Enzyme-coupled assays for proteases

We have developed a general strategy for assaying proteases that does not require the use of fluorogenic, chromogenic, or radiolabeled peptide substrates. The endo or exoproteolytic hydrolysis of simple peptides can be followed spectrophotometrically by coupling the proteolytic event via enzyme-catalyzed reactions to a chromo genic redox dye. The couple can be used directly to follow the action of carboxy or amino peptidases on peptide substrates or can be coupled by use of carboxy or amino peptidases to follow the action of endoproteases on peptide substrates that are blocked at the amino or carboxy terminus, respectively. Liberated amino acids are detected by use of amino acid oxidase, oxygen, horseradish peroxidase, and the redox dye 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid (epsilon(414 nm) = 36,000 M(-1) cm(-1)) (C) 1996 Academic Press, Inc.