Design of optimum refolding solution by combination of reagents classified by specific function
Design of optimum refolding solution by combination of reagents classified by specific function
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DOI：
10.1016/0922-338x(96)89159-8
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发表时间：
1996-01-01
期刊：
影响因子：
--
通讯作者：
Sakai, T
中科院分区：
文献类型：
--
作者：
Nohara, D;Matsubara, M;Sakai, T
氯化胍对蛋白质去折叠的影响被认为是对疏水相互作用或氢键等相互作用以及构建蛋白质高级结构的离子相互作用的影响的组合。假设尿素和氯化锂分别控制疏水相互作用或氢键和离子相互作用，而不是胍氯化物用于变性还原溶菌酶的复性。使用尿素和LiCl浓度的各种组合制备重折叠介质。巯基的再氧化在谷胱甘肽氧化还原体系中进行。在溶菌酶浓度为10 μ M时，由于不可逆的错误折叠和/或重折叠过程中的聚集，在50 mM Tris缓冲液中回收的酶活性通常小于20%。在高浓度的尿素和氯化锂的存在下，恢复的活性显着增加。完全复性可以在很宽的尿素和LiCl浓度范围内实现，最佳浓度为3 M尿素和1.5 M LiCl，而仅用盐酸胍则不能实现。通过对尿素和LiCl组合制备的复性介质的优化设计，证实了氯化胍的多功能性可以近似地分离为尿素和LiCl两个单功能性的概念的有效性。绘制了溶菌酶复性的特征图。该图有助于复性介质的优化设计和复性机理的研究。
The effect of guanidinium chloride on protein unfolding was regarded as a combination of the effects on interactions such hydrophobic interaction or hydrogen bond, and ionic interaction that construct a higher-order structure of the protein. Urea and LiCl instead of guanidinium chloride were used for refolding of denatured reduced lysozyme by assuming that urea and LiCl individually control hydrophobic interaction or hydrogen bond and ionic interaction, respectively. The refolding media were prepared using various combinations of urea and LiCl concentrations. Reoxidation of thiol groups was carried out in a glutathione redox system. The recovered enzyme activity at a lysozyme concentration of 10 mu M was usually less than 20% in 50 mM Tris buffer due to irreversible misfolding and/or aggregation during refolding. In the presence of urea and LiCl at high concentrations the recovered activity increased markedly. Complete renaturation could be achieved in a broad range of concentrations of urea and LiCl around the optimum of 3 M urea and 1.5 M LiCl, while it could not be achieved solely with guanidinium chloride. The availability of the concept that multifunction of guanidinium chloride can be separated approximately into two monofunctions of urea and LiCl was confirmed by the optimum design of refolding media prepared by combinations of urea and LiCl. A characteristic diagram for the refolding of lysozyme was depicted. This diagram will aid the optimum design of refolding media and study of the refolding mechanism.