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

The available methods for assaying of protease activity of unknown or partially defined specificity utilize long peptides, mostly denatured proteins, comprehending at least all coded amino acids in cleavable positions. In contrast, here we report on an alternative approach which utilizes a small synthetic ester substrate containing only one amino acid. The approach equally detects endo- and carboxypeptidases with a wide variety of specificities including enzymes specific for basic, acidic, aromatic, and nonaromatic hydrophobic amino acid moieties. The results further revealed that most proteases could be detected in activities considerably less than 1 U. In contrast to the methods used thus far, the enzymatic hydrolysis of the small substrate 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 additional 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 protease activity.