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

Anal Biochem. 1988 Aug 1;172(2):304-10. doi: 10.1016/0003-2697(88)90449-6.


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.

MeSH terms

  • Animals
  • Autoanalysis
  • Coumarins / metabolism
  • Cytochrome P-450 Enzyme System / analysis*
  • Fluorometry*
  • Male
  • Methylcholanthrene / pharmacokinetics
  • Microsomes, Liver / enzymology
  • Mixed Function Oxygenases / analysis*
  • Nitriles / metabolism
  • Phenobarbital / pharmacokinetics
  • Rats
  • Rats, Inbred F344


  • Coumarins
  • Nitriles
  • 3-cyano-7-ethoxycoumarin
  • 3-cyano-7-hydroxycoumarin
  • Methylcholanthrene
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Phenobarbital