Functional Screening of Cytochrome P450 Activity and Uncoupling by Capillary Electrophoresis

Cytochrome P450s (CYPs) are functionally diverse monooxygenases responsible for oxidation of endogenous and xenobiotic compounds. The function of nonmammalian CYPs are largely unknown and tools for characterization limited. CYPs critical for xenobiotic metabolism are prone to catalytic cycle uncoupling resulting in reactive oxygen species (ROS) generation that is highly dependent on the specific CYP isoform and substrate interaction. This study describes the rapid assessment of the activity and coupling efficiency of CYPs using capillary electrophoresis with UV detection. The coupling efficiency of five zebrafish (Danio rerio) CYP1 isoforms with a series of fluorogenic substrate probes was determined by the rate of NADP⁺ formation and compared with fluorescent product turnover rates. In most cases, NADP⁺ formation significantly overestimated CYP1 catalytic activity for substrate O-dealkylation suggesting uncoupling. ROS production was confirmed by elevated hydrogen peroxide generation in poorly coupled reactions. Reactions with β-estradiol confirmed that CYP1A, 1C1, and 1C2 have greater catalytic activity and coupling efficiency; CYP1B1 and 1D1 had coupling efficiencies under 4%. This work highlights the wide disparity in uncoupling induced by unproductive substrate binding among different CYP isoforms.