荧光光谱法研究盐酸胍浓度不同时变性胰蛋白酶的构象变化

蛋白变性过程中间体的存在是蛋白变性及复性动力学研究中不可缺少的证据。以胰蛋白酶为模型蛋白 ,用荧光光谱法系统地研究了在不同浓度变性剂盐酸胍存在时胰蛋白酶构象的变化 ,并与活性数据进行了对比。发现胰蛋白酶荧光光谱发射波长随变性剂盐酸胍浓度增大而逐渐增大 ,并且当盐酸胍浓度达到 2mol·L- 1 时胰蛋白酶的最大发射波长达到最大值 ,其后随盐酸胍浓度的增大最大发射波长反而逐渐减小 ,当盐酸胍浓度大于 3mol·L- 1 呈现不变的趋势。也就是说 ,在低浓度变性剂环境下 ,胰蛋白酶存在着一个与天然态和完全变性态的分子构象都不同的中间体状态 ,这个中间体状态的荧光发射波长最大 ,荧光发射强度也最大 ,而以此状态为复性起点 ,最终得到的复性产率也最低。对此原因从分子结构的基础上进行了探讨。

Research on Conformational Changes of Trypsin in Different Concentrations of GuHCl by Fluorescence Spectra

The existing of intermediate in the process of protein unfolding is necessary for studying the kinetic folding and unfolding pathway of protein. With trypsin as a model protein, the conformational changes of trypsin in the presence of different GuHCl concentrations were investigated by using fluorescence spectra (FR), and compared with the activity recovery of denatured trypsin. It was found that the maximum wavelength of FR of trypsin increases with the concentration of GuHCl increasing, and the maximum wavelength of FR of trypsin reaches the maximum when the concentration of GuHCl is 2 mol x L(-1), and then, the maximum wavelength of FR of trypsin decreases with the concentration of GuHCl increasing. There exists a intermediate state in the presence of low concentration of GuHCl. The conformation of the intermediate is different from the native and unfolding states. The maximum wavelength of FR of the intermediate is the maximum, and fluorescence intensity is the highest, while the bioactivity recovery is the lowest. The reasons for these were studied with the knowledge of the molecular structure.