Rapid and simple preparation of N-linked oligosaccharides by cellulose-column chromatography.
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 alpha1-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.
Shimizu
Y
Y
Department of Applied Biochemistry, Tokai University, Hiratsuka, 259-1292, Kanagawa, Japan.
Nakata
M
M
Kuroda
Y
Y
Tsutsumi
F
F
Kojima
N
N
Mizuochi
T
T
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D002482:Cellulose; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins / Q000737:chemistry*; D006026:Glycoside Hydrolases; D006801:Humans; D006834:Hydrazines; D007074:Immunoglobulin G / Q000737:chemistry*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000737:chemistry*; D009961:Orosomucoid / Q000737:chemistry; D010047:Ovalbumin / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry
D006023:Glycoproteins; D006834:Hydrazines; D007074:Immunoglobulin G; D009844:Oligosaccharides; D009961:Orosomucoid; D002482:Cellulose; D010047:Ovalbumin; D012260:Ribonucleases; C029600:ribonuclease B; D006026:Glycoside Hydrolases