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Measurement of cytochrome P450 and NADPH–cytochrome P450 reductase

Abstract

Cytochrome P450 (P450) enzymes are important in the metabolism of steroids, vitamins, carcinogens, drugs and other compounds. Two of the commonly used assays in this field are the measurements of total P450 and NADPH–P450 reductase in biological preparations. A detailed protocol is presented for the measurement of P450 by its spectral properties, along with a protocol for measuring NADPH–P450 reductase by its NADPH–cytochrome c reduction activity. Each assay can be completed in 5–10 min. Detailed explanations for the rationale of particular sequences in the protocols are provided, along with potential confounding problems.

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Figure 1: Ferrous·CO versus ferrous difference spectrum used for the quantitation of a preparation of (E. coli) recombinant P450 2C9.
Figure 2: NADPH–cytochrome c reduction assay and sample calculations.

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Acknowledgements

The research on cytochrome P450 and NADPH–P450 reductase in this laboratory is supported by United States Public Health Service Grant no. R37 CA090426. We thank K. Trisler for her assistance in preparation of the paper.

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F.P.G. and M.V.M. optimized the assays. F.P.G. wrote most of the paper, with the assistance of M.V.M. and C.D.S. Q.C. checked the protocols and participated in some of the optimization steps.

Corresponding author

Correspondence to F Peter Guengerich.

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Guengerich, F., Martin, M., Sohl, C. et al. Measurement of cytochrome P450 and NADPH–cytochrome P450 reductase. Nat Protoc 4, 1245–1251 (2009). https://doi.org/10.1038/nprot.2009.121

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