A CONTINUOUS FLUOROMETRIC METHOD TO MONITOR THE ENZYMATIC-HYDROLYSIS OF MEDICINAL ESTERS

This paper describes a new spectrofluorimetric assay for continuously monitoring the enzymatic hydrolysis of medicinal esters. The procedure is based on the stoichiometric quantity of protons generated by the h ydrolysis of the substrate, which produces changes in the fluorescence of a pH-sensitive dye. The pH indicator, 2', 7'-bis(carboxyethyl)-5(6 )-carboxyfluorescein, was selected due to its favourable pK(a) for stu dies under physiological conditions. Moreover, the presence of a domai n in the spectra (< 442 nm) where fluorescence intensities are indepen dent of pH allows measurements of wavelength ratios that cancel artifa cts and lower sample-to-sample variability, The indicator did not affe ct the catalytic activity of purified hog liver carboxylesterase or hu man serum albumin. This assay is easy to perform and appears to be esp ecially useful for studying enzymatic reactions with half-lives of the order of minutes or hours.