Heterogeneity of protein-polysaccharides of porcine articular cartilage. The sequential extraction of chondroitin sulphate-proteins with iso-osmotic neutral sodium acetate.

Protein-polysaccharides of knee-joint cartilage of 9-month-old pigs were extracted sequentially with neutral iso-osmotic sodium acetate after five repeated homogenizations. One-third of the uronic acid originally present in the tissue was brought into solution, about half being in the first extract. The protein-polysaccharides, which were purified by precipitation with 9-aminoacridine, were heterogeneous in size on gel chromatography. The smallest (retarded by 6% agarose) were the most easily extracted since they were most prevalent in the initial extracts and absent from later ones, whereas the proportion of larger molecules increased progressively in successive extracts. Nevertheless a small proportion of the largest molecules (excluded from Sepharose 2B) was present even in the first extract. None of the protein-polysaccharide preparations contained hydroxyproline, and the analyses of their constituent sugars were the same, although there was a progressive increase in the protein content and in the glucosamine/galactosamine molar ratio of successive extracts. In each preparation this molar ratio was invariably greater in larger than in smaller molecules separated by gel filtration. From galactosamine/pentose molar ratios it appeared that the chondroitin sulphate chains were on average about 29 disaccharide units in length in the protein-polysaccharides of each extract, although gel-chromatography and cetylpyridinium chloride elution profiles showed that a somewhat higher proportion of shorter chondroitin sulphate chains occurred in the larger protein-polysaccharides. In the last extract, where the largest molecules predominated, about half could be reversibly dissociated by urea, whereas this had no effect on the protein-polysaccharides of earlier extracts even though these contained some large molecules.