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

Abstract

Heparan sulphate by-products from the commercial manufacture of pig mucosal heparin were freed of chondroitin sulphate 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 analysed for their disaccharide composition by paper chromatography and by high-pressure liquid chromatography. The results show that the molar ratio of 2-O-sulpho-alpha-L-iduronosylanhydromannose to 6-O-sulpho-(2-O-sulpho-alpha-L-iduronosyl)anhydromannose decreased from 2.5 to 0.04 as the degree of sulphation of the fractions increased. In contrast, the molar ratio of 6-O-sulpho-(beta-D-glucuronosyl)anhydromannose to 6-O-sulpho-(alpha-L-iduronosyl)anhydromannose was approx. 2.4 in all heparan sulphate fractions and decreased to only half of this value in the most highly sulphated heparin fractions. These results are consistent with biosynthetic studies, which have shown that the N-sulpho-(2-O-sulpho-alpha-L-iduronosyl)D-glucosamine disaccharide is the metabolic precursor of the NO-disulpho-(2-O-sulpho-alpha-L-iduronosyl)-D-glucosamine disaccharide in heparin biosynthesis. The high-pressure liquid chromatography of the heparan sulphate oligosaccharides also revealed a number of unidentified oligosaccharides in the deamination mixtures.