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High-throughput fluorescence assay of cytochrome P450 3A4

Nature Protocols volume 4, pages 12581261 (2009) | Download Citation



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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Cytochrome P450 research in this laboratory is supported by United States Public Health Service grant R37 CA090426.

Author information


  1. Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee USA.

    • Qian Cheng
    • , Christal D Sohl
    •  & F Peter Guengerich


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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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Correspondence to F Peter Guengerich.

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