Carbohydrate composition analysis of bacterial polysaccharides: optimized acid hydrolysis conditions for HPAEC-PAD analysis.
The capsular polysaccharide from Haemophilus influenzae type b (polyribosyl ribitol-phosphate; PRP) and the capsular polysaccharides from Streptococcus pneumoniae types 6B, 14, 18C, and 23F (Pn6B, Pn14, Pn18C, and Pn23F) were subjected to acid hydrolysis using hydrofluoric (HF) and/or trifluoroacetic acid (TFA) and high-pH anion-exchange chromatography with pulsed amperometric detection in an effort to identify optimum hydrolysis conditions for composition analysis of their carbohydrate components. With the exception of PRP, composition analyses of polysaccharides containing a phosphate moiety in the repeating unit structure (Pn6B, Pn18C, and Pn23F) are significantly improved by subjecting the sample to HF hydrolysis (65 degrees C, 1 h) followed by TFA hydrolysis (98 degrees C, 16 h). This results in essentially quantitative hydrolysis of the phosphodiester bond to the carbohydrate components, which otherwise remained predominantly phosphorylated and poorly accounted for in the analysis. Optimum analysis of PRP was achieved following a 2-h hydrolysis with TFA at 80 degrees C, whereas Pn14 showed optimum results after a 16-h hydrolysis with TFA at 98 degrees C. These analyses also provide information about the relative susceptibility to acid hydrolysis of the various glycosidic and phosphodiester bonds in these polysaccharides, with evidence to suggest that the acid lability of a given bond can be dramatically different from one polysaccharide to another.
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Department of Cellular & Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
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Hepler
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Hennessey
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D001419:Bacteria / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006858:Hydrofluoric Acid; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D014269:Trifluoroacetic Acid
D002241:Carbohydrates; D011134:Polysaccharides; D014269:Trifluoroacetic Acid; D006858:Hydrofluoric Acid