Kinetic assay of fluorescein mono-beta-D-galactoside hydrolysis by beta-galactosidase: a front-face measurement for strongly absorbing fluorogenic substrates

A novel enzymatic assay method was developed for fluorogenic substrates that have significant intrinsic absorbance and fluorescence under the assay conditions. Fluorescein mono-beta-D-galactoside (FMG) was chosen as the substrate for the fluorescence enzymatic assay because of the high fluorescence of its hydrolytic product (fluorescein) and suitability of being hydrolyzed by beta-galactosidase. The fluorescence-concentration relationships for fluorescein and for FMG in both the right-angle detection mode of a fluorometer and the front-face detection mode of a fluorescence plate reader were exactly established and used to determine the kinetics of the enzyme assay. The results show that only front-face detection in the fluorescence plate reader can overcome the fluorescence concentration quenching that inevitably results from high absorbance by the intrinsically absorbing substrate in the conventional fluorometer, which utilizes right-angle detection. Only with front-face detection was the fluorescent assay of FMG hydrolysis under conditions of high optical density possible. The enzymatic measurements on the fluorescence plate reader were particularly efficient for determination of the enzyme kinetics because of the high rate of data collection. In this assay system, Michaelis-Menten constant Km and enzymatic catalysis rate k2 of FMG were determined as 117.6 microM and 22.7 mumol-(min.mg)-1, respectively. The results and methods described in this paper can be generalized for any assay using a fluorogenic substrate whether or not it has a high background absorbance.