Monooxygenase assays

Abstract

Cytochrome P-450 assay methods and kits for the methods are provided employing a cytochrome P-450 enzyme, substrates characterized by having an oxidizable methylene group oxidized to an aldehyde and a fluorescent hydrazine. A fluorescent hydrazine is added to the reaction mixture and the resulting hydrazone analyzed by capillary electrophoresis. The method finds use in evaluating compounds for enzyme modulating activity.

What is claimed is:  
     
         1 . A kit comprising a cytochrome P-450 enzyme, at least one P-450 enzyme substrate comprising an ether group bonded to a methylene, which is oxidized to an aldehyde by said P-450 enzyme, and a fluorescent hydrazine.  
     
     
         2 . A kit according to  claim 1 , wherein said P-450 enzyme is the 3A4 isoform and said fluorescent hydrazine is fluorescein hydrazine.  
     
     
         3 . A kit according to  claim 1 , wherein said P-450 enzyme substrate is at least one of 7-benzyloxyresorufin or 7-benzyloxyquinoline.  
     
     
         4 . A kit according to  claim 1  wherein said fluorescein hydrazine is N-(α-hydrazylacetyl) 5-aminofluorescein.