New method for determining the extent of proline hydroxylation by measuring changes in the ratio of [4-3H]:[14C]proline in collagenase digests

A sensitive colorimetric assay for proteases and certain polysaccharidases is based on the digestion of proteoglycan from bovine nasal cartilage. This high-molecular-weight substrate is trapped in the interstices of a polyacrylamide gel. The gel is then dispersed as small particles. Enzymes can diffuse into these particles and digestion products can diffuse out. Following digestion, the particles are centrifuged off and the digestion products in the supernatant are quantitated by reaction with the metachromatic dye 1,9-dimethylmethylene blue or by assay of their uronic acid content with carbazole reagent. Proteases from each of the four major classes can be quantitated at levels of 1–200 ng. The method is particularly suitable for the study of cartilage proteases that degrade matrix proteoglycans.