:Serum albumin is the most abundant protein in plasma. It has important physiological functions such as storing and transporting endogenous metabolites and small molecules of exogenous drugs. It can combine with many kinds of endogenous and exogenous molecules. The physiological functions of the plasma are accomplished by the formation of a complex. The study of the interaction between hypoglycemic drugs and serum albumin can provide some basic information in pharmacokinetic, pharmaceutical and new drug development. Blue copper protein (RUS) is a kind of copper-carrying protein, which is an important component of iron respiratory electron transport in thiobacillus ferrooxidans. The study of the properties of the protein provides a basis for the structure of other proteins in the copper protein family, such as plasma ceruloplasmin. In this paper, the interaction of pioglitazone hydrochloride (PIO) and metformin hydrochloride (MET) with bovine serum albumin (BSA) was studied by fluorescence spectroscopy and ultraviolet-visible light (UV-vis) techniques, and the interaction of pioglitazone hydrochloride and metformin hydrochloride with bovine serum albumin (BSA) was studied. The effects of these two hypoglycemic drugs on BSA were explored, which provided the basis for the research and development of hypoglycemic drugs and better guidance for drug use. At the same time, the role of RUS His143 in electron transfer was studied by spectroscopic and molecular structure simulation techniques, and further verified by site-directed mutation of His143. The main contents of the work are as follows: 1. The interaction between PIO and BSA was studied by spectroscopic method. By means of fluorescence and UV-vis spectroscopy, it was found that PIO had obvious quenching effect on BSA fluorescence, and its quenching mode was dynamic quenching. The binding constants K, binding site number and 螖 G, 螖 H, 螖 S of PIO and BSA were further determined, and the interaction between PIO and BSA was determined to be hydrophobic force according to the three thermodynamic constants. The interaction between MET and BSA was studied by spectroscopic method. The quenching effect of MET on BSA fluorescence was studied by fluorescence spectroscopy and other methods. The quenching mode was dynamic quenching. According to the binding constants K and the number of binding sites and thermodynamic constants of MET and BSA, the interaction between MET and BSA is determined as hydrogen bond and van der Waals force, and the conclusion is further verified by molecular docking method. Spectroscopic and molecular simulation techniques were used to study the properties of RUS His143. By site-directed mutation of His143 site, polyacrylamide gel electrophoresis (page) and spectroscopic data, the His143 site was determined to be an important site for electron transfer between RUS and the outside world. The results of molecular structure simulation show that the imidazole group at His143 site forms a hydrophobic barrier so that copper ions will not be exposed to the solution environment, which further illustrates the importance of RUS His143 in electron transport.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R96

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