Abstract
Cytochrome P450 mono-oxygenases (P450s) are the principal enzymes involved in the oxidative metabolism of drugs and other xenobiotics. In this protocol, we describe a fluorescence-based, high-throughput assay for measuring the activity of P450 3A4, one of the key enzymes involved in drug metabolism. The assay involves the oxidative debenzylation of a substituted coumarin, yielding an increase in fluorescence on reaction. The entire procedure can be accomplished in 1 h or less.
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Acknowledgements
Cytochrome P450 research in this laboratory is supported by United States Public Health Service grant R37 CA090426.
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Q.C. performed and optimized the assays. Q.C. wrote most of the paper, with the assistance of F.P.G. and C.D.S. C.D.S. checked the protocols and helped Q.C. with data processing.
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Cheng, Q., Sohl, C. & Guengerich, F. High-throughput fluorescence assay of cytochrome P450 3A4. Nat Protoc 4, 1258–1261 (2009). https://doi.org/10.1038/nprot.2009.123
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DOI: https://doi.org/10.1038/nprot.2009.123
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