p-Aminothiophenol modification on gold surface improves stability for electrochemically driven cytochrome P450 microsome activity.
p-Aminothiophenol modification on gold surface improves stability for electrochemically driven cytochrome P450 microsome activity.
复制标题
金表面上的对氨基苯硫酚修饰提高了电化学驱动的细胞色素 P450 微粒体活性的稳定性。
DOI：
10.1016/j.electacta.2013.10.170
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Y.
中科院分区：
文献类型：
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作者：
Mie;Y.;Tateyama;E. and Komatsu;Y.
人微粒体的电化学驱动的细胞色素P450（CYP 450）反应有望提高药物代谢测定的效率，并证明对药物研究有用。我们以前报道过，用苯硫酚（SPh）修饰的纳米结构的金电极，使电催化氧化微粒体反应。然而，重复测量导致活性显著降低。在本研究中，我们研究了固定和电化学测量的重组CYP 2C 9微粒体的金电极修饰4-氨基硫酚（SPh-NH 2），4-羟基硫酚，或4-羧基硫酚作为促进剂。在SPh-NH_2修饰的表面上观察到一对明显的伏安峰，归属于电极与微粒体之间的电子转移。在氧气和已知的底物甲苯磺丁脲存在下，观察到了由氧化反应产生的电催化电流。有趣的是，与SPh改性表面的那些相比，响应是稳定的并且被维持。研究表明，这种稳定的活性与SPh-NH_2改性表面产生的活性氧减少有关。我们还利用CYP 2C 9微粒体的等位基因变异体在SPh-NH_2修饰电极上测定了甲苯磺丁脲的代谢反应。估算的Kmandkcat值与从溶液体系中获得的值相当。因此，SPh-NH 2修饰提供了一个精致的表面，用于电化学分析的重组体的微粒体反应，表明该酶的快速测定的有用性。
The electrochemically driven cytochrome P450 (CYP) reaction of a human microsome is expected to increase efficiency of drug metabolism assays and as well as prove useful for drug research. We previously reported that a nanostructured gold electrode modified with thiophenol (SPh) enabled the electrocatalytic CYP microsome reaction. However, repeated measurements resulted in a significant decrease in the activity. In the present study, we examined the immobilization and electrochemical measurements of the recombinant CYP2C9 microsome on gold electrodes modified with 4-aminothiophenol (SPh–NH2), 4-hydroxythiophenol, or 4-carboxythiophenol as the promoter. A clear pair of peaks in the voltammogram, assigned to the electron transfer between the electrode and CYP microsome, was observed at the SPh–NH2modified surface. In the presence of oxygen and the well-known substrate, tolbutamide, the electrocatalytic current by the CYP reaction was observed. Interestingly, the responses were stable and were maintained compared with those at the SPh modified surface. It was suggested that this stable activity was related to less reactive oxygen species being produced at the SPh–NH2modified surface. We also measured the tolbutamide metabolism reactions by the allelic variants of the CYP2C9 microsome on SPh–NH2modified electrode. The estimatedKmandkcatvalues were comparable to those obtained from the solution system. Therefore, SPh–NH2modification gave an exquisite surface for electrochemically analyzing the recombinant CYP microsome reaction, indicating the usefulness for rapid assay of the enzyme.