A quantitative technique for determining proteases and their substrate specificities and pH optima in crude enzyme extracts

Abstract

A zymography technique based on native polyacrylamide gel electrophoresis (PAGE) has been devised, which enables the substrate specificities, content and pH profiles of proteolytic enzymes to be determined in an unfractionated tissue extract. Enzymes were visualized by exogenous application of small molecule substrates that fluoresce when hydrolyzed. The linearity of response, treatment of background fluorescence, and effects of diffusion of substrate and enzyme were taken into account. Based on these studies, successive application of different substrates on the same gel has enabled the presence and specificity of individual enzymes to be determined. Differences in the concentrations and profiles of enzymes, resulting from environmental factors or ontogeny of the organism, can be assessed from crude extracts on a single gel. The technique was applied to aminopeptidases and peptidases in crude Phaseolus vulgaris leaf extracts. One enzyme active against Ala–AMC (7-amino-4-methylcoumarin), one enzyme active against Z-Arg–AMC, several enzymes active against Leu–AMC, and (for the first time in plants) several enzymes active against Phe–AMC were identified. The technique is very sensitive, and microgram quantities of total protein led to picomoles of liberated AMC, with a linear response over a 32-fold range of concentration. The experimental procedure, including electrophoresis, is rapid, taking approximately 1 h.