Islam Tasneem, Butler Melissa, Sikkander Sulthan A, Toida Toshihiko, Linhardt Robert J
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.
Carbohydr Res. 2002 Nov 19;337(21-23):2239-43. doi: 10.1016/s0008-6215(02)00229-x.
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.
通过用氢氧化钠处理高碘酸盐氧化的肝素，随后用硼氢化钠还原并进行酸水解，将猪黏膜肝素片段化为低分子量（LMW）肝素。梯度聚丙烯酰胺凝胶电泳分析显示肝素片段混合物的平均大小为八个二糖单位。一维¹H NMR显示，在高碘酸盐裂解时，三分之二的N-乙酰基丢失，表明裂解发生在抗凝血酶III（ATIII）结合位点内及相邻的吡喃葡萄糖醛酸（GlcpA）和艾杜糖醛酸（IdopA）残基处。在处理过程中，N-乙酰葡糖胺（GlcpNAc）残基丢失。ATIII结合位点内的GlcpA残基位于N-磺基、3,6-O-磺基吡喃糖胺（GlcpNS3S）残基的非还原端。因此，高碘酸盐裂解的肝素应富含GlcpNS3S6S残基。二维相关光谱（2D COSY）证实，通过高碘酸盐裂解制备的LMW肝素在其非还原端含有GlcpNS3S6S。正如预期的那样，这种LMW肝素还显示出ATIII介导的抗因子IIa和抗因子Xa活性降低。
