A KINETIC ASSAY FOR P-NITROPHENOL HYDROXYLASE IN RAT-LIVER MICROSOMES

A real-time kinetic method for measuring the activity of microsomal p- nitrophenol hydroxylase, in which the rate is measured directly by uv- visible spectrophotometry, is described. The method is based on the fa ct that the reaction product, 4-nitrocatechol, absorbs at 480 nm and l onger wavelengths while the absorbance of the reactant, p-nitrophenol, decreases to baseline at these wavelengths. The conditions of the ass ay are similar to the incubation conditions of the Reinke and Moyer me thod. The advantages of the new method include simplicity, direct meas urement of the rate rather than use of a timed assay, and elimination of experimental steps such as changing pH and centrifugation before sp ectrophotometric reading. The new method produces results that are com parable to and may be more reproducible than those of the Reinke and M oyer method. (C) 1994 Academic Press, Inc.