A Competitive Fluorescence Assay to Measure the Reactivity of Compounds

Abstract

A sensitive competitive method was developed for assessing the reactivity of compounds toward glutathione and toward thiols in general. The method employs the reaction of the fluorogenic reagent fluorescein-5-maleimide (FM) with glutathione (GSH) to generate a large increase in fluorescence emission. When the reaction is measured in the presence of a compound that competes with FM toward GSH, the rate constant for fluorescent product formation increases while the total amount of product formed at the end of the reaction decreases. These changes in the presence of a series of competitor concentrations allow one to calculate the rate constant of the reaction of the competitor with GSH. At 23°C, pH 7.40 in PBS buffer the second-order rate constant of the FM–GSH reaction is k2 = (1.67 ± 0.32) × 104 M−1 · s−1. Two GSH-reactive compounds were evaluated: the second-order rate constant for the reaction of PNU-27707 with GSH under our experimental conditions is ki = 5660 ± 266 M−1 · s−1, while that of PNU-37802 is ki = 21,200 ± 1600 M−1 · s−1. The method is easily adaptable to a high-throughput screening format.