猪肝单胺氧化酶B的分离纯化及其与胞质蛋白相互作用研究

发布时间：2018-04-04 22:29

本文选题：蛋白质相互作用　切入点：单胺氧化酶B　出处：《大连理工大学》2010年硕士论文

【摘要】： 蛋白质相互作用是功能蛋白质组学研究的重要内容,酶作为生物体内的一类重要蛋白分子,几乎参入了所有的生命活动和生命过程,研究酶与其它蛋白或调节因子的作用将对人们理解生命体的生理及病理过程具有重要意义。在实验室前期研究的基础上,本论文继续研究单胺氧化酶B与猪肝细胞质蛋白间相互作用的色谱方法：基于底物与酶的相互作用,通过计算机模拟合理设计底物配基和间隔臂,优化合成材料的配基密度,结合串联质谱分析,分子对接计算,探索利用固定化底物吸附纯化单胺氧化酶B (MAOB)从而锁定底物与酶的结合状态的可行性。联合硫酸铵反抽提、苯胺疏水色谱、强阴离子交换色谱等方法从猪肝线粒体中分离纯化出MAOB,进一步研究了酶的固定化方法,并对固定化MAOB与其相关蛋白质间的相互作用进行了初步考察。实验结果表明：(1)通过计算机对接模拟,选择与MAOB有较高亲和性的DADPA-戊二醛为连接臂、多巴胺(DA)为配基进行材料合成；较高配基密度(40μmol／mL胶)的DA材料可以吸附MAOB,对其纯化倍数为2.16,但吸附选择性不好；低密度(10μmol／mL胶)的DA材料分离得到的蛋白较纯,经串联质谱鉴定及酶活测定,不是MAOB,而是以过氧化氢酶为主的蛋白,同时有少量的肝羧酸酯酶、血清白蛋白；进一步的分子对接实验说明固定化DA对MAOB的亲和性低于对过氧化氢酶、肝羧酸酯酶、血清白蛋白的亲和力,且固定化DA与MAOB的优势结合部位并非酶的活性口袋部位,而是酶表面膜结合端螺旋结构附近的一处空穴。(2)探索出一种有效分离纯化MAOB的方法,即用Triton X-100裂解液制备粗酶、硫酸铵反抽提、苯胺色谱、联合Sepharose Q High Performance离子交换色谱,得到纯化倍数为18.2,酶比活为135 U／mg的MAOB。SDS-PAGE显示为分子量约60 kDa的单一蛋白质带,LC-ESI-MS/MS证实该蛋白为MAOB。(3)盐梯度洗脱考察固定化DA材料对纯化的MAOB吸附研究表明,固定化DA与MAOB不能形成稳定的吸附,无法进一步实现相互作用蛋白的捕捉和分离。(4)选择共价偶联法固定MAOB,通过对连接臂、醛基密度的优化,选择乙二胺、戊二醛为连接臂,醛基密度为5.7μmol／mL胶的材料固定MAOB,固定蛋白量为0.53 mg／mL胶,得到酶活收率为40.98%；MAOB固定化后最适温度、最适pH升高,温度及pH稳定性增强。(5)用固定化MAOB材料吸附猪肝细胞质蛋白,SDS-PAGE没有检测到相互作用蛋白。以上研究结果表明：通过固定化DA吸附纯化MAOB并形成底物-酶复合物的设想很难实现,且通过SDS-PAGE方法,没有检测到共价固定的MAOB材料吸附的猪肝细胞质中的相互作用蛋白。
[Abstract]:Protein interaction is an important part of functional proteomics. As a kind of important protein molecules in organism, enzyme is involved in almost all life activities and processes.It is important to study the action of enzyme and other proteins or regulatory factors in understanding the physiological and pathological processes of organisms.Based on the previous research in laboratory, the chromatographic method of interaction between monoamine oxidase B and porcine liver cytoplasmic protein was studied. Based on the interaction between substrate and enzyme, the substrate ligand and spacer were designed by computer simulation.The ligand density of the synthesized materials was optimized, and the binding state of the substrate and the enzyme was determined by using the immobilized substrate to adsorb and purify monoamine oxidase B (MAOB) by means of tandem mass spectrometry and molecular docking calculation.Combined with ammonium sulfate reverse extraction, aniline hydrophobic chromatography and strong anion exchange chromatography, MAOB was isolated and purified from pig liver mitochondria.The interaction between immobilized MAOB and its related proteins was investigated.The results showed that by computer simulation, DADPA-glutaraldehyde, which had high affinity to MAOB, was selected as the connecting arm and dopamine was used as ligand to synthesize.The DA material with a high ligand density of 40 渭 mol/mL) could adsorb MAOB, and its purification multiple was 2.16, but the adsorption selectivity was not good, and the protein isolated from the DA material with low density of 10 渭 mol/mL was relatively pure, which was identified by tandem mass spectrometry and determined by enzyme activity.Not MAOB, but a catalase protein with a small amount of hepatic carboxylesterase and serum albumin. Further molecular docking experiments showed that the affinity of immobilized DA to MAOB was lower than that to catalase and hepatic carboxylesterase.The affinity of serum albumin, and the dominant binding site of immobilized DA to MAOB is not the active pocket site of enzyme, but a hole near the helical structure of enzyme surface membrane.That is, the crude enzyme was prepared from Triton X-100 pyrolysis solution, ammonium sulfate was extracted back, aniline chromatography was used, and Sepharose Q High Performance ion exchange chromatography was used.A single protein band LC-ESI-MS / MS / MS with a specific enzyme activity of 135 U/mg and an enzyme specific activity of 18.2 U/mg was obtained. The protein was confirmed as MAOB.T3 by salt gradient elution. The adsorption of purified MAOB by immobilized DA material was studied.Immobilized DA and MAOB could not form a stable adsorption and could not further realize the capture and separation of interacting proteins. The method of covalent coupling was chosen to immobilize MAOB.According to the optimization of the connecting arm and aldehyde density, ethylenediamine and glutaraldehyde were selected as the connecting arm.The immobilized MAOB was immobilized with a density of 5.7 渭 mol/mL and the amount of protein was 0.53 mg/mL. The optimum temperature and pH of the immobilized MAOB were 40.98% and 40.98 渭 mg/mL, respectively.The interaction protein was not detected by SDS-PAGE when the immobilized MAOB material was used to adsorb porcine liver cytoplasmic protein.The results showed that the idea of purification of MAOB by immobilized DA and the formation of substrate-enzyme complex were difficult to realize, and no interaction protein was detected in porcine liver cytoplasm adsorbed by covalently immobilized MAOB material by SDS-PAGE method.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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