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 chromogenic 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-ethyl-benzthiazoline-6-sulfonic acid (epsilon 414nm = 36,000 M-1 cm-1).
Cathers
B E
BE
Department of Medicinal Chemistry, University of Kansas, Lawrence 66045, USA.
Schloss
J V
JV
D001681:Biological Assay; D004796:Clinical Enzyme Tests; D010450:Endopeptidases / Q000032:analysis*
D010450:Endopeptidases