体积排阻色谱法研究变性溶菌酶分子复性过程中集聚体的形成

Studies on Formation of Aggregates from Denatured Lysozymes upon Renaturing with Size Exclusion Chromatography

在体积排阻色谱柱上研究了还原剂存在时脲和盐酸胍变性的 3种溶菌酶溶液的复性和分离过程。当变性液中原始溶菌酶浓度大于 10g/L时,变性溶菌酶在体积排阻色谱柱上除了复性为与未变性溶菌酶出峰时间相同的复性态溶菌酶分子外,还形成了溶菌酶折叠中间体的二分子集聚体。这个结果得到了用稀释法复性时溶菌酶的蛋白电泳检测结果的支持。与稀释法复性相比较,用体积排阻色谱法复性时所形成的折叠中间体二分子集聚体的量要远远低于用稀释法所形成的集聚体的量。

In order to study the renaturation mechanism of denatured protein in denaturant (solution,) the renaturation and separation process for three kinds of lysozyme molecules, which were (separately) denatured by urea and guanidine hydrochloride in the presence of reducing (agents,) was studied by size exclusion chromatography. When initial lysozyme concentration in denaturant solution was more than 10 (g/L), the denatured lysozyme molecules were renatured and isolated in a size exclusion chromatographic column. A refolded lysozyme intermediate, a bi-molecular aggregate, was found. This result was confirmed by non-reducing sodium dodecyl sulfate-polyacrylamide gel electrohoresis (SDS-PAGE) analysis of renatured lysozyme molecules with the dilution method. Compared with the dilution method, the amount of the (bi-molecular) aggregate found by size exclusion chromatography was far less than that found in the dilution (method). This result shows that the process for the renaturing of denatured lysozyme molecules in solution can be well described with three-state model in the presence of reducing agents.