Liquid chromatographic method for the determination of the carbohydrate moiety of glycoproteins. Application to alpha 1-acid glycoprotein and tissue plasminogen activator.
A rapid procedure is described for the qualitative and quantitative analysis of the carbohydrate composition of glycoproteins by liquid chromatography with light-scattering detection. The analysis was carried out in three steps. First, the glycoprotein samples were purified by a two-step purification on a Sephadex G-25 column with a 90% yield. Second, the selectivity of the separation and the sensitivity of detection of monosaccharides, as methyl glycosides obtained by direct methanolysis of glycoproteins, were improved by modified simplex optimization of the methanolysis parameters (temperature, methanolic hydrochloric acid strength and reaction time) determined at 66 degrees C, 1.2 M and 8.1 h for alpha 1-acid glycoprotein (alpha-AGP) and 73 degrees C, 1.5 M and 12.5 h for tissue plasminogen activator (tPA). Finally, the method was applied to the determination of the carbohydrate moiety of the two N-glycosylated glycoproteins alpha-AGP and tPA.
Taverna
M
M
Centre d'Etudes Pharmaceutiques, Laboratoire de Chimie Analytique, Chatenay Malabry, France.
Baillet
A
A
Werner
R
R
Baylocq-Ferrier
D
D
D000117:Acetylglucosamine / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods*; D005643:Fucose / Q000032:analysis; D005690:Galactose / Q000032:analysis; D006851:Hydrochloric Acid; D008027:Light; D008358:Mannose / Q000032:analysis; D000432:Methanol; D009961:Orosomucoid / Q000032:analysis*; D012542:Scattering, Radiation; D010959:Tissue Plasminogen Activator / Q000032:analysis*
D002241:Carbohydrates; D009961:Orosomucoid; D005643:Fucose; D010959:Tissue Plasminogen Activator; D008358:Mannose; D006851:Hydrochloric Acid; D000117:Acetylglucosamine; D005690:Galactose; D000432:Methanol