大分子拥挤环境中溶菌酶的去折叠过程  

Studies on unfolding of lysozyme Induced by guanidine hydrochloride at different macromolecular crowding

作　　者：王莉衡[1] 钦传光[1] 尚晓娅[1] 牛卫宁[1] 

机构地区：[1]西北工业大学生命科学院,陕西西安710072

出　　处：《西北农林科技大学学报（自然科学版）》2009年第9期187-192,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：西北工业大学科技创新基金项目(2008KJ02038)

摘　　要：【目的】研究不同大分子拥挤体系对盐酸胍诱导的溶菌酶去折叠过程的影响。【方法】以葡聚糖70（Dextran70）、菲可70（Ficoll70）、聚乙二醇2000（PEG2000）作为大分子拥挤试剂，模拟体内高度拥挤的环境，用荧光相图法研究不同大分子拥挤试剂中，有无还原剂2－巯基乙醇存征时，盐酸胍诱导溶菌酶去折叠过程的变化及其特点，探讨大分子拥挤环境对蛋白质去折叠的影响。【结果】Dextran’70、Ficoll70和PEG20003种大分子拥挤试剂对溶菌酶活性几乎没有影响。无大分子拥挤试剂条件下，溶菌酶从天然态转变为去折叠态的过程中（盐酸胍浓度为0～6．0mol／L），当无还原剂2－巯基乙醇时，溶菌酶的去折叠过程符合“三态模型”，有1个中间态；当存在还原剂2-巯基乙醇时，该变性过程则符合“二态模型”，不存在任何中间态，溶菌酶可直接从天然态转变为去折叠态。溶菌酶在大分子拥挤体系中变性时，当不存在还原剂2巯基乙醇，变性过程符合“四态模型”，存在2个中间态；而当存在还原剂2-巯基乙醇时，变性过程则符合“三态模型”，有1个中间态。【结论】：通过添加大分子拥挤试剂，增加了溶菌酶变性过程的中间态，改变了蛋白由天然态到完全去折叠态的变性过程。[Objective] The effect of different macromolecular crowding agents on the unfolding of lysozyme induced by guanidine hydrochloride was studied. [Method] Three nonspecific high molecular mass crowding agents, dextran 70,Ficoll 70 and poly（ethylene glycol） 2000,were used to mimic a crowded environment in vivo. Lysozyme was denatured from native state to unfolded state by GuHCI in this crowded solutions with or without 2-mercaptoethanol and studied by ＂phase diagram＂ method of fluorescence in order to investigate the effect of different macromolecular crowding agents on the unfolding of lysozyme. [Result] Three crowding agents, dextran 70,Ficoll 70 and poly（ethylene glycol） 2000,had no effect on the activity of lysozyme. In the range of GuHC1 concentration （0-- 6.0 mol/L）, lysozyme was denatured from native state to unfolded state. In absence of crowding agents and 2-mercaptoethanol, the denaturation procedure of lysozyme confromed with the three-state model and one folded intermediate was detected, and in the presence of 2-mercaptoethanol it conformed with the two-state model and no folded intermediate was detected. However,in crowded solutions it conformed wilh the four-state model （having two intermediates） and the three-state model （having one intermediate） respectively at the same conditions. [Conclusion] Experimental results showed that the unfolding process of lysozyme was changed obviously by the addition of macromolecular crowding agent and increased the folded intermediates.

关 键 词：大分子拥挤 溶菌酶 去折叠 荧光相图法 盐酸胍 

分 类 号：Q518.4[生物学—生物化学]