Electrochemical determination of (S)-7-hydroxywarfarin for analysis of CYP2C9 catalytic activity

Abstract

An electrochemical method for the quantitative determination of (S)-7-hydroxywarfarin as a metabolite of the vitamin K antagonist warfarin has been developed to analyze the catalytic activity of cytochrome P450 2C9 (CYP2C9). The electrochemical properties of (S)-7-hydroxywarfarin were investigated using screen-printed carbon electrodes. We have shown by cyclic voltammetry that irreversible (S)-7-hydroxywarfarin electrochemical oxidation, in contrast to (S)-warfarin, can be registered by an oxidation peak at approximately 0.6 V (vs. Ag pseudo-reference electrode). A linear dependence of the oxidation peak amplitude in the range of 0.57–0.61 V on (S)-7-hydroxywarfarin concentration (in the range of 0.1–1 μM) was shown by the square-wave voltammetry method. The limit of (S)-7-hydroxywarfarin detection and the sensitivity were calculated as 0.091 μM and 0.0075 A/M, respectively. Using an electrochemical system based on recombinant human CYP2C9 immobilized on screen-printed carbon electrodes modified by didodecyldimethylammonium bromide, we have shown the possibility of (S)-7-hydroxywarfarin electrochemical quantification without separation of the enzymatic system component. The (S)-7-hydroxywarfarin quantitative electrochemical determination was used to calculate the kinetic parameters of CYP2C9 immobilized on electrode toward (S)-warfarin: the values of the maximal reaction rate (Vmax) and the Michaelis constant (KM) were calculated as 0.1 ± 0.002 min−1 and 3.03 ± 0.38 μM, respectively. The usage of an enzymatic CYP2C9-containing electrode as a catalyst and an indicator electrode as a sensor for the formation of metabolite allows to effectively register the kinetic parameters of the process. Such a two-electrode system can be applied to assess drug-drug interactions.