Separation of capsular polysaccharide K4 and defructosylated K4 derived disaccharides by high-performance capillary electrophoresis and high-performance liquid chromatography.
A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of disaccharides present in the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, DeltaHexAFrc-GalNAc for K4 and deltaHexA-GalNAc for defructosylated K4, are separated and readily determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 230 nm. Comparison was made by separation of these two disaccharides in isocratic strong-anion exchange HPLC. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from approximately 0.5 to 5 micro g for high-performance liquid chromatography (HPLC) and from approximately 0.06 to 0.3 micro g for HPCE. The HPCE separation produced a greater detection sensitivity (about 10 times greater) than HPLC. The described methods were used to evaluate the defructosylation process of K4 under drastic acid conditions. Good correspondence was found for the amount of unsaturated disaccharides for the two techniques.
Volpi
Nicola
N
Dipartimento di Biologia Animale, University of Modena and Reggio Emilia, Modena, Italy. volpi@unimo.it
D016667:Bacterial Capsules; D019765:Chondroitin ABC Lyase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004187:Disaccharides / Q000737:chemistry / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D011135:Polysaccharides, Bacterial / Q000737:chemistry* / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004187:Disaccharides; D011135:Polysaccharides, Bacterial; C063323:capsular polysaccharide K4; D019765:Chondroitin ABC Lyase