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 pepti de substrates. The endo or exoproteolytic hydrolysis of simple peptide s can be followed spectrophotometrically by coupling the proteolytic e vent via enzyme-catalyzed reactions to a chromo genic redox dye. The c ouple can be used directly to follow the action of carboxy or amino pe ptidases on peptide substrates or can be coupled by use of carboxy or amino peptidases to follow the action of endoproteases on peptide subs trates that are blocked at the amino or carboxy terminus, respectively . Liberated amino acids are detected by use of amino acid oxidase, oxy gen, horseradish peroxidase, and the redox dye 2,2'-azino-bis-(3-ethyl benzthiazoline-6-sulfonic acid (epsilon(414 nm) = 36,000 M(-1) cm(-1)) (C) 1996 Academic Press, Inc.