【作者】 谢光；

【导师】 刘克武；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 胃蛋白酶是动物胃液中主要的蛋白水解酶，具有较强的专一性，催化具有苯丙氨酸，酪氨酸，色氨酸等肽键的断裂，使大分子的蛋白质变为较小的多肽。作为一种重要的蛋白水解酶，胃蛋白酶的酶学性质，底物专一性，抑制作用等都与其它的蛋白酶不同，具有一定的理论研究价值和应用价值。生物碱是一类含氮杂环的碱性天然有机物，广泛存在于植物体内，在医药上有重要的用途。本文基于生物碱的药用价值，借助近年来发展迅速的光谱分析的办法探讨了三种茶叶生物碱小分子与胃蛋白酶的相互作用。通过这三种生物碱对胃蛋白酶的动力学性质的影响以及与胃蛋白酶的相互作用的紫外光谱和荧光光谱的研究，对胃蛋白酶在三种小分子影响下的变化及作用机理作了初步推测。 首先对胃蛋白酶的酶学性质进行研究，本实验采用2000药典方法，以血红蛋白作底物，在275nm下以紫外分光光度法测定胃蛋白酶的活力，以每分钟酶水解血红蛋白生成一微摩尔的酪氨酸为一个活力单位。以血红蛋白为底物作酶学性质分析，测得Km值为0.86mmol／L,最适温度为40℃，最适pH2.0。 茶碱，咖啡因，可可碱对胃蛋白酶的动力学影响主要表现在对胃蛋白酶有抑制作用，在不同的底物浓度下分别作三种生物碱对胃蛋白酶的抑制动力学，求得茶碱ki值为1.08×10^-3mol／L，咖啡因ki值为1.46×10^-3mol／L，可可碱ki值为1.48×10^-3mol／L，三种生物碱的抑制剂类型为非竞争性抑制。抑制作用大小依此为：茶碱＞咖啡因＞可可碱。 通过对茶碱，咖啡因，可可碱与胃蛋白酶在200-400nm下分别作紫外浓度和时间差谱，发现茶碱，咖啡因与胃蛋白酶在3：1的摩尔浓度比，可可碱与胃蛋白酶在4：1的摩尔浓度比具有最明显的紫外吸收差谱，同时发现更多还原

【Abstract】 The pepsin is the main digestive ferment in animal, which have stronger uniqueness in the gastric juice, it catalysis the peptide hydrolysis consisted by Phe, Tyr and Trp, making the protein turn into smaller polypeptide. As an important protease, pepsin s enzymic nature, substrate uniqueness, inhibition, et al. are different from other protease, therefore it has theoretical research value and applied value .The alkaloid is a kind of alkali natural organic containing nitrogen heterocyclic compound, which exist in the plant extensively, there are many important applications on medicine. Based on alkaloid medical value, in view of the method of developed spectral analysis this article discussed the interaction between three kinds of tea alkaloid and pepsin in recent years. Through the effect of three kinds of alkaloid on pepsin enzyme kinetics and interactive ultraviolet spectrum and fluorescence spectrum of the pepsin, we inferred the change and act mechanism of pepsin under influence of three kinds of alkaloid.First we study nature of the pepsin by adopting 2000 pharmacopoeia .we make substrate with the hemoglobin, determining the pepsin activity with ultraviolet spectrum under 275nm, enzyme hydrolysis hemoglobin turn into a umol tyrosine as a activity unit with every minute, using hemoglobin as the substrate and study enzymes property , get Km value is 0. 86mmol/L, the optimum temperature is 40°C, the optimum pH2. 0.It is found that the effect of theophylline, caffeine , theobromine on pepsin enzyme kinetics exhibited inhibition , the inhibition dynamics of three kinds of alkaloid in pepsin is made respectively under the different concentration. The result is that: the ki of theophylline is 1.08×10^-3 mol/L,the ki of caffeine is 1.46×0^-2 mol/L, the ki of theobromine is 1.48× 10^-3 mol/L, Three kinds of alkaloid belong to in-competition type of inhibitor. The order of更多还原