Combined lectin-affinity and metal-interaction chromatography for the separation of glycophorins by high-performance liquid chromatography.
Human erythrocyte sialoglycoproteins, or glycophorins, were chromatographed by lectin-affinity and metal-interaction chromatography on high-performance liquid chromatographic columns. Glycophorins A, B and C were separated from other proteins and from glycophorin E by using a column containing wheat germ agglutinin, immobilized on a microparticulate silica support. The glycophorins were adsorbed on the lectin column from a mobile phase containing 0.25 M sodium chloride and recovered by stepwise desorption with 0.2 M N-acetylglucosamine solution. Glycophorins A, B and C were separated into the individual components on a silica-bound iminodiacetic acid stationary phase in the copper(II) chelate form. The separation of the glycophorins by metal-interaction chromatography was accomplished by decreasing salt gradient elution. Retention times and resolution of the individual glycophorins were sensitive to the initial sodium chloride concentration and the pH of the eluent. Addition of methanol to the eluent increased the resolution. The effects of linear, decreasing gradients of pH and methanol in 25 mM phosphate buffer on the resolution of glycophorins were also investigated. In both types of chromatography the mobile phases contained 0.05% (w/v) sodium dodecyl sulfate. With octylglycoside or CHAPS in the eluent glycophorins A and C could not be eluted. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze all the chromatographic results.
Corradini
D
D
Department of Chemical Engineering, Yale University, New Haven, CT 06520.
el Rassi
Z
Z
Horváth
C
C
Guerra
G
G
Horne
W
W
D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000032:analysis; D006021:Glycophorins / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D037102:Lectins; D008670:Metals; D012795:Sialoglycoproteins / Q000032:analysis*
D006021:Glycophorins; D037102:Lectins; D008670:Metals; D012795:Sialoglycoproteins