Quantitation of l- glycero- d- manno-heptose and 3-deoxy- d- manno-octulosonic acid in rough core lipopolysaccharides by partition chromatography

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 R e and R dP − 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 R e or R dP − 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 R dP − 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 R e LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the R dP − LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed.