Purification of the Escherichia coli K5 capsular polysaccharide and use of high-performance capillary electrophoresis to qualitative and quantitative monitor the process.
A rapid, highly sensitive, and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the polysaccharide from the uropathogenic Escherichia coli K5 bacteria Bi8337/41 010:K5:H4. This natural polysaccharide having the structure of a desulfo-heparin composed of -4)-alphaGlcUA-(1,4)-alpha-GlcNAc-(1- is separated (GlcUA = D-glucuronic acid; GlcNAc = D-glucosamine) and qualitatively and quantitatively determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship (correlation coefficient > approximately 0.99) was found for the polymer over a wide range of concentrations, from approx. 60 to 1500 ng, with a detection sensitivity of < approximately 60 ng. Furthermore, this qualitative and quantitative HPCE approach was applied to the K5 extraction and purification process from cultured bacteria in several stages. HPCE was also able to separate several molecular species mainly due to the presence of polysaccharides of distinct and increasing mean chain lengths. A linear relationship was found for migration time and log molecular mass of different K5 polysaccharide species, and this model was used to calculate the molecular mass of the main K5 species producing a result of approx. 17,000, also confirmed by high-performance size-exclusion chromatography analysis, yielding approx. 17 200.
Volpi
Nicola
N
Department of Biologia Animale, University of Modena and Reggio Emilia, Modena, Italy. volpi@unimo.it
D004587:Electrophoresis, Agar Gel; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D011135:Polysaccharides, Bacterial / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D011135:Polysaccharides, Bacterial