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.
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MRC Toxicology Unit, Surrey, United Kingdom.
D000818:Animals; D001322:Autoanalysis; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D005470:Fluorometry; D008297:Male; D008748:Methylcholanthrene / Q000493:pharmacokinetics; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000032:analysis*; D009570:Nitriles / Q000378:metabolism; D010634:Phenobarbital / Q000493:pharmacokinetics; D051381:Rats; D011916:Rats, Inbred F344
D003374:Coumarins; D009570:Nitriles; C057714:3-cyano-7-ethoxycoumarin; C057715:3-cyano-7-hydroxycoumarin; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010634:Phenobarbital