Protein redox potential measurements based on kinetic analysis with mediated continuous-flow column electrolytic spectroelectrochemical technique. Application to TTQ-containing methylamine dehydrogenase.
Protein redox potential measurements based on kinetic analysis with mediated continuous-flow column electrolytic spectroelectrochemical technique. Application to TTQ-containing methylamine dehydrogenase.
复制标题
基于介导的连续流柱电解光谱电化学技术的动力学分析的蛋白质氧化还原电位测量。
DOI：
10.1021/ac990944a
复制
发表时间：
2000
影响因子：
7.4
通讯作者：
T. Ikeda
中科院分区：
文献类型：
--
作者：
A. Sato;M. Torimura;K. Takagi;K. Kano;T. Ikeda
本文介绍了一种介导的连续流柱电解光谱电化学技术（CFCESET）对蛋白质氧化还原电位的动力学测定。在该方法中，介质的氧化还原状态完全由连续流柱电解调节，并且在柱的下游用光谱监测介质与柱中蛋白质样品之间的均相氧化还原反应。蛋白质/介质反应为准一级动力学，然后解析求解速率方程。动力学分析提供了蛋白质氧化还原电位和均匀速率常数。在动力学测量中，系统在柱内的平衡是不需要的，这允许使用更多种类的介质。该方法成功应用于以色氨酸色氨酸醌（TTQ）为假基的醌蛋白甲胺脱氢酶。动力学方面也有价值的热力学分析与介导CFCESET。动力学的半衰期可以用来优化系统以达到柱内的平衡状态，并可以保证系统处于平衡状态。
Kinetic determination of protein redox potentials with a mediated continuous-flow column electrolytic spectroelectrochemical technique (CFCESET) is described. In this method, the redox state of the mediator is completely regulated by the continuous-flow column electrolysis, and the homogeneous redox reaction between the mediator and a protein sample in the column is monitored spectroscopically at the downstream of the column. The protein/mediator reaction is in the pseudo-first-order kinetics, and then the rate equation is analytically solved. The kinetic analysis provides the protein redox potential as well as the homogeneous rate constant. In the kinetic measurements, equilibration of the system within the column is not required, which allows the use of increased kinds of mediators. This method was successfully applied to quinoprotein methylamine dehydrogenase containing tryptophan tryptophylquinone (TTQ) as a prosthetic group. The kinetic aspect is also valuable for the thermodynamic analysis with the mediated CFCESET. The half-life time of the kinetics can be utilized to optimize the system for the attainment of the equilibrated state within the column and can provide the assurance that the system is in equilibrium.