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.
Kawaji
A
A
Department of Toxicology, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
Ohara
K
K
Takabatake
E
E
D000818:Animals; D001591:Benzydamine / Q000378:metabolism*; D005470:Fluorometry; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity
D001591:Benzydamine; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)