Anal Chem. 2012 Jan 17;84(2):862-6. doi: 10.1021/ac202787n. Epub 2011 Dec 21.
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.
细胞色素 P450 酶（CYPs）是功能多样的单加氧酶，负责氧化内源性和外源性化合物。非哺乳动物 CYP 的功能在很大程度上是未知的，并且表征工具也很有限。对异生物质代谢至关重要的 CYP 容易发生催化循环解偶联，导致活性氧（ROS）的产生，这高度依赖于特定的 CYP 同工型和底物相互作用。本研究描述了使用带有紫外检测的毛细管电泳快速评估 CYP 的活性和偶联效率。通过 NADP(+)形成的速率来确定 5 种斑马鱼（Danio rerio）CYP1 同工型与一系列荧光底物探针的偶联效率，并与荧光产物周转率进行比较。在大多数情况下，NADP(+)的形成显著高估了 CYP1 对底物 O-脱烷基化的催化活性，表明发生了解偶联。在偶联效率低的反应中，通过过氧化氢生成的增加证实了 ROS 的产生。与β-雌二醇的反应证实 CYP1A、1C1 和 1C2 具有更高的催化活性和偶联效率；CYP1B1 和 1D1 的偶联效率低于 4%。这项工作强调了不同 CYP 同工型之间由于非生产性底物结合而导致的解偶联的广泛差异。
