STRUCTURAL STUDIES ON THE TRISACCHARIDES AND TETRASACCHARIDES ISOLATED FROM PORCINE INTESTINAL HEPARIN AND CHARACTERIZATION OF HEPARINASE HEPARITINASES USING THEM AS SUBSTRATES/

We prepared a series of oligosaccharides from porcine intestinal hepar in after extensive digestion with a mixture of Flavobacterium heparina se as well as heparitinases I and II. Previously, we reported the stru ctures of the two glycoserines derived from the carbohydrate-protein l inkage region [Sugahara et al., J. Biol. Chem., 267, 1528-1533 (1992)] and three tetrasaccharides derived from the antithrombin III-binding site [Yamada et al., J. Biol. Chern., 268, 4780-4787 (1993)]. In this study, we determined the structures of 10 other tetrasaccharides and a trisaccharide by enzymatic digestion, fast atom bombardment mass spec trometry and 500-MHz H-1 NMR spectroscopy. These tetrasaccharides shar e the common disulphated structure, Delta HexA alpha 1-4GlcN(N-sulphat e)alpha 1-4IdoA(2-sulphate)alpha 1-4GlcN (where HexA is hexuronic acid and IdoA is L-iduronic acid), and their structural variations are bas ed upon the positions of additional sulphate groups. Eight among the 1 0 have never been isolated as discrete structures. The structure of th e trisaccharide is GlcN(N-sulphate)alpha 1-4IdoA(2-sulphate)alpha 1-4G lcN(N,6-disulphate) and is derived from the non-reducing terminus of h eparin chains. This structure may represent the terminus of a biosynth etically formed native heparin chain or a newly formed non-reducing te rminus exposed by a tissue endo-beta-glucuronidase which may be involv ed in the intracellular post-synthetic fragmentation of macromolecular heparin. The 11 structures characterized in the present study and 6 a dditional tetrasaccharides were used to investigate the substrate spec ificities of heparinase, as well as heparitinases I and II. The result s indicate that modification of the adjacent glucosamine on the reduci ng side of the disaccharide cleavage site influences the enzymatic act ion of the lyases, whereas the adjacent uronic acid on the non-reducin g side is not recognized by these enzymes.