Quantitation of l-glycero-d-manno-heptose and 3-deoxy-d-manno-octulosonic acid in rough core lipopolysaccharides by partition chromatography |
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Authors: | Robert C Seid Herman Schneider Sophia Bondarew Robert A Boykins |
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Institution: | 1. Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D. C. 20012 USA;2. Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20205 USA |
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Abstract: | A partition chromatographic procedure utilizing a cationic exchange resin column in the Li+ form and 90% ethanol as the mobile phase was employed to quantify 3-deoxy-d-manno-octulosonic acid (KDO) and l-glycero-d-manno-heptose in the lipopolysaccharides (LPS) of Re and RdP? rough mutants of Salmonella minnesota. In a standard mixture of monosaccharides, KDO eluted shortly after the void volume and heptose eluted after the neutral hexoses. Mild acid treatment of either the Re or RdP? LPS with 0.16 n methanesulfonic acid in the presence of Dowex 50-X8 resin (H+ form) released more than 80% of the KDO residues within 15 min. The heptose of the RdP? LPS, first detected after 90 min of hydrolysis, increased gradually to a maximum level at 12 h. A secondary gradual increase in KDO became apparent during the heptose release. The weight contents of these two monosaccharides based upon aheir maximum values detected during hydrolysis were 20.3 ± 0.6% KDO, for the Re LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the RdP? LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed. |
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