material with a specific gravity of 1 53 and greater containing proteoglycan subunits was retained and recentrifuged as above again the gradient material with a specific gravity of 1 53 and greater was saved the isolated proteoglycan subunits were dialized exhaustively against deionized water containing 0 02 sodium azide for 24 to 36 hr and then lyophilized the proteoglycan polyacrylamide beads were prepared as described in nagase et al supra the bead assay of enzyme activity was modified as follows the assay solutions in the tubes contained 100 u03bcl enzyme preparation and 100 u03bcl buffer tris hcl ph 7 4 or inhibitor in the buffer incubation with the beads was carried out at 37 u00b0 c for 6 or 20 hr the degraded proteoglycan released from the polyacrylamide beads was determined assaying 100 u03bcl spectrophotometrically at 535 nm with dimethylmethylene blue dye as described in farndale et al conn tissue res 9 247 248 1982 chondroitin sulfate was used as a standard one unit of proteoglycan degrading activity is defined as the amount of enzyme required to release 1 u03bcg chondroitin sulfate ml hr at 37 u00b0 c chondrocyte collagenase inhibition acid soluble collagen was extracted from rat tail tendon by the method of birkedal hansen et al anal biochem 115 18 26 1981 rats were killed using co 2 asphyxiation tails were removed and stripped of skin the tendons were removed and washed several times in cold distilled water the salt soluble collagen was removed by extracting the tendons twice 24 hr extraction in 1m nacl 50 mm tris and 5 mm cacl 2 ph 7 4 at 4 u00b0 c the residue was washed.