【作者】 苏少景；

【导师】 倪永年；

【作者基本信息】 南昌大学 ， 分析化学， 2007， 硕士

【摘要】 本论文共分为四个部分，主要对近年来小分子与血清白蛋白相互作用的研究现状进行了综述，同时尝试将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。第一部分：本节首先对血清白蛋白的化学和生物学性质进行了简介，然后依次对小分子与蛋白质相互作用的研究方法、存在的问题等方面展开了评述。在分子水平上深入研究小分子化合物与蛋白质的键合与识别机理能够帮助人们理解蛋白质与小分子的作用原理，为寻找、筛选及研制开发药效高、抗癌或抗菌谱广及毒副作用小的新药提供丰富的理论依据。最后还展望了小分子与白蛋白作用研究的发展趋势，将开发新的探针、各种仪器方法的联用及化学计量学方法的引入作为本研究的重点。第二部分：本节在实验条件接近人体生理环境的pH 7.4的Tris-HCl缓冲液中，应用荧光光谱法并结合SiteⅠ标记药物华法林和SiteⅡ标记药物布洛芬来研究水杨酸与牛血清白蛋白（BSA）的作用，从相应的二维荧光图上只能推测出在结合BSA过程中，布洛芬和水杨酸可能存在着竞争作用，但难以推测华法林与水杨酸是否存在竞争结合．另外，从各个组分的三维荧光等高线图上可以看出混合物溶液中的各个组分的最佳激发波长之间变化很大，如BSA、水杨酸、华法林和布洛芬分别为278、295、306和218 nm，难以选择一个固定的最佳激发波长来扫描。因此，我们采集了三维荧光激发—发射数据矩阵（EEMs），解决了复杂体系最佳激发波长选择的问题，同时还可以得到足够多的信息量。最后，我们结合平行因子算法（PARAFAC）对三维数据荧光激发—发射数据矩阵进行解析。由PARAFAC分辨出的光谱曲线与真实谱线能很好地吻合，保证了其分辨出的相对浓度曲线的合理性和准确性。由解析得到的各个组分的相对浓度趋势图可以推测出：1)水杨酸在BSA的siteⅠ内存在一个高亲和结合区，但不同于华法林的高亲和结合区，可能与其低亲和结合区有一点重叠：2)水杨酸在BSA上可能存在两个低亲和结合区，分别位于布洛芬在BSA的siteⅠ内的低亲和结合区和siteⅡ内的高亲和结合区。第三部分：本节同时应用荧光光谱法和紫外可见光谱法来研究人体生理条件下牛血清白蛋白与氧氟沙星及恩诺沙星间结合作用的机制。实验中计算了Stern-Volmer猝灭常数K_SV，相应的沙星-蛋白质的结合常数和结合位点数以及相应的热力学参数ΔH，ΔS和ΔG，依据F（?）rster非辐射能量转移理论，计算了授体-受体间的结合距离，并同时用吸收光谱和同步荧光研究了沙星对牛血清白蛋白构象的影响。结果表明，沙星与蛋白质之间的荧光猝灭机理符合生成沙星-BSA络合物的静态猝灭机制，沙星与BSA之间的作用力均主要是氢键和范德华力，它们不仅能与牛血清白蛋白结合，通过血液循环达到作用部位，而且对蛋白质分子的构象都有一定的影响。实验结果还表明，氧氟沙星和恩诺沙星在体内与蛋白结合直到运输到受体部位这一环节基本相同。第四部分：本文采用紫外-可见光谱法和荧光光潜法研究盐酸小檗碱（BC）与牛血清白蛋白（BSA）相互作用。实验表明温度为37℃时，在pH为7.4的Tris-HCl缓冲溶液中，BC与BSA在反应前后的光谱变化虽然明显，但反应各组分的光谱重叠比较严重，难以直观地进行深入研究。本文中我们对原有测量矩阵进行扩展，并结合交替最小二乘（ALS）对数据进行迭代计算，能较好地获得复杂体系的各项信息，同时解决了用交替最小二乘法解析单个矩阵时存在的旋转不确定性问题。本文方法能较好地分辨出各个组分的纯光谱图，与组分的实际光谱相吻合（保证了分辨的浓度趋势图的可靠性）：另外还能解析得到各个组分的浓度变化趋势图，使我们可以很直观地监测到反应过程中各个组分的浓度变化，可用于定量地算出BC和BSA表观结合常数和结合比。更多还原

