Previously we isolated a tetrasaccharide-serine and a hexasaccharide-serine from the carbohydrate-protein linkage region of porcine intestinal heparin after digestion with a mixture of Flavobacterium heparinase and heparitinases I and II (Sugahara, K., Yamada, S., Yoshida, K., de Waard, P., and Vliegenthart, J.F.G.(1992) J. Biol. Chem. 267, 1528-1533). In this study four longer carbohydrate sequences (I-IV) attached to Ser or a dipeptide (Ser-Gly or Gly-Ser), which accounted for at least 18.2% of the total linkage region, were isolated from the same heparin preparation after digestion with heparinase only. IV was successfully isolated only after subsequent digestion with glycuronate-2-sulfatase. Their structures were determined by chemical and enzymatic analyses and H NMR spectroscopy and found to be the following octa- and decasaccharide sequences attached to Ser in a molar ratio of 1.1:2.3:1.0:1.3: HexA(2S)1-4GlcN(NS,6S)1-4GlcA1-4GlcNAc1-4GlcA1-3Gal1-3Gal1-4Xyl1-O-Ser (I), HexA(2S)1-4GlcN(NS,6S)1-4IdoA1-4GlcNAc1-4GlcA1-3Gal1-3Gal1-4Xyl1-O-Ser (II), HexA(2S)1-4GlcN(NS,6S)1-4IdoA1-4GlcNAc1-4GlcA1-4GlcNAc1-4GlcA1-3Gal1-3Gal1-4Xyl1-O-Ser (III), HexA1-4GlcN(NS,6S)1-4IdoA1-4GlcNAc(6S)1-4GlcA1-3Gal1-3Gal1-4Xyl1-O-Ser (IV) (HexA, GlcA, IdoA, and GlcN represent 4,5-unsaturated hexuronic acid, D-glucuronic acid, L-iduronic acid, and D-glucosamine, whereas 2S, 6S, and NS stand for 2-sulfate, 6-sulfate, and N-sulfate, respectively). I and II contained 1 mol of Gly in addition to Ser. The four structures indicate that sulfation in heparin chains takes place on the monosaccharide residues located in closer vicinity to the core protein than found for heparan sulfate chains and that there exist at least several heparin subclass chains with different linkage region structures. The significance of the isolated structures is discussed in relation to the biological functions and the biosynthetic mechanisms of heparin.

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