Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site.
Porcine mucosal heparin was fragmented into low-molecular-weight (LMW) heparin by treatment of periodate-oxidized heparin with sodium hydroxide, followed by reduction with sodium borohydride and acid hydrolysis. Gradient polyacrylamide gel electrophoresis analysis showed a mixture of heparin fragments with an average size of eight disaccharide units. 1D 1H NMR showed two-thirds of the N-acetyl groups were lost on periodate cleavage, suggesting cleavage had occurred at the glucopyranosyluronic acid (GlcpA) and idopyranosyluronic acid (IdopA) residues located within and adjacent to the antithrombin III (ATIII) binding site. The N-acetyl glucopyranose (GlcpNAc) residue was lost on workup. The GlcpA residue, within the ATIII binding site, is on the non-reducing side of the N-sulfo, 3, 6-O-sulfo glycopyranosylamine (GlcpNS3S6S) residue. Thus, periodate cleaved heparin should be enriched in GlcpNS3S6S residues. Two-dimensional correlation spectroscopy (2D COSY) confirmed that LMW heparin prepared through periodate cleavage contained GlcpNS3S6S at its non-reducing end. As expected, this LMW heparin also showed reduced ATIII mediated anti-factor IIa and anti-factor Xa activities.
Islam
Tasneem
T
Department of Chemistry, Division of Medicinal and Natural Products Chemistry and Department of Chemical and Biochemical Engineering, PHAR S 328, University of Iowa, Iowa City, IA 52242, USA.
Butler
Melissa
M
Sikkander
Sulthan A
SA
Toida
Toshihiko
T
Linhardt
Robert J
RJ
D000818:Animals; D000990:Antithrombin III; D001665:Binding Sites; D001780:Blood Coagulation Tests; D004591:Electrophoresis, Polyacrylamide Gel; D065427:Factor Xa Inhibitors; D006493:Heparin / Q000737:chemistry*; D006495:Heparin, Low-Molecular-Weight / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D010504:Periodic Acid / Q000737:chemistry*; D011516:Prothrombin / Q000037:antagonists & inhibitors; D012972:Sodium Hydroxide; D013552:Swine
D065427:Factor Xa Inhibitors; D006495:Heparin, Low-Molecular-Weight; D010504:Periodic Acid; D012972:Sodium Hydroxide; D000990:Antithrombin III; D011516:Prothrombin; D006493:Heparin; C009288:metaperiodate