Selective hydrolysis of chondroitin sulfates by hyaluronidase

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Abstract
Chondroitin 4-sulfate and chondroitin 6-sulfate were incubated with testicular hyaluronidase in the presence of excess .beta.-glucuronidase. The .beta.-glucuronidase caused rapid removal of the nonreducing terminal .beta.-D-glucuronosyl residues from the oligosaccharides formed by the action of the hyaluronidase, destroying the oligosaccharide acceptors required for the transglycosylation activity of hyaluronidase and releasing free D-glucuronic acid at a rate that was equal to the rate of the hyaluronidase-catalyzed hydrolysis. When hyaluronidase was assayed at 37.degree. C in the presence of 0.05 M NaCl, 0.05 M Na2SO4 and 0.1 M sodium acetate at pH 5, chondroitin 4-sulfate was hydrolyzed at 1.5 times the rate found for chondroitin 6-sulfate. When hyaluronidase was assayed at 45.degree. C in 0.06 M sodium acetate at pH 6, chondroitin 4-sulfate was hydrolyzed at 8 times the rate observed for chondroitin 6-sulfate. Under the pH 5 conditions, the chondroitin 4-sulfate was converted to a mixture of tri- and pentasaccharides, while the chondroitin 6-sulfate was converted primarily to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide. Under the pH 6 conditions, the chondroitin 4-sulfate was converted to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide, but the products from chondroitin 6-sulfate were a mixture of oligosaccharides ranging in degree of polymerization from 7-25 monosaccharides per oligosaccharide. End-group analyses of the products formed at pH 6 showed that both substrates were cleaved preferentially at the glycosidic bonds of the 4-sulfated disaccharides. The use of the enhanced substrate selectivity of hyaluronidase at the higher pH in the analysis of chondroitin sulfate structures is discussed.