Structural Determination of the O‐Linked Sialyl Oligosaccharides Liberated from Fetuin with Endo‐α‐N‐Acetylgalactosaminidase‐S by HPLC Analysis and 600‐MHz ¹H‐NMR Spectroscopy

The endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) hydrolyzed the O-glycosidic linkage between GalNAc and Ser (Thr) in fetuin, liberating oligosaccharides. The O-linked oligosaccharides liberated from the fetuin with endo-GalNAc-ase-S were pyridylaminated following fractionation on a Bio-Gel P-4 column. The structure of the pyridylaminated O-linked oligosaccharides from fetuin has been determined by reverse-phase HPLC and 600-MHz 1H-NMR spectroscopy. The chemical shifts and the coupling constants of pyridylaminated (PA) NeuAc alpha2-3Gal beta1-3GalNAc were refined by computer simulation of the spectrum. The structures of NeuAc alpha2-3Gal beta1-3(NeuAc alpha2-6)GalNAc-PA and NeuAc alpha2-3Gal beta1-3(NeuAc alpha2-3Gal beta1-4GlcNAc beta1-6)GalNAc-PA were determined by their structural reporter groups.