High-throughput fluorescence assay of cytochrome P450 3A4

Nat Protoc. 2009;4(9):1258-61. doi: 10.1038/nprot.2009.123. Epub 2009 Aug 6.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Coumarins / chemistry*
  • Cytochrome P-450 CYP3A / metabolism*
  • Humans
  • Inhibitory Concentration 50
  • Ketoconazole / chemistry
  • Oxidation-Reduction
  • Software
  • Spectrometry, Fluorescence / methods*


  • Coumarins
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human
  • Ketoconazole