G.l.c. of O-methyloxime and alditol acetate derivatives of neutral sugars, hexosamines, and sialic acids: "one-pot" quantitative determination of the carbohydrate constituents of glycoproteins and a study of the selectivity of alkaline borohydride reductions.
A new procedure for the quantification by g.l.c. of the carbohydrate constituents of glycoproteins is proposed which involves (a) simultaneous action of neuraminidase and neuraminic acid aldolase, (b) hydrolysis with 4M trifluoroacetic acid at 125 degrees for 1 h, and (c) conversion of the products into O-methyloxime acetates and g.l.c. The procedure has been successfully tested on fetuin, transferrin, alpha 1-acid glycoprotein, and mucin. The g.l.c. conditions used also enabled the complete separation of O-methyloxime and alditol acetate derivatives in one run, so that the release of carbohydrate chains from glycoproteins by treatment with alkaline borohydride can be investigated conveniently. There was complete release of O-linked oligosaccharides from fetuin on treatment with 0.1M NaOH/0.8M NaBH4 (68 h, 37 degrees) or 0.05M KOH/M KBH4 (24 h, 45 degrees) and also release of approximately 75% and 35-40%, respectively, of N-asparagine-linked chains. Reduced oligosaccharides were formed only from O-linked chains; the mechanism by which N-linked chains were released is still not clear.
D000818:Animals; D001894:Borohydrides; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis*; D002849:Chromatography, Gas / Q000379:methods; D006023:Glycoproteins; D006595:Hexosamines / Q000032:analysis*; D007202:Indicators and Reagents; D010084:Oxidation-Reduction; D010091:Oximes; D012794:Sialic Acids / Q000032:analysis*; D013402:Sugar Alcohols
D001894:Borohydrides; D002241:Carbohydrates; D006023:Glycoproteins; D006595:Hexosamines; D007202:Indicators and Reagents; D010091:Oximes; D012794:Sialic Acids; D013402:Sugar Alcohols