Formation of Three Types of Disulfated Disaccharides from Chondroitin Sulfates by Chondroitinase Digestion

Abstract

Abstract By means of enzymatic digestion with chondroitinase-ABC, a novel unsaturated disaccharide bearing two sulfate residues has been derived from chondroitin sulfate of squid cartilage. Various data, particularly the formation of 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose and its 6-O-sulfate isomer by hydrolysis with chondro-6-sulfatase and chondro-4-sulfatase, respectively, and the formation of 2-acetamido-2-deoxy-4,6-di-O-sulfo-d-galactose by mild acid hydrolysis, indicate that the two sulfate residues are located at positions 4 and 6 of the hexosamine moiety. A distinct unsaturated disaccharide also bearing two sulfate residues has been obtained from chondroitin sulfate B preparations of bovine lung and pig skin. One of the sulfate residues has been shown to be substituted at position 4 of the hexosamine moiety. The resistance of the second residue to both chondro-4-sulfatase and chondro-6-sulfatase and of the uronic acid moiety to Flavobacterium heparinum glucuronidase suggests that the sulfate residue is substituted at position 2 or 3 of the uronic acid moiety. These disulfated disaccharides are, therefore, isomers of 2-acetamido-2-deoxy-3-O-(2- or 3-O-sulfo-β-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose, the compound previously found in the digest of chondroitin sulfate from shark cartilage. The three isomeric disulfated disaccharides were separated from nonsulfated and monosulfated homologues and from one another by paper chromatography in 1-butyric acid-0.5 n ammonia (5:3). The separation of disaccharides from chondroitinase digests by paper chromatography permits comparison of chondroitinase digests from different chondroitin sulfate preparations and detection of slight variations in the types of sulfate linkage.