本文選題：熒光光譜法 + 牛血清蛋白��； 參考：《河北大學》2017年碩士論文

【摘要】：目前,傳統(tǒng)熒光猝滅法和同步熒光法在生物化學和藥學領域被廣泛應用于研究蛋白質與小分子藥物之間的相互作用。本文主要以鹽酸頭孢吡肟(CH)為小分子藥物代表,利用多種熒光光譜法研究了蛋白與藥物之間相互作用的反應機理,并在此基礎上利用多種光譜法聯用更深入的研究了兩者反應過程中的氨基酸殘基微環(huán)境的變化。同時還研究了小分子藥物本身的熒光特性,和一系列小分子藥物和蛋白的作用情況。本研究內容共包含以下五部分:第一章:綜合介紹了藥物小分子本身熒光特性以及蛋白質與藥物小分子間相互作用的一般研究方法。對采用多種光譜法研究蛋白與藥物小分子相互作用的研究進展進行了綜述,并在此基礎上提出了本文的立題思路。第二章:利用熒光光譜對沙拉沙星(SAR)在不同pH條件下的結構及其變化進行了研究并計算其解離常數及熒光量子產率,研究結果可以為喹諾酮類藥物理化性質的比較研究提供參考。研究結果表明,在強酸性條件下沙拉沙星以H3L2+形式存在,最大熒光發(fā)射波長約455 nm;當pH 3~5時,H3L2+失去喹啉環(huán)1位N上結合的質子而以H2L+形式存在,熒光強度較大且基本不變,最大熒光發(fā)射波長位于458 nm;當pH5時,隨著pH升高,熒光發(fā)射波長藍移至430 nm,H2L+失去羧基質子而以雙極離子HL形式存在;當pH進一步升高時,HL失去哌嗪環(huán)N上結合的質子,轉化成為陰離子L-,導致熒光強度降低,最大發(fā)射波長紅移至約466 nm。在強堿性條件下,由于介質環(huán)境的影響,L-的熒光強度降低,但最大發(fā)射峰位置基本不變。第三章:利用熒光光譜法(包括熒光猝滅法、同步熒光法,共振光散射法三種方法)和紫外吸收光譜法研究298 K時牛血清白蛋白(BSA)和硫酸粘桿菌素,硫酸頭孢匹羅,頭孢匹胺鈉三種不同的藥物之間的相互作用機理。結果表明:對于相同的體系利用四種方法計算得到的結合常數的數量級一致;三個體系主要的猝滅方式均為生成新物質的靜態(tài)猝滅;三種藥物與蛋白作用時以1:1的比例結合;利用實驗數據計算的Hill系數相似。文章中對四種方法所得實驗結果進行了分析和比較,表明所用方法都可用于蛋白與藥物反應機理的研究。第四章:利用熒光猝滅法和同步熒光法,分別研究了不同溫度下鹽酸頭孢吡肟與牛血清白蛋白之間的反應機理。兩種研究方法結果均表明鹽酸頭孢吡肟對牛血清蛋白的熒光發(fā)生了猝滅作用,二者之間通過靜態(tài)猝滅的方式相互作用;牛血清蛋白與鹽酸頭孢吡肟反應的結合位點數約為1,表明它們相互作用形成1:1復合物;利用熱力學參數計算得到牛血清蛋白與鹽酸頭孢吡肟之間主要的作用力類型為范德華力或是氫鍵;根據F?rster非輻射能量轉移理論確定了鹽酸頭孢吡肟與牛血清蛋白的結合距離,進一步說明二者通過靜態(tài)猝滅方式相互作用并伴有非輻射能量轉移。第五章:利用靈敏度較高的四階導數紫外光譜法,結合內源熒光法、同步熒光法、近紫外圓二色光譜法和位點試劑法多種方法共同研究牛血清白蛋白與鹽酸頭孢吡肟體系作用過程中氨基酸殘基微環(huán)境極性及蛋白質大分子的構象變化。研究發(fā)現,隨著CH濃度的增大,CH分子首先聚集在結構域IIA的Trp-213位置附近,導致色氨酸(Trp)殘基所處微環(huán)境極性增強。形成聚集體后,誘導結構域IIA展開暴露出更多殘基,導致酪氨酸(Tyr)和苯丙氨酸(Phe)殘基所處微環(huán)境極性增強。CH增大到一定濃度后,各殘基微環(huán)境極性保持不變,BSA大分子構象趨于穩(wěn)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.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96;O657.3

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本文編號：1964683