p-Aminothiophenol modification on gold surface improves stability for electrochemically driven cytochrome P450 microsome activity

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-NH2 modified 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-NH2 modified surface. We also measured the tolbutamide metabolism reactions by the allelic variants of the CYP2C9 microsome on SPh-NH2 modified electrode. The estimated Km and kcat values were comparable to those obtained from the solution system. Therefore, SPh-NH2 modification gave an exquisite surface for electrochemically analyzing the recombinant CYP microsome reaction, indicating the usefulness for rapid assay of the enzyme.