Structural peculiarity and antithrombin binding region profile of mucosal bovine and porcine heparins

• NMR and LC/MS differentiation of bovine and porcine mucosal heparin is provided.
• LC/MS profiles of high ATIII affinity fractions are characteristic fingerprints.
• 2-O-Sulfated GlcA was found to be a new NMR marker of bovine mucosal heparin.
• BMH and PMH significantly differ in their content of structural variants of the ATBR.
• ΔU2,4,0 and ΔU4,6,0 fragments are highly represented in bovine affinity fractions.

The major compositional differences between bovine mucosal heparin (BMH) and the currently employed porcine mucosal heparin (PMH) have been reported to essentially consist of reduced 6-O-sulfation of the glucosamine residues in BMH and somewhat lower 2-O-sulfation of the iduronate residues in PMH. The present work is based on direct comparison of several BMH and PMH commercial preparations. A combined study by 2D (heteronuclear single quantum coherence, HSQC) NMR and ion-pair reversed-phase high performance liquid chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) on the heparins, extended to the analysis of their heparinases digests and fractions separated by affinity chromatography on antithrombin (AT), confirmed the previously reported lower degree of 6-O-sulfation and showed lower 3-O-sulfated glucosamine content in BMH. More detailed studies allowed the identification of structural variants of AT-binding region (ATBR) structural variants, showing higher content of the N-sulfated components in BMH than in PMH.

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