| 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（Dextran 70）、菲可70（Ficoll 70）、聚乙二醇2000（PEG 2000）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-巯基乙醇存在时，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、Ficoll 70和PEG 2000 3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0～6.0 mol/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。 |
| 关键词: 大分子拥挤 溶菌酶 去折叠 荧光相图法 盐酸胍 |
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| 基金项目:西北工业大学科技创新基金项目(2008KJ02038) |
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| Studies on unfolding of lysozyme Induced by guanidine hydrochloride at different macromolecular crowding |
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| Abstract: |
| 【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 GuHCl 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 GuHCl 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 with 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. |
| Key words: macromolecular crowding lysozyme unfolding “phase diagram” method of fluorescence guanidine hydrochloride |
