Precipitation of Lipopolysaccharides from Rough Mutants of Salmonella typhimurium by an A‐Hemagglutinin from Helix pomatia

Purified Helix pomatia A‐hemagglutinin precipitated lipopolysaccharides of Salmonella typhimurium rough mutants of chemotype Ra and Rb. No precipitation was obtained with lipopoly‐saccharide of the parent smooth strain or of Rc, Rd and Re mutants or with isolated lipid A. Reactivity of lipopolysaccharide from some of the Ra and Rb mutants was increased when precipitated in a mixture of tetrahydrofuran—phosphate‐buffered saline (1:3, by vol.). However lipopolysaccharide from nonreactive mutants was still negative in the latter medium. Precipitating ability of lipopolysaccharide from Ra and Rb mutants was retained after treatment of the lipopolysaccharide with sodium borohydride (or NaOH). Precipitation was inhibited by N ‐acetyl‐D‐galactosamine but not as easily by D‐galactose or D‐mannose. The lipopolysaccharide from the Ra mutant TV160 displaced a labelled blood‐group‐A‐active pentasaccharide from the combining site of the hemagglutinin. Inhibition studies indicated that the interaction was weak. Core polysaccharides prepared by acid hydrolysis and gel filtration from the lipopolysaccharide of the Ra mutant SH180 and the Rb mutant TV160 inhibited precipitation. On a molar basis the SH180 core polysaccharide was approximately as active as α‐D‐Gal‐(1→6)‐D‐Glc while the TV160 fragment was five times more active. The precipitating ability of TV160 lipopolysaccharide was abolished after treatment with the enzyme D‐galactose oxidase. The structural element in the core region in common for all reactive lipopolysaccharide is nonreducing D‐Gal linked α(1→6) to D‐Glc. It is suggested that this residue is the “immunodominant” sugar responsible for precipitation. In the TV160 lipopolysaccharide there is an additional nonreducing D‐Gal residue linked α(1→3) to the same D‐Glc. It is possible that the presence of this residue increases the binding. The experiments suggest that precipitation occurs when both reagents are polyvalent although each individual interaction may be very weak.