Abstract
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 am. 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 ( K m = 197 μM, V max = 2.8 nmol/mg protein/min).
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