Determination of p-Nitrophenol Hydroxylase Activity of Rat Liver Microsomes by High-Pressure Liquid Chromatography

✍ Scribed by R.J. Duescher; A.A. Elfarra

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 294 KB
• Volume: 212
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1335

✦ Synopsis

p-Nitrophenol hydroxylation to p-nitrocatechol is a useful metabolic marker for the presence of functional cytochrome (P 4502 E 1) 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 (\boldsymbol{p})-nitrocatechol determination, is described. The method involves monitoring the presence of p-nitrocatechol in the trifluoroacetic acid-quenched reaction mixtures at (345 \mathrm{~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 \mathrm{~min})) and protein concentrations (0.03-0.48 mg/incubation), and exhibited typical Michaelis-Menton kinetics (\left(K_{m}=197 \mu \mathrm{M}, V_{\max }=2.8 \mathrm{nmol} /\right.) mg protein/min). 1993 Academic Press, Inc.