脲和盐酸胍诱导溶菌酶去折叠的荧光相图法研究

用荧光相图法分别研究了脲和盐酸胍诱导卵清溶菌酶去抓叠的过程。当变性体 系中无还原剂2－巯基乙醇存在、脲浓度从0变化至4.0 mol/L（或盐酸胍浓度从0变 化至3.0 mol/L）时,溶菌酶从天然态转变为部分折叠中间态,当脲浓度从4.0 mol/L变化至8.0 mol/L（或盐酸胍浓度从3.0 mol/L变化至6.0 mol/L）时,溶菌 酶从中间态转变为去折叠态,此时该蛋白的变性过程符合“三态模型”。而当变性 体系中有该还原剂存在时,溶菌酶则由天然态直接转变为去折叠态,此时脲诱导该 蛋白去折叠的过程符合曲型的“二态模型”。实难结果表明荧光相图法可以检测蛋 白南去抓叠的中间态。

Unfolding of Lysozyme Induced by Urea and Guanidine Hydrochloride Studied by "Phase Diagram" Method of Fluorescence

The unfolding of hen egg-white lysozyme induced by urea and guanidine hydrochloride (GuHCl) has been studied by "phase diagram" method of fluorescence. In the absence of 2-mercaptoethanol, the conformational transi-tion from the native state to a partially folded intermediate of this protein occurs in the range of urea concentration (0～4.0 mol/L) and GuHGl concentration (0～3.0 mol/L), and the transition from the intermediate to the unfolded state of this protein occurs in the range of urea concentration (4.0～8.0 mol/L) and GuHCl concentration (3.0～6.0 mol/L) , indicating that lysozyme unfolding follows a three-state model under such conditions. In the presence of this reducing agent, however, the denaturation of lysozyme induced by urea obeys a typical two-state model and there is only one conformational transition from the native to the unfolded states during the unfolding. The experimental results show that the "phase diagram" method of fluorescence can be used to detect unfolding intermediates of proteins.