High-throughput fluorescence assay of cytochrome P450 3A4.
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.
Cheng
Qian
Q
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee USA.
Sohl
Christal D
CD
Guengerich
F Peter
FP
D003374:Coumarins / Q000737:chemistry*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D007654:Ketoconazole / Q000737:chemistry; D010084:Oxidation-Reduction; D012984:Software; D013050:Spectrometry, Fluorescence / Q000379:methods*
D003374:Coumarins; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D007654:Ketoconazole