The development of an assay for determination of monooxygenase of human foetal livers based on rate of decrease in substrate concentration.

A simple monooxygenase assay based on the rate of decrease in the concentration of the substrate "supona' [2-chloro-1(2,4-dichlorophenyl)vinyl diethyl phosphate] (1) was investigated spectrophotometrically at E246nm in the presence of oxygen and NADPH without the enzyme. The assay was found reliable for incubations at 37 degrees C for periods up to 10 minutes. Incubations in excess of 10 minutes were unreliable due to an unexpected increase in E246nm. Mechanisms of hydrolysis (Scheme 1) and of reduction (Scheme 2) of the substrate were considered, and it was concluded that hydrolysis is the cause of the increase. Using 5 and 10 minute incubations, specific activity values for monooxygenase in 13 and 16 week old human foetuses were determined as 6.47 +/- 0.84 and 5.26 +/- 0.46, respectively; no monooxygenase could be detected in 24 week old foetuses.