【作者】 汪双双；

【导师】 倪永年；

【作者基本信息】 南昌大学 ， 分析化学， 2010， 硕士

【摘要】 本论文分别采用紫外-可见吸收光谱、荧光光谱为研究手段,研究了几种有机小分子与牛血清白蛋白(bovine serum albumin, BSA)的相互作用。主要包括两个方面的内容：一、综述了有机小分子与血清白蛋白相互作用的研究现状及意义；二、在模拟人体生理条件下研究了几种有机小分子与BSA相互作用的光谱信息,并结合化学计量学对其数据进行解析。光谱法在小分子与生物大分子相互作用的研究领域的应用日益广泛,但是光谱法结合化学计量学来探讨小分子与血清白蛋白的结合作用的文章还比较少。本论文尝试将多种化学计量学方法用于解析实验得到的光谱数据,讨论了方法的原理和实际应用,并探讨了化学计量学在复杂的生物化学体系中实现同时解析多组分平衡浓度和纯光谱的可行性。论文第一部分首先简要的介绍了蛋白质的定义、结构、功能和性质等,然后依次对小分子与血清白蛋白相互作用的研究方法、研究内容和存在的问题等方面展开了评述,探讨了化学计量学在小分子与血清白蛋白相互作用研究中的应用。最后还展望了小分子与血清白蛋白相互作用研究的发展趋势,将新探针的开发、各种仪器方法的联用及化学计量学方法的引入作为研究重点。论文第二部分运用荧光,紫外-可见以及同步荧光光谱研究了山姜素(APT)与BSA的相互作用。把所测得的混合物的一系列光潜数据用化学计量学方法—多元曲线解析-交替最小二乘法(MCR-ALS)来解析,得到混合物中纯物质光谱图和浓度图,这是一般方法所得不到的信息。而且,体系中生成的APT-BSA复合物被解析出来,这也进一步说明APT对BSA的内源荧光的猝灭是属于静态猝灭,并且形成了APT-BSA复合物。本文还计算出反应的结合常数(Ka(ave)＝2.34×106 L mol-1)、结合位点数(n=1)以及热力学常数(ΔH0,ΔS0和ΔG0),数据表明APT与BSA之间的作用力主要是疏水作用。通过把APT与BSA相互作用的热力学参数与具有类似分子结构的柯因和wogonin与BSA相互作用的热力学参数进行比较,结果表明焓变随分子结构的变化影响不大,此为熵驱动反应。BSA与APT相互作用的同步荧光光谱表明APT与BSA结合之后对BSA的空间构象有一定影响。APT与位点探针的竞争结合实验说明APT在BSA上的结合位点是在Ⅰ位。论文第三部分是运用荧光光谱法来研究溴鼠灵(BDF)与牛血清白蛋白(BSA)的相互作用。所测得的荧光光谱用SIMPLISMA进行解析,这也是SIMPLISMA首次被用于小分子与BSA相互作用的研究,从中抽取了BDF-BSA混合体系中单个组分的纯光谱图。解析所得的光谱图中含有BDF-BSA复合物的光谱,这也就说明BDF与BSA相互作用机制是静态猝灭。通过做BSA对BDF荧光敏化作用的实验,结果表明BDF浓度越高,体系中的荧光量子产率就越大,导致BSA对BDF的敏化效应越强。同步荧光光谱和三维荧光光谱是用于研究BSA构象变化较好的方法,实验发现BDF的加入很明显的改变了BSA的空间构象,使得BSA分子中色氨酸残基的极性增加,疏水性下降,而BDF的加入并不会导致BSA分子中酪氨酸残基周围的环境发生变化。曙红Y是一种新的Ⅰ位探针,本文利用曙红Y来研究BDF在BSA上的结合位点,结果表明曙红Y与BDF并不会同时结合在BSA上,而是发生竞争作用,曙红Y把BDF竞争来。论文第四部分用荧光光谱法研究了克百威(carbofuran, CF)及其降解产物(carbofuran catabolite,CFP)与BSA结合反应的光谱行为。实验发现,CFP比CF对BSA有更强的荧光猝灭作用。本为采用了MCR-ALS来解析所得到的荧光光谱图,得出溶液中存在三个组分,也就是说CF(CFP)与BSA都发生了静态猝灭作用产生了复合物。同时用三维荧光光谱和同步荧光光谱法探讨了CF及CFP对BSA构象的影响,为预防和医治CF及CFP中毒提供了重要的依据。最后本文还研究了金属离子对它们之间作用的影响。论文第五部分在生理pH条件下用荧光光谱法和紫外光谱法结合化学计量学来研究10-羟基喜树碱(10-HCPT)与BSA的相互作用,结果发现10-HCPT对BSA的内源荧光有静态猝灭作用。本文以华法林作为标记药物,采用三维荧光技术,分别对竞争实验过程进行扫描,进而用平行因子法(PARAFAC)处理所得三维数据,并根据蛋白质结合位上药物的置换作用确定了10-HCPT的结合位置是BSA的siteⅠ。利用同步荧光光谱,考察了10-HCPT对BSA构象的影响,结果发现,10-HCPT的加入使BSA构象发生变化,BSA内部氨基酸残基所处环境的疏水性下降。更多还原