【Abstract】 This thesis is divided into four parts. The research actuality of interaction between small moleculeswith serum albumin in recent years has been reviewed. The application of chemometrics in analysis ofspectral data also had been developed. And the application of chemometrics in complicated biochemicalsystem can solve some problems and obtain the equilibrium concentration and pure spectra of eachcomponent.PartⅠIn this section, the chemical and biological characters of serum albumin were introduced briefly first,then the research methods and existent problems of interaction between small molecules and proteinswere reviewed in turns. Serum albumin is the most abundant of all proteins in blood plasma of manyspecies, and study on it has become an important research field of life science. Along with more attentionhas been given on the research of the interaction between serum albumin and small molecules, themechanisms of the action of some drugs and origins of some diseases have generally been understood.Moreover, the investigation based on serum albumin interactions with small molecular compounds alsohas great significance to design new protein-targeted drugs and to screen these drugs in vitro. At last, thedevelopment trend was forecasted and exploiting new probes, using various techniques simultaneouslyand the use of chemometrics were chosen as the emphasis of this thesis.PartⅡIn part two, the interactions of salicylic acid (SL) and two different site markers (warfarin for siteⅠand ibuprofen for siteⅡ) with bovine serum albumin (BSA) in pH 7.4 Tris-HCl buffer have beeninvestigated with the use of spectrofluorimetry. An equilibrium solution of BSA and SL was titratedseparately with the two markers. This initial work showed that the binding of SL with BSA could be quitecomplex, and that there was probably a competitive interaction occurring between ibuprofen and SL.However, the spectral results were difficult to interpret clearly for the interaction of warfarin and SL insimilar circumstances.To extract more information from the resolution of fluorescence excitation-emission spectra, thecontour plots of the fluorescence spectra indicated that the optimal excitation wavelengths for BSA, SL,warfarin and ibuprofen were different, and were found to be at 278, 295, 306 and 218 nm, respectively.The spectral information was arranged into three-way EEM stack arrays, and was submitted for analysisby the PARAFAC algorithm. Firstly, it was demonstrated that the estimated excitation and emissionspectral responses for SL, BSA and the site markers, warfarin and ibuprofen, agreed well with themeasured spectra. Then, the interpretation of the plots of simultaneously extracted (by PARAFAC)equilibrium concentrations for the above four reactants, showed that:ⅰ) the SL primarily appears to bindin siteⅠbut at a different location from the high-affinity binding site (HAS) for warfarin, and theinteraction partially overlaps with the low-affinity binding site (LAS) for warfarin,ⅱ) the SL may havetwo LAS, -one in siteⅡwhere the HAS for ibuprofen is located, and the other in siteⅠat the LAS foribuprofen.PartⅢIn part three, the interaction between fluoroquinolones (FQ, here, ofloxacin and enrofloxacin) andBSA was investigated by fluorescence and UV-vis absorbance spectroscopy. The Stern-Volmerquenching constant Ksv, the binding constant K, the number of binding sites n and corresponding thermodynamic parameters AH, AS and AG were calculated. The distance r between donor (BSA) andacceptor (FQ) was obtained according to the theory of F(o|¨)rster’s nonradiative energy transfer. The effectof FQ on the conformation of BSA has been analyzed by means of both UV-vis absorbance spectra andsynchronous fluorescence spectroscopy. It was proved that the fluorescence quenching of BSA by FQ ismainly a result of the formation of FQ-BSA complex and hydrogen bonds and van der Waals play majorrole in the reaction. All the results showed that the mechanism of the interaction between ofloxacin withthe BSA is similar to that of enrofloxacin.PartⅣIn part four, the interaction between berberine chloride (BC) and BSA was studied by fluorescenceand UV-vis absorbance spectroscopy in pH 7.4 Tris-HC1 buffer at 37℃. The spectra of the mixturesolutions varied in evidence through the process of the interaction, but the spectrum of each component inthe BC-BSA system showed significant overlapping between each other. Moreover, the analysis ofindividual data matrices by the MCR-ALS method might exist possible unresolved underlying factoranalysis ambiguities. Therefore, the whole set of spectroscopic data matrices was simultaneouslyanalyzed by the MCR-ALS method. This procedure allowed the recovery of the concentration profilesand pure spectra for each species and the calculation of the BSA:BC ration in the complex and theapparent equilibrium constant.更多还原