HIGHLY SENSITIVE AND RAPID FLUOROMETRIC ASSAY FOR UDP-GLUCURONYLTRANSFERASE USING 3-HYDROXYBENZO(A)PYRENE AS SUBSTRATE

A rapid and highly sensitive fluorometric assay for UDP-glucuronyltransferase (EC 2.4.1.17) has been devised using 3-hydroxybenzo(a)pyrene as substrate. The sensitivity of the procedure is based on (a) the high coefficient of fluorescence of the product, benzo(a)pyrene-3-glucuronide, and (b) the very low background which is obtained by an efficient differential extraction of substrate and product and their widely differing fluorescence characteristics in alkaline solution. As little as 5-10 pmol of product can be determined. The procedure involves essentially a single extraction and transfer step. The method may be applicable in measuring transferase activity in a few micrograms of tissue protein or of cultured cells as well as in the routine processing of large numbers of samples. Some of the properties of glucuronyltransferase activity directed toward 3-hydroxybenzo(a)pyrene are described such as kinetic constants and the sensitivity of the reaction to detergents and organic solvents. © 1979.