Methods in Molecular Biology 2006-01-01

Spectrofluorometric analysis of CYP2A6-catalyzed coumarin 7-hydroxylation.

David J Waxman, Thomas K H Chang

Index: Methods Mol. Biol. 320 , 91-6, (2006)

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Abstract

A spectrofluorometric method is described for the determination of CYP2A6-catalyzed coumarin 7-hydroxylation. Following acidification of the reaction mixture, the enzymatic product, 7-hydroxycoumarin, is recovered by a double-extraction procedure and assayed using an excitation wavelength of 370 nm and an emission wavelength of 450 nm. This assay is applicable to enzymatic studies of cDNA-expressed CYP2A6 and can be used to monitor coumarin 7-hydroxylation activity in microsomes prepared from liver and other tissues and in isolated hepatocytes and cultured cells that express this cytochrome P450 activity.