Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin.
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TLDR
3-sulfated glucosamine is a unique component of high-affinity heparin, located at a specific position in the antithrombin-binding sequence of the molecule.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.read more
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References
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Journal ArticleDOI
Formation of anhydrosugars in the chemical depolymerization of heparin.
John E. Shively,H. Edward Conrad +1 more
TL;DR: It is demonstrated here that only low pH reaction conditions favor the deamination of N-sulfated D-glucosamine residues; the reaction proceeds very slowly at pH 3.5 or above and at room temperature solutions of nitrous acid lose one-fourth to one-third of their capacity to deaminate amino sugars in 1 h at all pHs.
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The separation of active and inactive forms of heparin
TL;DR: Heparin has been fractionated into two distinct forms and the mucopolysaccharide component was clearly separated from the remaining 2 3 of the heparin which could not form a stable complex with antithrombin-heparin cofactor and had minimal anticoagulant activity.
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Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin iii and by gel filtration.
TL;DR: Gel filtration of the material showed that there was a definite molecular size dependency of the specific activities of heparin anticoagulant action and the activity profiles were markedly different when assay -ed by different methods.
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Anticoagulant activity of heparin: Separation of high-activity and low-activity heparin species by affinity chromatography on immobilized antithrombin
TL;DR: In the present study heparin was separated by affinity chromatography on antithrombin-substituted Sepharose into two distinct fractions, one with high affinity and one with little or no affinity for the protein.
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Structure of the antithrombin-binding site in heparin
TL;DR: Compositional analysis based on separation and identification of deamination products reduced with sodium boro[3H]hydride showed that nonsulfated L-iduronic acid occurred in larger amounts in high-affinity heparin than in low-Affinity heParin; furthermore, this component was concentrated in the antithrombin-binding regions of the high- Affinity Heparin molecules.