A Rapid Micro-Assay Method for Gelatinolytic Activity Using Tritium-Labeled Heat-Denatured Polymeric Collagen as a Substrate and Its Application to the Detection of Enzymes Involved in Collagen Metabolism

Abstract

A rapid micro-assay method for gelatinolytic activity has been developed using ³H-labeled heat-denatured polymeric collagen (gelatin) as a substrate to investigate enzymes involved in the post-collagenase catabolism of collagen. The method is based on the incubation of gelatin with enzyme followed by determination of the enzyme digestion products soluble in 67% dioxane.
It is sensitive enough to detect microgram levels of gelatin fragments, and can be employed over wide ranges of pH and ionic strength. By applying the method to an embryonic chick skin culture system, three gelatinolytic enzyme fractions which showed high, limited and no caseinolytic activities were demonstrated to be separable by gel chromatography.