High-performance liquid chromatographic identification of disaccharides generated from heparan sulphate isomers using heparitinases.

Specific heparan sulphate-lyases, heparitinases I and II, were used to identify unsaturated disaccharide constituents generated from heterogeneous heparan sulphate isomers. All determinations were made using high-performance liquid chromatography with a column containing a sulphonized styrene-divinylbenzene copolymer. Unsaturated disaccharides generated from variously sulphated heparan sulphate isomers after simultaneous digestion with heparitinases I and II facilitated separation of the individual disaccharides, based on sulphate groups at the specific position of the uronic acid and glucosamine residues. The simultaneous digestion with heparitinases I and II produces unsaturated disaccharides from heparan sulphate isomers with the structure of 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-amino-deoxy-D-glucose, 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-deoxy-2-sulphamido-D-glucose, 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-aminodeoxy-6-O-sulpho-D-glucose, 4-deoxy-2-O-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-deoxy-2-sulphamido-6-O-sulpho-D-glucose, 4-deoxy-2-O-sulpho-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-amino-2-deoxy-6-O-sulpho-D-glucose and 4-deoxy-2-O-sulpho-alpha-L-threo-hex-4-enepyranosyluronic acid (1----4)-2-deoxy-2-sulphamido-6-O-sulpho-D-glucose.