DIRECT FLUOROMETRIC ASSAY OF BENZO[A]PYRENE HYDROXYLASE

The fluorescence of benzo[a]pyrene is greatly enhanced upon binding to microsomes. The excitation and emission maxima are 387 and 407 nm, respectively. At this wavelength setting, the fluorescence of the benzo[a]pyrene metabolites is very low. Therefore, the oxidative metabolism of benzo[a]pyrene can be assayed by following the NADPH-dependent deerease in fluorescence with a recording spectrofluorometer. Under optimal conditions, the decrease in fluorescence follows first-order kinetics and the rate of the reaction is proportional to the amount of microsomes in the assay system. The rate of metabolism of some benzo[a]pyrene derivatives can also be assayed by a similar method. In comparison with the commonly used benzo[a]pyrene hydroxylase assay, this direct fluorometric assay employs lower substrate concentrations (0.1-2.0 μm benzo[a]pyrene) and reveals a larger difference between the activities of control and 3-methylcholanthrene-induced microsomes. The method is also easier and allows the entire course of the reaction to be measured. © 1978.