Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin.

TLDR

The authors isolated a high‑affinity octasaccharide from heparin by partial deaminative cleavage, converted its 2,5‑anhydro‑D‑mannose end group, and released a labeled pentasaccharide via periodate‑alkali treatment, subsequently recovering the terminal glucosamine residue through deamination, reduction, and chromatographic separation. Incubation with a 3,O‑sulfatase and electrophoretic analysis revealed a 3‑sulfated terminal glucosamine, a disulfated anhydro[3H]mannitol (C3/C6) as the major component, and the absence of such disulfation in low‑affinity tetrasaccharides, indicating that 3‑sulfated glucosamine is a unique, position‑specific component of high‑affinity heparin.

Abstract

An octasaccharide with high affinity for antithrombin was isolated after partial deaminative cleavage of heparin with nitrous acid. After conversion of the 2,5-anhydro-D-mannose end group to anhydro[1-3H]mannitol, labeled pentasaccharide was released from the octasaccharide by periodate-alkali treatment. Incubation of the pentasaccharide with a recently discovered 3,O-sulfatase from human urine resulted in desulfation, suggesting the occurrence of a 3-sulfate group on the terminal glucosamine residue. The same glucosamine residue was recovered as a 2,5-anhydro[1-3H]mannitol derivative by a procedure involving deamination of the octasaccharide with nitrous acid, reduction of the products with sodium boro[3H]hydride, isolation of 3H-labeled tetrasaccharide by gel chromatography, and release of the labeled end-group by periodate-alkali treatment. Paper electrophoresis indicated disulfated anhydro[3H]mannitol, presumably sulfated at C3 and C6, as a major component, along with smaller amounts of monosulfated (presumably 3-sulfated) anhydro[3H]mannitol. Similar treatment of an analogous tetrasaccharide derived from heparin with low affinity for antithrombin failed to produce any disulfated anhydromannitol. These results suggest that 3-sulfated glucosamine is a unique component of high-affinity heparin, located at a specific position in the antithrombin-binding sequence of the molecule.