光谱法研究蛋白与头孢吡肟的作用机理及沙拉沙星荧光性质

发布时间：2018-06-01 15:32

本文选题：荧光光谱法 + 牛血清蛋白　；参考：《河北大学》2017年硕士论文

【摘要】：目前,传统荧光猝灭法和同步荧光法在生物化学和药学领域被广泛应用于研究蛋白质与小分子药物之间的相互作用。本文主要以盐酸头孢吡肟(CH)为小分子药物代表,利用多种荧光光谱法研究了蛋白与药物之间相互作用的反应机理,并在此基础上利用多种光谱法联用更深入的研究了两者反应过程中的氨基酸残基微环境的变化。同时还研究了小分子药物本身的荧光特性,和一系列小分子药物和蛋白的作用情况。本研究内容共包含以下五部分:第一章:综合介绍了药物小分子本身荧光特性以及蛋白质与药物小分子间相互作用的一般研究方法。对采用多种光谱法研究蛋白与药物小分子相互作用的研究进展进行了综述,并在此基础上提出了本文的立题思路。第二章:利用荧光光谱对沙拉沙星(SAR)在不同pH条件下的结构及其变化进行了研究并计算其解离常数及荧光量子产率,研究结果可以为喹诺酮类药物理化性质的比较研究提供参考。研究结果表明,在强酸性条件下沙拉沙星以H3L2+形式存在,最大荧光发射波长约455 nm;当pH 3~5时,H3L2+失去喹啉环1位N上结合的质子而以H2L+形式存在,荧光强度较大且基本不变,最大荧光发射波长位于458 nm;当pH5时,随着pH升高,荧光发射波长蓝移至430 nm,H2L+失去羧基质子而以双极离子HL形式存在;当pH进一步升高时,HL失去哌嗪环N上结合的质子,转化成为阴离子L-,导致荧光强度降低,最大发射波长红移至约466 nm。在强碱性条件下,由于介质环境的影响,L-的荧光强度降低,但最大发射峰位置基本不变。第三章:利用荧光光谱法(包括荧光猝灭法、同步荧光法,共振光散射法三种方法)和紫外吸收光谱法研究298 K时牛血清白蛋白(BSA)和硫酸粘杆菌素,硫酸头孢匹罗,头孢匹胺钠三种不同的药物之间的相互作用机理。结果表明:对于相同的体系利用四种方法计算得到的结合常数的数量级一致;三个体系主要的猝灭方式均为生成新物质的静态猝灭;三种药物与蛋白作用时以1:1的比例结合;利用实验数据计算的Hill系数相似。文章中对四种方法所得实验结果进行了分析和比较,表明所用方法都可用于蛋白与药物反应机理的研究。第四章:利用荧光猝灭法和同步荧光法,分别研究了不同温度下盐酸头孢吡肟与牛血清白蛋白之间的反应机理。两种研究方法结果均表明盐酸头孢吡肟对牛血清蛋白的荧光发生了猝灭作用,二者之间通过静态猝灭的方式相互作用;牛血清蛋白与盐酸头孢吡肟反应的结合位点数约为1,表明它们相互作用形成1:1复合物;利用热力学参数计算得到牛血清蛋白与盐酸头孢吡肟之间主要的作用力类型为范德华力或是氢键;根据F?rster非辐射能量转移理论确定了盐酸头孢吡肟与牛血清蛋白的结合距离,进一步说明二者通过静态猝灭方式相互作用并伴有非辐射能量转移。第五章:利用灵敏度较高的四阶导数紫外光谱法,结合内源荧光法、同步荧光法、近紫外圆二色光谱法和位点试剂法多种方法共同研究牛血清白蛋白与盐酸头孢吡肟体系作用过程中氨基酸残基微环境极性及蛋白质大分子的构象变化。研究发现,随着CH浓度的增大,CH分子首先聚集在结构域IIA的Trp-213位置附近,导致色氨酸(Trp)残基所处微环境极性增强。形成聚集体后,诱导结构域IIA展开暴露出更多残基,导致酪氨酸(Tyr)和苯丙氨酸(Phe)残基所处微环境极性增强。CH增大到一定浓度后,各残基微环境极性保持不变,BSA大分子构象趋于稳定。
[Abstract]:At present, the traditional fluorescence quenching and synchronous fluorescence method are widely used in the field of Biochemistry and pharmacy to study the interaction between protein and small molecular drugs. In this paper, the reaction mechanism of the interaction between proteins and drugs is studied by using CH as the representative of small molecular drugs. On the basis of this, the changes in the microenvironment of the amino acid residues in the reaction process are further studied by using a variety of spectroscopic methods. The fluorescence characteristics of the small molecular drugs themselves and the effects of a series of small molecular drugs and proteins are also studied. The contents of this study include the following five parts: Chapter 1: a comprehensive introduction of the drug A general study of the fluorescence characteristics of small molecules and the interaction between proteins and small molecules of drugs. A review of the research progress on the interaction of proteins and small molecules by a variety of spectroscopic methods is reviewed. On the basis of this, the second chapter: the use of fluorescence spectroscopy to be used in the study of salfloxacin (SAR) The structure and its change under the same pH condition were studied and calculated the dissociation constant and the fluorescence quantum yield. The results can provide reference for the comparative study of the physicochemical properties of quinolones. The results show that in the strong acidic condition, the maximum fluorescence emission wavelength of salfloxacin is about 455 nm; when pH 3~5, H3L2 The proton combined with 1 bit N lost quinoline ring exists in the form of H2L+, the fluorescence intensity is large and basically unchanged, the maximum fluorescence emission wavelength is 458 nm. When pH5, as pH rises, the fluorescence emission wavelength is blue to 430 nm, H2L+ loses the carboxyl