A continuous fluorometric assay for cytochrome P-450-dependent mixed function oxidases using 3-cyano-7-ethoxycoumarin.

A direct fluorometric procedure for the continuous determination of cytochrome P-450-dependent mixed function oxidases, using 3-cyano-7-ethoxycoumarin substrate, is described. The reaction product, 3-cyano-7-hydroxycoumarin, is fluorescent at neutral pH values (excitation and emission wavelength maxima: 408 and 450 nm, respectively). Using hepatic microsomal preparations from control rats, the enzyme(s) had an apparent Km of 16 microM. Vmax values (0.5 nmol/min/mg protein) were induced 6- and 21-fold by pretreatment of rats with phenobarbitone and about 50- to 100-fold more sensitive than the ethoxyresorufin deethylase assay. Reaction rates using 3-cyano-7-pentoxycoumarin as substrate were generally much lower than with the ethoxy analog. 3-Cyano-7-ethoxycoumarin can also be used as a substrate to measure mixed function oxidases in isolated hepatocytes. However, 3-cyano-7-hydroxycoumarin shows a time- and concentration-dependent loss of fluorescence when incubated with such cells. This causes an approximately 5% underestimate of the true reaction rates.