A convenient fluorescent assay for vertebrate collagenases.
A versatile, convenient assay for vertebrate collagenases has been developed using the fluorescent peptide substrate dansyl-Pro-Gln-Gly-Ile-Ala-Gly-D-Arg. This sequence resembles that of collagen at the site of cleavage but includes modifications designed to eliminate nonspecific hydrolysis by contaminating peptidases. Both human skin fibroblast and bovine corneal cell collagenases cleave the substrate specifically at the Gly-Ile bond. Plasmin, thrombin, trypsin, alpha-chymotrypsin, carboxypeptidase B, and bacterial collagenase do not cleave the substrate. Elastase and angiotensin converting enzyme display 20- and 400-fold less activity than the vertebrate collagenases, respectively, and cleave the peptide at different positions. The assay is performed by incubating a 5- to 25-microliters aliquot of trypsin-activated sample with an equal volume of 2 mM substrate overnight at 33 degrees C and pH 7.5. Thin-layer chromatography then separates the fluorescent product from the substrate in less than 20 min and allows the detection of subnanogram levels of collagenase. The assay is applicable to the screening of large numbers of samples under different conditions of pH and ionic strength and is readily adaptable for use in a variety of collagenase-dependent systems, such as assays for collagenase activating and/or inducing factors.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003315:Cornea / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D003012:Microbial Collagenase / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D003012:Microbial Collagenase