Weak affinity chromatography of small saccharides with immobilised wheat germ agglutinin and its application to monitoring of carbohydrate transferase activity.
In this work we have evaluated the potential to use wheat germ agglutinin (WGA) for weak affinity chromatography (WAC) of N-acetyl derivatives of mono-, di-, tri- and tetrasaccharides. WGA was used as a ligand in a high performance liquid affinity chromatography (HPLAC) system. Isocratic affinity chromatography was conducted where similar N-acetyl saccharides were separated according to their binding strength to WGA. Affinities are weak and lie typically in the mM range. For example, for 3'sialyllactose, the dissociation constant (Kd) was found to be 2.4 mM at 8 degrees C. It was interesting to note that the WGA-HPLC column can distinguish between the anomeric forms of N-acetylglucosamine. Weak affinity chromatography with immobilised WGA was used in an enzyme assay to detect the activity of GlcNAc-transferases.
University of Kalmar, Department of Natural Sciences, Sweden.
S
S
Ohlson
M
M
Bergström
L
L
Leickt
D
D
Zopf
D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis*; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D014909:Wheat Germ Agglutinins
D009844:Oligosaccharides; D014909:Wheat Germ Agglutinins; D017351:N-Acetylglucosaminyltransferases