Changes in disaccharide composition of heparan sulphate fractions with increasing degrees of sulphation

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Abstract
Heparan sulfate by-products from the commercial manufacture of pig mucosal heparin were freed of chondroitin sulfate and fractionated according to anionic density. The fractions were treated with HNO2 at pH 1.5, and the resulting mixtures of oligosaccharides were reduced with NaB3H4 and analyzed for their disaccharide composition by paper chromatography and by high-pressure liquid chromatography (HPLC). The molar ratio of 2-O-sulfo-.alpha.-L-iduronosylanhydromannose to 6-O-sulfo-(2-O-sulfo-.alpha.-L-iduronosyl)anhydromannose decreased from 2.5 to 0.04 as the degree of sulfation of the fractions increased. In contrast, the molar ratio of 6-O-sulfo-(.beta.-D-glucuronosyl)anhydromannose to 6-O-sulfo-(.alpha.-L-iduronosyl)anhydromannose was .apprx. 2.4 in all heparan sulfate fractions and decreased to only half of this value in the most highly sulfated heparin fractions. These results are consistent with biosynthetic studies, which have shown that the N-sulfo-(2-O-sulfo-.alpha.-L-iduronosyl)-D-glucosamine disaccharide is the metabolic precursor of the NO-disulfo-(2-0-sulfo-.alpha.-L-iduronosyl)-D-glucosamine disaccharide in heparin biosynthesis. HPLC of the heparan sulfate oligosaccharides also revealed a number of unidentified oligosaccharides in the deamination mixtures.