Assay of diverse protease activities on the basis of a small synthetic substrate

The available methods for assaying of protease activity of unknown or parti ally defined specificity utilize long peptides, mostly denatured proteins, comprehending at least all coded amino acids in cleavable positions. In con trast, here we report on an alternative approach which utilizes a small syn thetic ester substrate containing only one amino acid. The approach equally detects endo- and carboxypeptidases with a wide variety of specificities i ncluding enzymes specific for basic, acidic, aromatic, and nonaromatic hydr ophobic amino acid moieties, The results further revealed that most proteas es could be detected in activities considerably less than 1 U, In contrast to the methods used thus far, the enzymatic hydrolysis of the small substra te can be easily and rapidly assayed by a shift in absorption resulting in a change in color of the assay mixture at visible wavelengths. Thus, no add itional instrumental efforts are generally required. On the basis of these characteristics, the approach presented here could be particularly valuable for monitoring the purification of enzymes or as a rapid check for proteas e activity. (C) 2000 Academic Press.