Structural determination of the O-linked sialyl oligosaccharides liberated from fetuin with endo-alpha-N-acetylgalactosaminidase-S by HPLC analysis and 600-MHz H-1-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-GalNAcase-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 H-1-NMR spectroscopy. The chemical shifts and the coupling constants of pyridylaminated (PA) NeuAc alpha 2-3Gal beta 1-3GalNAc were refined by computer simulation of the spectrum. The structures of NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc-PA and NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-6)GalNAc-PA were determined by their structural reporter groups.