Enzymatic Analysis of cDNA-Expressed Human CYP1A1, CYP1A2, and CYP1B1 With 7-Ethoxyresorufin as Substrate
Cytochrome P450 (P450) enzymes belonging to the CYP1 family are highly inducible by polycyclic aromatic hydrocarbons and other environmental chemicals and play a major role in the metabolism of many foreign chemicals and endogenous substances. We describe a spectrofluorometric method for determining 7-ethoxyresorufin O-dealkylation catalyzed by CYP1A1, CYP1A2, and CYP1B1. The formation of the enzymatic product, resorufin, is monitored continuously by fluorescence using an excitation wavelength of 530 nm and an emission wavelength of 580 nm. This method can be applied to assay P450-catalyzed formation of resorufin from other alkoxyresorufins, such as 7-methoxyresorufin, 7-benzyloxyresorufin, and 7-pentoxyresorufin. It can also be used to assay 7-ethoxyresorufin O-dealkylation activity in isolated hepatocytes and cultured cells that express this P450 activity.
Key WordsCytochrome P450 CYP1A1 CYP1A2 CYP1B1 7-ethoxyresorufin 7-ethoxyresorufin O-dealkylation 7-methoxyresorufin, 7-benzyloxyresorufin 7-pentoxyresorufin
This work was supported in part by the Canadian Institutes of Health Research (grant MOP-42385) and by the National Institutes of Health (grant 5 P42 ES07381).
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