proton and is deposited in the form of bipolar ion HL; when pH is further elevated, HL loses the N binding of piperazine ring. The proton, converted into anionic L-, leads to the decrease of fluorescence intensity and the maximum emission wavelength red shift to about 466 nm. in strong alkaline conditions. The fluorescence intensity of L- is reduced because of the influence of the medium environment, but the maximum peak position of the emission peak is basically unchanged. Third chapter: fluorescence spectrometry (including fluorescence quenching, synchronous fluorescence, resonance light scattering three) The interaction mechanism between the three kinds of drugs, such as bovine serum albumin (BSA) and Mycobacterium sulfate, cefriol sulfate and cefpimiamine sodium, was studied by UV absorption spectroscopy. The results showed that the number of binding constants calculated by the same system using four methods was consistent with the 298 K methods; the three systems were mainly sudden. The quenching methods are static quenching of the new substances; the three drugs and proteins are combined with the proportion of 1:1; the Hill coefficient calculated by the experimental data is similar. The results of the experimental data obtained from the four methods are analyzed and compared, which shows that the methods used can be used in the study of the mechanism of protein and drug reaction. The fourth chapter: using fluorescence. The reaction mechanism between cefepime hydrochloric acid and bovine serum albumin at different temperatures was studied at different temperatures. The results of the two methods showed that cefepime HCl had a quenching effect on the fluorescence of bovine serum proteins, and the interaction between the two groups by static quenching, bovine serum protein and hydrochloric acid head. The number of binding sites of the cyclosporine oxime reaction is about 1, indicating that they interact to form 1:1 complexes. The main types of interaction between bovine serum protein and cefepime are van Edward force or hydrogen bond, and the cefepime hydrochloric oxime and bovine serum protein are determined according to the F? Rster non radiation energy transfer theory. Combined with distance, the two groups were further explained by the interaction of static quenching and non radiation energy transfer. The fifth chapter: using the four derivative UV Spectrometry with high sensitivity, combined with internal source fluorescence, synchronous fluorescence, near ultraviolet two color spectroscopy and loci reagents, the study of bovine serum albumin and hydrochloric acid In the process of cefepime system, the microenvironmental polarity of amino acid residues and the conformation changes of protein macromolecules are found. It is found that with the increase of CH concentration, CH molecules first gather near the Trp-213 position of IIA in the domain, leading to the increasing polarity of the microenvironment of the tryptophan (Trp) residue. After the formation of the aggregates, the induced domain IIA is exposed. The microenvironmental polarity of the Tyr and Phe residues increased to a certain concentration, and the microenvironmental polarity of the residues remained unchanged, and the conformation of the BSA macromolecules tended to be stable.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96;O657.3

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