Z及logI对离子交换色谱中脲变溶菌酶分子构象变化的表征

Characterization of the Changes in the Molecular Conformation of Lysozyme with Various Urea Concentrations by Parameters Z and log I in Ion Exchange Chromatography

以溶菌酶(Lys)为目标蛋白,用计量置换保留理论(SDT R)中的参数Z和logI对弱阳离子交换色谱(WCX)中"准天然态"和脲变还原与非还原的两种变性状态的Lys的分子构象变化进行了表征。发现在流动相中含有脲时,蛋白的保留仍服从SDT理论,可以准确测定在特定脲浓度条件下Lys的Z及logI值。结果表明,3种分子构象状态的Lys的Z值均随脲浓度改变呈现不连续变化;"准天然态"Lys在同一脲浓度条件下的Z值比变性状态的大,logI比变性状态的小,而非还原变性态和"准天然态"Lys的Z和logI值比较接近。还对不同脲浓度条件下Lys的活性回收率与Z值之间的关系进行了研究。

The parameters Z and logI of lysozyme (Lys), which serves as a model protein for the stoichiometric displacement theory for retention (SDTR), were used to characterize the molecular conformational changes of Lys for different molecular conformation states (pseudonative, ureaunfolded, ureareducedunfolded) with various urea concentrations in weak cation exchange chromatography (WCX). It was found that the retention of the three molecular conformations of Lys behaved as predicted by the SDTR theory provided that the urea concentration remained constant in the mobile phase. The Z value of the Lys in its pseudonative state decreased with the increase of the urea concentration in the mobile phase and was the largest of the three molecular conformational states, while its corresponding logI was the smallest. The values of both Z and logI of Lys in their pseudonative and ureaunfolded states were closed with each other. The changes of Z of Lys in the three molecular conformational states as a function of urea concentration in mobile phase were found to be discontinuous. The relationship between the Z value and bioactivity recovery of Lys with different urea concentrations was also investigated. 