【Abstract】 The reaction mechanism of small molecules with Protein was studied by fluorescence spectroscopy (FS) and UV spectroscopy (UV) and interaction of several organic molecules with bovine serum albumin (BSA) were investigated. There are two parts in this work:1. the recent developments and its significance of interaction between organic molecules and serum albumin were summarized; 2. the binding interaction of several organic molecules with BSA were studied by kinds of spectroscopy methods under simulated physiological conditions and the spectral data were resolved by chemometrics methods in turns. Spectroscopic techniques are widely used in analytical chemistry, however, the spectroscopic investigation combined with chemometrics, particularly for the interaction of small molecules and macromolecules, are relatively few. The application of chemometrics method in processing of spectral data also has been developed. And the application of chemometrics in complicated biochemical systems can be used to solve some problems and the equilibrium concentration and pure spectra of each component can be obtained.In the first section, the definition, structure, function and nature of proteins were introduced briefly. Furthermore, the research methods and existent problems of interaction between small molecules and proteins were reviewed in turns. The application of chemometrics in the field of protein research was discussed. At last, forecasting the development trends and exploiting new probes, applying various techniques simultaneously and the use of chemometrics were chosen as the emphasis of this thesis.In the second section, the binding interaction of Alpinetin (APT) with BSA was studied by fluorescence, UV-visible and synchronous fluorescence spectroscopy (SFS). The measured complex spectra were resolved by multivariate curve resolution-alternating least squares (MCR-ALS), which yielded a host of data and information, which otherwise would have been impossible to obtain. The extracted profiles corresponded to the spectra of the single species in the APT/BSA mixture. In addition, the presence of the APT-BSA complex was demonstrated, and it was shown that the associated quenching of the fluorescence from the BSA protein resulted from the formation of APT-BSA complex via a static mechanism. The binding constant (Ka(ave)= 2.34×106 L mol-1) and the number of sites (n=1) were obtained by fluorescence methods as were the thermodynamic parameters (ΔH0,ΔS0 andΔG0). This work suggested that the principal binding between APT to BSA was facilitated by hydrophobic interactions. The thermodynamic parameters for APT were compared to those from the structurally similar Chrysin and Wogonin molecules. It appeared that the entropy parameters were relatively more affected by the small structural changes. SFS from the interaction of BSA and APT showed that the ligand affected the conformation of BSA. The competitive interaction of APT and site makers with BSA indicated site I as the binding area of APT in BSA.In the third section, the association of brodifacoum (BDF) with BSA was monitored with the using of fluorescence spectroscopy. The measured spectra were resolved by SIMPLISMA, which was the first application to the system concerned with the interaction of BSA with small molecules, and the extracted profiles corresponded to the spectra of the single species in the BDF/BSA mixture. In addition, the presence of the BDF-BSA complex was demonstrated, and it was shown that the associated quenching of the fluorescence from the BSA protein resulted from the formation of BDF-BSA complex via a static mechanism. Fluorescence sensitization of BSA to BDF also was studied by adding different concentration of BSA into different concentration of BDF. The results showed that the higher concentration corresponds the more quantum efficiency resulting in stronger sensitization. Synchronous fluorescence spectra three-dimensional fluorescence spectroscopy was used to study the conformation change of BSA invoked by BDF. As a result, BDF could lead to the conformation change of BSA apparently and the polarity around the Trp residues on BSA increased and the hydrophobicity decreased, while the interaction of BSA with BDF did not have a distinct effect on the conformation of the microenvironment around Tyr. The binding sites of BDF on BSA were studied by using Eosin Y which is a new site marker and has been found to bind to site I. The conclusion was obtained that the two molecules, BDF and Eosin Y, did not readily share a common binding site on the BSA protein rather than the BDF been replaced by EY absolutely.In the forth section, The binding interaction of carbofuran (CF) and its degradation product, 2,3-dihydro-2,2-dimethyl-7-hydroxy benzofuran (carbofuranphenol, CFP), with BSA was studied by spectrofluorimetry using chemometrics approaches. It was shown that both CF and CFP quenched the intrinsic fluorescence of BSA via a static mechanism. By making use of the chemometrics techniques, SVD and MCR-ALS, to solve the data obtained from the fluorescence spectrum of the interaction of either CF or CFP with BSA, we can draw a conclusion that there were three components exiting in the mixture solutions of BSA and either carbofuran or its degradation product, in other words, they interacted each other and gave birth to a complex. Synchronous and three-dimensional fluorescence spectroscopy of the interaction of BSA with either CF or CFP showed that the molecular structure of the BSA was changed significantly, which is consistent with the known toxicity of the pesticide i.e., the protein is denatured. The value of the binding constant K of BSA with its degradation product is bigger than that of BSA with CF which illuminated its degradation product is more toxic than its parent, CF. In addition, the effect of some metal ions on the binding constants of either CF or CFP with BSA was also investigated.In the fifth section, the binding interaction of the 10-hydroxycamptothecin (10-HCPT) with BSA, using warfarin as a fluorescence probe, was studied by spectrofluorimetry and UV-vis spectroscopy with the aid of chemometrics. The result indicated that the endogenetic fluorescence quenching of BSA by 10-HCPT is static quenching. In this work, the competitive interaction of the 10-HCPT and warfarin with BSA was also studied by three-way excitation-emission fluorescence by parallel factor analysis (PARAFAC). The estimated relative concentration profiles of the components showed that the binding site of the 10-HCPT on BSA is site I. The effect of 10-HCPT on the conformation of BSA was studied by synchronous fluorescence technique, and the results indicated that the conformation of BSA was changed when 10-HCPT was added, and the hydrophobic properties of the environment of residues in BSA decreased.更多还原