An assay of flavin-containing monooxygenase activity with benzydamine N-oxidation

An assay system of flavin-containing monooxygenase was developed by fluorometric determination of benzydamine (BZY) N-oxidation with HPLC. The apparent Km value for the formation of BZY N-oxide from BZY by rat liver microsomes was similar to that by purified FMO. The Km and Vmax values for the formation of N-desmethylbenzydamine (Nor-BZY) by rat liver microsomes were about 50 times greater and 2000 times less, respectively, than those of BZY N-oxide. Nor-BZY was not formed upon incubation with purified enzyme. BZY N-oxidation activity was completely inhibited both in the absence of NADPH and by heat inactivation. The reaction was inhibited in the presence of 0.5 mM thiourea, but 2 mM SKF-525A did not affect BZY N-oxidation. Moreover, rabbit antibody raised against the rat enzyme inhibited BZY N-oxidation. These results are in accord with a simple, rapid, and sensitive assay for the enzyme.