Determination of p-nitrophenol hydroxylase activity of rat liver microsomes by high-pressure liquid chromatography.
p-Nitrophenol hydroxylation to p-nitrocatechol is a useful metabolic marker for the presence of functional cytochrome P450 2E1 in mammalian cell microsomes, but the assay is limited by the sensitivity of the spectrophotometric method used to monitor p-nitrocatechol formation. In this paper, a reverse-phase high-pressure liquid chromatography method, which is nearly 20 times more sensitive than the spectrophotometric method and more specific for p-nitrocatechol determination, is described. The method involves monitoring the presence of p-nitrocatechol in the trifluoroacetic acid-quenched reaction mixtures at 345 nm. The utility of the method was demonstrated with rat liver microsomes, where p-nitrocatechol formation was found to be NADPH dependent, was linear with incubation times (2.5 to 30.0 min) and protein concentrations (0.03-0.48 mg/incubation), and exhibited typical Michaelis-Menton kinetics (Km = 197 microM, Vmax = 2.8 nmol/mg protein/min).
Duescher
R J
RJ
Department of Comparative Biosciences, University of Wisconsin, Madison 53706.
Elfarra
A A
AA
D000818:Animals; D002396:Catechols / Q000032:analysis / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013053:Spectrophotometry
D002396:Catechols; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; C001833:4-nitrocatechol