Quantitation of dermatan sulfate active site for heparin cofactor II by 1H nuclear magnetic resonance spectroscopy

The sequence (IdoA2SO3-GalNAc4SO3)n contributes to the HCII-mediated inhibition of thrombin by dermatan sulfate (DS). This sequence clearly results from the 13C NMR spectrum and can be quantified by the signal C1-H of IdoA2SO3 in the 1H NMR spectrum. A linear correlation has been found between the content in the disulfated disaccharide ΔDi-2,4diS obtained by enzymatic demolition with ABC lyase, the percentage content in IdoA2SO3 quantified by 1H NMR, and the HCII-mediated activity of dermatan sulfates from beef mucosa and pig skin. DSs have been obtained also from pig mucosa and contain an amount, not negligible, of ΔDi-4, 6diS. This disulfate disaccharide contributes to the activity expressed by the IdoA2SO3-GalNAc4SO3 sequence. The analytical techniques HPLC and 1H NMR, applied to the currently performed analyses of DS, are described and discussed.