A versatile assay for gelatinases using succinylated gelatin.
A spectrophotometric assay using succinylated gelatin as substrate is described for measuring the catalytic activity of gelatinases. The assay is based on measurement of primary amines exposed as a result of hydrolysis of the substrate by gelatinases. Comparison of hydrolysis by matrix metalloproteinase (MMP) 1, 2, 3, 7, 9 indicated that succinylated gelatin was primarily digested by MMP-2 and -9. The assay is rapid (<60 min), specific, suitable for measuring gelatinolytic activity of enzymes and high volume screening of MMP-2 and -9 inhibitors. Sensitivity of the assay is comparable to that of gelatin zymography, under similar experimental conditions. Thus, the assay combines ease and rapidity of assays based on synthetic peptide substrates with specificity of the gelatin zymography technique.
Baragi
V M
VM
Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI 48105, USA. vijaykumar.baragi@wl.com
Shaw
B J
BJ
Renkiewicz
R R
RR
Kuipers
P J
PJ
Welgus
H G
HG
Mathrubutham
M
M
Cohen
J R
JR
Rao
S K
SK
D005780:Gelatin / Q000378:metabolism*; D018093:Gelatinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D013053:Spectrophotometry; D013379:Substrate Specificity; D013386:Succinates / Q000378:metabolism*; D019802:Succinic Acid / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D013386:Succinates; D013876:Thiophenes; C060085:succinylated gelatin; D010649:Phenylalanine; D005780:Gelatin; D019802:Succinic Acid; C080985:batimastat; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9