A simple vertebrate collagenase assay using soluble radioactive collagen substrate.
A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.
D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D002642:Chick Embryo; D003094:Collagen / Q000302:isolation & purification / Q000378:metabolism*; D046508:Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D003012:Microbial Collagenase / Q000032:analysis*; D010446:Peptide Fragments / Q000302:isolation & purification; D011392:Proline / Q000378:metabolism; D011817:Rabbits; D012588:Scintillation Counting; D012995:Solubility; D013696:Temperature
D010446:Peptide Fragments; D003094:Collagen; D011392:Proline; D003012:Microbial Collagenase