Yokoyama K, Kayanuma Y
School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan.
Anal Chem. 1998 Aug 15;70(16):3368-76. doi: 10.1021/ac9711807.
A cyclic voltammetric simulation that can be applied to an electrochemically mediated enzyme reaction involving any substrate and mediator concentrations was developed. Concentration polarization of the substrate in the vicinity of an electrode was considered as well as mediator concentration. Reversible and quasi-reversible electrochemical reactions with one electron followed by an enzyme reaction with two electrons were modeled. The differential equations for the mediator and substrate were solved using digital simulation techniques. The calculated cyclic voltammograms showed prepeaks when there was a low substrate concentration, high mediator concentration, and high enzyme activity. Digital simulation was applied to the determination of the kinetic constants of glucose oxidase (GOx). Cyclic voltammetry was carried out experimentally in a phosphate buffer solution containing GOx, ferrocene derivatives, and glucose. The ratio of the catalytic to the diffusion-controlled current, ik/id, was evaluated. The kcat, KMM, and KMS values were determined from the current values obtained by simulation and by experimentation at various enzyme, mediator, and substrate concentrations. The kcat, KMM, and KMS values for GOx, ferrocenedimethanol, and glucose were 340 s-1, 110 microM, and 30 mM, respectively.
开发了一种循环伏安模拟方法，该方法可应用于涉及任何底物和介质浓度的电化学介导酶反应。考虑了电极附近底物的浓度极化以及介质浓度。对单电子可逆和准可逆电化学反应以及随后的双电子酶反应进行了建模。使用数字模拟技术求解介质和底物的微分方程。当底物浓度低、介质浓度高且酶活性高时，计算得到的循环伏安图显示出预峰。数字模拟应用于葡萄糖氧化酶（GOx）动力学常数的测定。在含有GOx、二茂铁衍生物和葡萄糖的磷酸盐缓冲溶液中进行了循环伏安实验。评估了催化电流与扩散控制电流的比值ik/id。通过模拟和在各种酶、介质和底物浓度下的实验获得的电流值确定了kcat、KMM和KMS值。GOx、二茂铁二甲醇和葡萄糖的kcat、KMM和KMS值分别为340 s-1、110 microM和30 mM。
