盐酸胍诱导的变性卵清溶菌酶分子重折叠过程及其各稳定构象态的分布和过渡  

Distribution and Transition of Stable Conformations of Denatured Hen Egg White Lysozymes During Their Refolding in Guanidine Hydrochloride Solutions

作　　者：冯瑄[1] 张潭[1] 边六交[1] 

机构地区：[1]西北大学生命科学学院,西安710069

出　　处：《高等学校化学学报》2013年第2期336-345,共10页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金(批准号:21075097)资助

摘　　要：采用变性和非变性电泳、高效凝胶排阻色谱、内源荧光发射光谱和荧光相图以及生物活性测定等方法,研究了盐酸胍诱导的变性卵清溶菌酶分子的重折叠过程及此过程中卵清溶菌酶分子各稳定构象态的分布和过渡.结果表明,当复性液中盐酸胍浓度分别约为5.0和2.4 mol/L时,变性卵清溶菌酶分子的重折叠过程各存在1个稳定折叠中间态,重折叠过程符合'四态模型'.在卵清溶菌酶分子四态重折叠过程基础上,结合盐酸胍与卵清溶菌酶分子之间的缔合-解离平衡,给出了一个定量描述变性剂诱导的蛋白质分子复性过程中蛋白质分子复性率随溶液中变性剂浓度变化的方程.该方程包含2个特征折叠参数,一个是蛋白质分子从一个稳定构象态过渡到另一个稳定构象态的热力学过渡平衡常数k;另一个是在此过程中平均每个蛋白质分子所结合的变性剂分子数目m.通过这2个特征折叠参数能够定量描述盐酸胍诱导的变性卵清溶菌酶完全去折叠态、折叠中间态和天然态分子随复性液中盐酸胍浓度变化的分布和过渡情况.The refolding of denatured hen egg white lysozymes induced by guanidine hydrochloride and the distribution and transition of stable conformations of denatured hen egg white lysozymes during the refolding procedure were studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and native-PAGE,high-performance size-exclusion chromatography,intrinsic fluorescence emission spectra and fluorescence phase diagram,and biological activity assay.The results show that during the refolding procedure of denatured hen egg white lysozymes induced by guanidine hydrochloride,two stable intermediates of hen egg white lysozymes separately existed at about 5.0 and 2.4 mol/L of guanidine hydrochloride in renaturation solution,and this refolding followed a four-state model.Based on the four-state model,an equation which described the renaturation rate of denatured protein molecules under different denaturant concentrations in renaturation solution was presented and through this equation two characteristic refolding parameters,the thermodynamic refolding equilibrium constant k of the conformational transition of denatured protein molecules from their one conformational states to their another ones and the number m of denaturant molecules associated with an protein molecule during the conformational transitions,can be simultaneously derived.And further,by means of these two characteristic refolding parameters,the distribution and transition of stable conformations of egg white lysozyme molecules under different guanidine hydrochloride concentrations in denaturation solution can be well described.

关 键 词：卵清溶菌酶 盐酸胍 重折叠 特征折叠参数 荧光光谱 

分 类 号：O657.3[理学—分析化学]