Degradative enzymes of cartilage. Effects of freeze-thawing of the tissue prior to extraction, and of protease inhibitors, on proteoglycans extracted with iso-osmotic neutral salt and 4 M guanidinium chloride.

The effects of freeze-thawing of the tissue, and of protease inhibitors, on proteoglycans extracted sequentially from pig laryngeal cartilage with 0.15 M sodium acetate and 4 M guanidinium chloride were examined. Freeze-thawing of the tissue prior to extraction resulted in an increase in the proportion of smaller-sized proteoglycans in the sodium acetate extracts and a decrease in the proportion of aggregated proteoglycans in 4 M guanidinium extracts. In addition, a slight decrease in the hydrodynamic size of purified disaggregated proteoglycans was noted after freeze-thawing of the cartilage. When the protease inhibitors EDTA, 6-aminohexanoic acid and benzamidine hydrochloride were added to the sodium acetate buffer the yields of proteoglycans from fresh and freeze-thawed cartilage were diminished, but the inhibitors had no effect on the hydrodynamic size of the proteoglycans extracted with sodium acetate. Addition of the protease inhibitors to the 4 M guanidinium solvent increased the proportion of proteoglycans present in aggregates. The highest proportion of aggregated proteoglycans was obtained when fresh tissue was extracted in the presence of the inhibitors.