STRUCTURAL STUDIES ON THE OLIGOSACCHARIDES ISOLATED FROM BOVINE KIDNEY HEPARAN-SULFATE AND CHARACTERIZATION OF BACTERIAL HEPARITINASES USEDAS SUBSTRATES

We prepared a series of oligosaccharides from commercial bovine kidney heparan sulphate after limited digestion with heparitinase I from fla vobacterium heparinum, and determined the structures of eight tetrasac charides and a hexasaccharide by enzymatic analysis, fast atom bombard ment mass spectrometry and 500 MHz H-1 NMR spectroscopy. The tetrasacc harides share the common core structure Delta(4,5)HexA alpha 1-4GlcN a lpha 1-4HexA1-4GlcN (where Delta(4,5)HexA is 4-deoxy-alpha-L-threo-hex -4-enopyranosyluronic acid and HexA is hexuronic acid), with zero, one or two sulphate groups. Seven of them contain non-sulphated glucuroni c or iduronic acid, and the other, 2-O-sulphated iduronic acid at the internal position. Although they contain ordinary structures which sho uld be widely distributed in the relatively low-sulphated region of he paran sulphate, five of the tetrasaccharides were isolated for the fir st time as discrete structures. The structure of the hexasaccharide wa s determined as Delta(4,5)Hex alpha 1-4GlcNAc alpha 1-4GlcA beta 1-3Ga l beta 1-3 Gal beta 1-4Xyl and is derived from the carbohydrate-protei n linkage region of the heparan sulphate chains. The hexasaccharide se ems to have been released by the alkaline treatment used to prepare th e heparan sulphate. The Gal residues were non-sulphated as are those i n the porcine intestinal heparin chains, but in contrast to the sulpha ted Gal structures previously demonstrated in the carbohydrate-protein linkage region of chondroitin sulphate chains. These oligosaccharides were used to investigate the substrate specificity of heparitinases I and II from F.heparinum. The results revealed that heparitinase I cle aves hexosaminidic bonds linked to non-sulphated glucuronic or iduroni c acid residues. The glucosaminidic linkage of the hexasaccharide was sensitive to heparitinase I, but resistant to heparitinase II, demonst rating the differential specificity of these enzymes towards the carbo hydrate-protein linkage region.