NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) is a ~78 kDa membrane-bound flavoenzyme that catalyzes the transfer of electrons from NADPH to members of the cytochrome P450 monooxidase (CYP) enzyme family in the endoplasmic reticulum. CPR contains two tightly bound flavin cofactors, FAD and FMN, which participate in the sequential transfer of electrons from NADPHADMNYP, oxidizing NADPH to NADP+ and reducing the CYP heme moiety to the substrate- and oxygen-binding ferrous state. As CPR is required for the function of all CYP isozymes, it plays a critical role in the metabolism of drugs, organic pollutants and other xenobiotic compounds, in addition to its role in biosynthesis of certain vitamins and steroid hormones. Assay Genie's cytochrome P450 reductase activity assay kit couples oxidation of NADPH by CPR to reduction of a nearly colorless probe into a brightly colored product with an absorbance peak at 460 nm, with the rate of color generation being directly proportional to CPR activity. The NADPH utilized by CPR is generated in situ from Beta-NADP+ via oxidation of glucose-6-phosphate (G6P) to 6-phospho-D-glucono-1,5-lactone by glucose-6-phosphatase dehydrogenase (G6PDH). The kit can be used to determine CPR activity in a variety of samples, with a detection limit of ~0.2 mU of CPR activity per reaction. For assessment of CPR activity in crude biological samples that may have extraneous reductases capable of reducing the substrate, an inhibitor of NADPH-dependent flavoproteins is included. In this case, the specific CPR activity may be calculated by running parallel reactions in the presence and absence of inhibitor and subtracting any residual activity detected with the inhibitor present. The kit contains sufficient reagents for performing 100 reactions in a 96-well plate format.

Figure: (a) G6P Standard curve. One mole G6P corresponds to one mole of β-NADP + reduced to NADPH, which subsequently generates one mole of reduced substrate. (b) Reaction kinetics of recombinant human CPR (positive control) and rat microsomal CPR (with and without inhibitor). (c) Relative CPR activity detected in rat liver microsomes (RLM, 25 µg total protein) and HepG2 cell lysate (40 µg total protein). Assays were performed according to the kit protocol.