Characteristics of proteoglycans extracted from the Swarm rat chondrosarcoma with associative solvents

Proteoglycans were extracted from Swarm rat chondrosarcoma tissue with solutions of 0.5 M guanidine hydrochloride containing protease inhibitors at pH 5.8 and 7.4. In this associative solvent, the noncovalent interactions between the components of the proteoglycan aggregates are not dissociated before subsequent isolation steps. Aggregates were purified by density gradient centrifugation and different preparations were compared by chromatography on Sepharose 2B. Almost 75% of the tissue proteoglycan could be extracted in 24 h with the associative solvent, although most of the extractable material was in solution after 4 h. the proportion of the extracted proteoglycan in the aggregate form was less in the pH 7.4 solvent and decreased in both solvents after 4 h, suggesting that there may be some proteolysis during the extraction step. for comparison, proteoglycans were first extracted with a dissociative solvent containing 4.0 M guanidine hydrochloride and aggregates reassociated by dialysis into an associative solvent, 0.5 M in guanidine hydrochloride. In this case, about 95% of the proteoglycans were extracted, but smaller proportions of aggregate were obtained than for the preparations extracted directly with the associative solvents, suggesting that reaggregation may not be completely efficient under the conditions employed. The aggregates prepared by the associative and dissociative methods were compared by electron microscopy. For a 4-h, pH 5.8, associative extract, the average monomer length was about 250 nm and the distribution was skewed slightly toward shorter lengths. Monomers in aggregates from the dissociative preparation showed a similar, skewed distribution with a somewhat shorter average length of 225 nm. The distribution of monomers in aggregates from a 16-h, pH 7.4, associative extract was bimodal with an average length of 202 nm, indicating that limited proteolysis occurred during the extraction step.