Artigo Revisado por pares

Rapid and simple preparation of N-linked oligosaccharides by cellulose-column chromatography

2001; Elsevier BV; Volume: 332; Issue: 4 Linguagem: Inglês

10.1016/s0008-6215(01)00113-6

ISSN

1873-426X

Autores

Yoshitaka Shimizu, Munehiro Nakata, Yasuhiro Kuroda, Fumihiko Tsutsumi, Naoya Kojima, Tsuguo Mizuochi,

Tópico(s)

Protein purification and stability

Resumo

As a means of preparing N-linked oligosaccharides from hydrazinolysates of glycoproteins in a rapid and simple manner, a method has been developed using cellulose-column chromatography. Hydrazinolysates of human IgG, containing a series of biantennary complex type oligosaccharides, were applied to a cellulose column equilibrated with (4:1:1, v/v) 1-butanol–ethanol–water. The N-linked oligosaccharides were eluted with (1:1, v/v) ethanol–water, and analyzed by HPLC in combination with sequential glycosidase digestion. The oligosaccharides, with or without sialic acid, were quantitatively recovered in the fraction eluted with (1:1, v/v) ethanol–water without UV-detectable contamination by impurities derived from protein or the cellulose. Other types of N-linked oligosaccharides of α1-acid glycoprotein (tetraantennary complex-type), ovalbumin (hybrid-type), and ribonuclease B (high mannose-type) were also quantitatively prepared from the hydrazinolysates by elution of the cellulose column with (1:1, v/v) ethanol–water and these had as high a quality as those prepared by conventional paper chromatography.

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