A rapid and sensitive microassay for bacterial collagenase and other proteolytic activities on collagenous substrates

Many of the common substrate digestion assays have the disadvantage of being tedious and expensive, thus aggravating serial determinations of collagenase activity. In the assay presented here, the proteolytic reaction proceeds on a sheet of chromatographic paper bearing spots of dried gelatin or collagen solutions. After application of the enzyme sample, the sheets are incubated in a water-saturated atmosphere for 45 min and the reaction products are visualized by spraying with chromatographic, ninhydrin-containing peptide reagents. By its specific yellow staining, the activity of bacterial collagenase can be discriminated from the gray, brown, red, or violet spots which are produced by eight tested noncollagenolytic proteases. The detection limit for collagenase activity reaches down to 0.002 U in a sample volume of 1-2 microliters dropped onto a spot of 25 microliters gelatin solution (3%), thus corresponding with the sensitivity of a conventional radioassay. The sensitivity for noncollagenolytic proteases is better than 2 ng per sample, herewith exceeding a common casein-agar diffusion test. The method is also evaluated for native collagen. Within 1.5 h, 100 samples can be assayed. An example for the application in a collagenase purification step (ion exchange chromatography) is also presented.