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目的研究辣根过氧化物酶(HRP)在盐酸胍中的变性过程,并建立荧光参数与酶活力的函数模型。方法采用可见光谱法,荧光光谱法及荧光相图法对盐酸胍诱导的HRP变性过程进行研究,并采用SPSS和Origin软件对变性过程中的构象参数与酶活力参数进行函数模型拟合。结果盐酸胍诱导的HRP去折叠经历了一个三态过程,其中,引起酶分子从部分折叠中间态向去折叠态转变,导致其疏水内核构象剧烈变化,酶活性中心结构及酶活力开始变化的临界盐酸胍浓度为1.5 mol·L-1;同时建立了构象参数与酶活力的函数模型。结论所建模型可以表征HRP的变性过程并用于酶活力的预测。
Objective To study the denaturation of horseradish peroxidase (HRP) in guanidine hydrochloride and to establish a functional model of fluorescence parameters and enzyme activity. Methods The degeneration of HRP induced by guanidine hydrochloride was investigated by visible spectroscopy, fluorescence spectroscopy and fluorescence phase diagram. The conformational parameters and enzyme activity parameters were characterized by SPSS and Origin software. Results The guanidine hydrochloride-induced de-folding of HRP underwent a three-state process in which the enzymatic molecule was caused to shift from the partially folded intermediate state to the unfolded state, resulting in a drastic change in the conformation of the hydrophobic core, the critical structure of the enzyme center, The concentration of guanidine hydrochloride was 1.5 mol·L-1. At the same time, a functional model of conformational parameters and enzyme activity was established. Conclusion The constructed model can characterize the process of HRP degeneration and be used for the prediction of enzyme activity.