Evaluation of the conformational equilibrium of reduced hen egg lysozyme by antibodies to the native form
Evaluation of the conformational equilibrium of reduced hen egg lysozyme by antibodies to the native form
复制标题
DOI：
10.1016/j.abb.2009.11.024
复制
发表时间：
2010-02-15
影响因子：
3.9
通讯作者：
Azuma, Takachika
中科院分区：
文献类型：
--
作者：
Oda, Masayuki;Kitai, Aki;Azuma, Takachika
为了评价还原型HEL的构象，利用识别天然鸡蛋溶菌酶（HEL）上不同构象表位的单克隆抗体HyC1和HyC2，利用表面等离子体共振技术评估了它们与天然HEL、s -1,2-二羧基乙基化HEL （DCE-HEL）、羧甲基化Cys6和Cys127 HEL （cm6127 -HEL）的相互作用动力学。虽然它们的结合仪式常数相差10(5)倍，但它们的解离速率常数基本相同，这表明DCE-HEL和cm6127 -HEL在结合抗体时具有与天然HEL相似的构象。我们认为，还原HEL的关联速率常数与原始HEL的比值代表了平衡状态下原始格式决定因素的比例。Cys6-Cys127二硫键的还原会将HyC1识别的表位转化为类似于DCE-HEL的非天然构象。我们发现单克隆抗体为评价蛋白质的结构和流体动力学变化提供了一种敏感的工具。(C) 2009爱思唯尔公司版权所有。
To evaluate the conformation of reduced HEL, the monoclonal antibodies HyC1 and HyC2, which recognize different conformational epitopes on native hen egg lysozyme (HEL), were used, and the kinetics of their interactions with native HEL, S-1,2-dicarboxyethylated HEL (DCE-HEL), and carboxymethylated Cys6 and Cys127 HEL (CM6,127-HEL) were assessed using surface plasmon resonance. Although their association rite constants differed 10(5)-fold, their dissociation rate constants were essentially the same, suggesting that DCE-HEL and CM6,127-HEL possess conformations similar to that of native HEL when they bind antibodies. We considered that the ratio of the association rate constant of reduced HEL to native HEL represents the proportion of the native format determinant in equilibrium. Reduction of the Cys6-Cys127 disulfide bond would transform the epitope recognized by HyC1 into a non-native conformation similar to that of DCE-HEL. We show that monoclonal antibodies provide a sensitive tool for evaluation of the structural and hydrodynamic changes of proteins. (C) 2009 Elsevier Inc. All rights reserved.