CTAB辅助溶菌酶复性过程动力学

KINETICS OF CTAB-ASSISTED LYSOZYME REFOLDING in vitro

研究了溶菌酶在十六烷基三甲基溴化铵 (CTAB)溶液中的复性过程 ,通过测定溶液表面张力与酶活力的变化证实变性溶菌酶首先与CTAB形成复合物 ,进而在氧化 还原剂作用下开始复性并与CTAB发生解离 .非还原型SDS PAGE分析结果表明 ,复性反应的主要产物有三类 :具有天然结构的溶菌酶单体、溶菌酶多聚体及溶菌酶单体与CTAB形成的无活性复合物 .不同产物的含量取决于溶液中CTAB与溶菌酶的摩尔比 .当变性溶菌酶浓度为 0 1～ 0 4mg·ml-1,CTAB与溶菌酶摩尔比为 5～ 2 0时 ,采用“变性 复性”二态复性模型分析了CTAB辅助溶菌酶复性过程的宏观动力学 .结果表明 ,随CTAB与溶菌酶摩尔比的增大 ,变性反应的速率常数显著增大 ,而折叠反应的速率常数先增大后减小 ,CTAB与溶菌酶的摩尔比为 10时 ,复性率最高 .而蛋白质浓度提高则导致折叠反应速率常数减小 ,变性反应速率常数增大 ,复性率缓慢下降 .

The investigation of lysozyme refolding in vitro in the presence of CTAB was carried out with emphasis on the interaction of denatured lysozyme and CTAB characterized by the formation of various protein products. The changes in surface tension as well as the lysozyme activity were determined as a function of the initial concentration of denatured lysozyme and the molecular ratio of CTAB to denatured lysozyme. It was concluded from the experimental results presented that the denatured lysozyme firstly formed a complex with CTAB upon dilution into the refolding buffer, and then refolded at the input of the oxidative-reductive pair, leading to the dissociation of denatured lysozyme-CTAB complex. The non-reductive SDS-PAGE showed three kinds of protein products obtained in the above refolding process, i.e., native lysozyme, denatured lysozyme aggregate, and CTAB-denatured lysozyme. Similar result was obtained for native lysozyme incubated with CTAB for 24 hours at 37°C. The kinetics of CTAB-assisted refolding of lysozyme were investigated at 0.1-0.4 mg &middot ml-1 lysozyme with a molecular ratio of CTAB to protein equating to 5-20. The apparent kinetics of refolding could be described by a two-state refolding model, i.e., a reversible reaction between unfolded and refolded lysozyme. The apparent rate constants were shown to be a function of the molar ratio of CTAB to protein but where slightly dependent on protein concentration. The recovery of lysozyme activity, an index of the refolding performance, reached its optimum at a molecular ratio of CTAB to denatured lysozyme of 10 and decreased against an increase of the initial concentration of denatured lysozyme.