Separation of capsular polysaccharide K4 and defructosylated K4 derived disaccharides by fluorophore-assisted carbohydrate electrophoresis (FACE)

An inexpensive, fast, simple, sensitive and reproducible fluorophore-assisted carbohydrate electrophoresis (FACE) method is described for the quantitative analysis of microgram amounts of the Escherichia coli K4 bacterium capsule polysaccharide and its defructosylated polymer. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, Delta HexAFru-GalNAc for K4 and Delta HexAGalNAc for defructosylated K4, are fluorotagged by 2-aminoacridone (AMAC) and the products separated on a polyacrylamide gel electrophoresis. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from 0.5 to 5 mu g for the Delta-disaccharide of K4 and from 0.2 to 5 mu g for the Delta-disaccharide of K4d. The detection limit was found to be approx. 0.5-1 mu g for K4 and 0.1-0.2 mu g for K4d. The FACE procedure described is especially useful when many samples need to be analyzed