A simple and very sensitive method for the separation of 4-hydroxyacetanilide, 3-hydroxyacetanilide, 2-hydroxyacetanilide, and acetanilide was developed with the use of high-pressure liquid chromatography. Each of these phenolic derivatives can be separated completely from acetanilide and from one another. A simple assay for "acetanilide 4-hydroxylase activity" is thus described. The limit of sensitivity for cytochrome P-450mediated acetanilide 4-hydroxylase activity is estimated to be 1 .O pmof/min/mg microsomal protein, thereby allowing this assay to be useful in detecting monooxygenase activity in "low level" nonhepatic tissues. Hepatic acetanilide 4-hydroxylase activity is induced about fourfold in C57BL/6N mice by 3-methylcholanthrene.
Although acetanilide 2hydroxylase activity is about seven times lower than the 4-hydroxylase activity, the 2-hydroxylase is also induced about three-or fourfold in C57BL/6N mice by 3-methylcholanthrene. The "Zhydroxylase activity" cannot, however, be strictly quantitated under the conditions described herein. The K, values of both the 3-methylcholanthrene-induced and control 4-hydroxylase activity are about 0.55 mM; V,,, values for 3-methylcholanthrenetreated and control mice, respectively, are 4.9 2 1.1 and 1.1 + 0.31 nmoUmin/mg microsomal protein. The 4-hydroxylase in the liver of both 3-methylcholanthrene-treated and control mice appears to represent two or more catalytic activities, i.e., two or more forms of P-450 having widely differing affinities for the substrate acetanilide.