A Coomassie brilliant blue G-250-based colorimetric assay for measuring activity of calpain and other proteases.
The activity of cytosolic calcium-dependent neutral protease (calpain) is commonly measured using casein as a substrate. A novel, modified caseinolysis assay is now developed. It involves incubation of calpain with substrate casein, followed by removal of an aliquot to which Coomassie brilliant blue G-250 dye reagent is added. The assay is based on the observation that the dye interacts only with protein but not the proteolytic products (small peptides and amino acids). Unlike the existing caseinolysis assay, this novel assay does not require separation of substrate from products and measurement is done in the visible range (595 nm). It is also more sensitive than existing colorimetric assays which use dye-conjugated protein substrates. The assay can also be used to measure the activity of other proteases such as trypsin and papain.
Buroker-Kilgore
M
M
Department of Pharmacology, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, Michigan 48105.
Wang
K K
KK
D000595:Amino Acid Sequence; D002154:Calpain / Q000032:analysis*; D002364:Caseins; D003124:Colorimetry / Q000379:methods* / Q000706:statistics & numerical data; D010450:Endopeptidases / Q000032:analysis*; D005069:Evaluation Studies as Topic; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D012394:Rosaniline Dyes; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D002364:Caseins; D009842:Oligopeptides; D012394:Rosaniline Dyes; D010450:Endopeptidases; D002154:Calpain; C004692:coomassie Brilliant Blue