Fluorometric continuous kinetic assay of alpha-chymotrypsin using new protease substrates possessing long-wave excitation and emission maxima

Abstract

A direct and continuous kinetic method for the fluorometric determination of alpha-chymotrypsin and trypsin is described, and 2-aminoacridone (2-AA) is introduced as a promising new fluorophore in analytical biochemistry. N-Succinyl- and N-glutaryl-phenylalanine as well as N-benzoylarginine were coupled to 2-AA via a peptide bond and the resulting fluorogenic substrates are shown to be cleaved by the two enzymes. Since the substrate and product of hydrolysis have quite different spectral properties, the increase in the long-wave fluorescence of 2-AA (measured at 570 nm under 450-nm excitation) is a parameter for the enzyme activity. Chymotrypsin (0.5 microgram/ml) and trypsin (0.1 microgram/ml) were detectable in a 3-min assay. The major advantages of the new substrates over existing ones are the analytical wavelengths which are distinctly outside the background fluorescence of most biological matter and the somewhat faster reaction rates which can reduce the time of analysis.