脲和盐酸胍诱导过氧化氢酶去折叠的研究  

Studies on the Unfolding of Catalase Induced by Urea and Guanidine Hydrochloride

作　　者：焦铭[1] 梁毅[1] 李洪涛[1] 王曦[1] 

机构地区：[1]武汉大学生命科学学院,武汉430072

出　　处：《化学学报》2003年第9期1362-1368,共7页Acta Chimica Sinica

基　　金：国家重点基础研究发展规划 (No.G1 9990 7560 8);国家自然科学基金 (No.39970 1 64);湖北省自然科学基金 (No.2 0 0 1abb0 4 6)资助项目

摘　　要：用荧光相图法分别研究了脲和盐酸胍诱导牛肝过氧化氢酶去折叠的过程 .当脲浓度从 0依次增大至 0 5 0 ,4 5和8 0mol/L时 ,过氧化氢酶从天然四聚体依次转变为蓬松的四聚体、部分折叠的无活性二聚体和去折叠态 ,而当盐酸胍浓度从 0依次变化至 0 65 ,2 5和 6 0mol/L时 ,过氧化氢酶则从天然四聚体依次转变为部分折叠的激活二聚体、部分折叠的单体和去折叠态 ,这表明无论是用脲还是用盐酸胍作为变性剂 ,该蛋白的变性过程都符合“四态模型” ,但这两种变性剂诱导该蛋白去折叠的途径和机制有较大差异 .实验结果表明荧光相图法可以检测蛋白质去折叠的中间态 .用等温滴定量热法研究了盐酸胍诱导过氧化氢酶去折叠过程的热力学 ,2 5 0℃时低浓度盐酸胍诱导该蛋白从天然四聚体转变为部分折叠的激活二聚体的本征摩尔构象变化焓、Gibbs自由能和熵分别为 -69 2kJ·mol-1,6 43kJ·mol-1和 -2 5 4J·K-1·mol-1,据此推断盐酸胍通过熵效应和静电效应来稳定和激活该二聚体 .The unfolding of bovine liver catalase induced by urea or guanidine hydrochloride (GuHCl) has been studied by 'phase diagram' method of fluorescence. With increasing the concentration of urea from 0 to 0.50, 4.5 and 8.0 mol/L, the conformation of catalase is changed from the native tetramer to an expanded tetramer, a partially folded inactive dimer and the unfolded state, respectively. With increasing the concentration of GuHCl from 0 to 0.65, 2.5 and 6.0 mol/L, however, the conformation of catalase is changed from the native tetramer to a partially folded activated dimer, a partially folded monomer and the unfolded state, respectively. This indicates that the unfolding of catalase induced by urea or GuHCl follows a four-state model, but both the pathway and the mechanism for the unfolding of catalase induced by urea are different from those induced by GuHCl. The experimental results show that the 'phase diagram' method of fluorescence can be used to detect unfolding intermediates of proteins. The unfolding of catalase induced by GuHCl has been studied by isothermal titration calorimetry. The intrinsic enthalpy, Gibbs free energy and entropy changes for formation of the partially folded activated dimer of the protein in the presence of low GuHCl concentrations at 25.0 degreesC were measured as -69.2 kJ(.)mol(-1), 6.43 kJ(.)mol(-1) and -254 J(.) K-1 . mol(-1) respectively. It is thus indicated that the stabilization and activation of this partially folded dimer by GuHCl originate from the entropic and the electrostatic effect of this denaturant.

关 键 词：脲 盐酸胍 诱导 过氧化氢酶 去折叠 蛋白质 热力学 等温滴定量热法 荧光相图法 

分 类 号：O621[理学—有机化学]