多方法组合分析脲诱导的淀粉液化芽孢杆菌α-淀粉酶去折叠和重折叠过程的构象转化

Combinatorial Analysis of the Conformational Transition of Bacillus Amyloliquefaciensα-Amylase during Urea-induced Unfolding and Refolding

以变性和非变性电泳、体积排阻色谱、内源荧光发射光谱、荧光相图、荧光猝灭以及活性测定等组合分析方法,研究了脲诱导的淀粉液化芽孢杆菌α-淀粉酶分子的去折叠和重折叠过程。结果表明,在脲诱导的芽孢杆菌α-淀粉酶分子的去折叠和重折叠过程中,芽孢杆菌α-淀粉酶分子始终以单分子形式存在,不会形成分子间的聚集体或聚集体沉淀。当变性液或复性液中脲浓度约为4.0mol/L时,芽孢杆菌α-淀粉酶分子的去折叠和重折叠过程中均出现一个部分折叠中间体,两个过程均符合"三态模型"。脲诱导的芽孢杆菌α-淀粉酶分子重折叠过程的复性曲线几乎与芽孢杆菌α-淀粉酶分子去折叠过程的残余活性率曲线重合。通过这些结果,并结合盐酸胍诱导的芽孢杆菌α-淀粉酶分子的去折叠和重折叠过程,推断脲和盐酸胍诱导的芽孢杆菌α-淀粉酶分子的去折叠和重折叠过程分别是相互可逆的。

The urea-induced unfolding and refolding of Bacillus amyloliquefaciensα-amylases was investigated through the combination of protein electrophoresis,size exclusion chromatography,intrinsic fluorescence emission spectroscopy,fluorescence phase diagram,fluorescence quenching and biological activity assay.The results showed that during the unfolding and refolding ofα-amylase induced by urea,theα-amylase molecules existed only in the unimolecular form instead of aggregate or aggregate precipitation,apartially folded intermediate separately occurred at about 4.0mol/L urea in the unfolding and refolding processes and both of the unfolding and refolding followed a three-state model.Additionally,the deactivation curve was nearly identical to the renaturation curve in urea solutions.Combining with previous studies about the unfolding and refolding of Bacillus amyloliquefaciensα-amylases induced by guanidine hydrochloride,it can be inferred that the unfolding and refolding ofα-amylase induced by urea and guanidine hydrochloride are separately a mutually reversible procedure.