SPECTROPHOTOMETRIC ASSAY FOR ACETANILIDE 4-HYDROXYLASE, AN ESTIMATE OF CYP1A2 ENZYME ACTIVITY

Acetanilide 4-hydroxylase provides the most reliable enzyme activity estimate for cytochrome P450 1A2. Current methods used to assess CYP1A2 activity employ high performance liquid chromatography (HPLC) or radiolabeled substrates. Drawbacks of the HPLC method include the need for expensive equipment and the slow throughput, while radioisotope use is associated with health hazards as well as extensive record-keeping. We describe a new spectrophotometric assay for acetanilide 4-hydroxylase using acid cleavage of the N-acetyl moiety from the reaction product, followed by derivatization with a phenol reagent. The assay detects 0.1 nmol 4-hydroxyacetanilide.