Physical, Chemical, and Immunological Properties of Lipopolysaccharide Released from Escherichia coli by Ethylenediaminetetraacetate

When Escherichia coli O111:B4 is exposed briefly to ethylenediaminetetraacetate (EDTA) it releases 30 to 50% of its lipopolysaccharide, determined not only by measuring percentage release of colitose, a sugar unique to this polymer, but also by measuring the amount of phenol-extractable lipopolysaccharide remaining with the cell. The fraction released cannot be significantly increased above 50% by several variations in procedure. The high molecular weight material released by EDTA consists of 85 to 90% lipopolysaccharide, 5 to 10% protein, and 5% phospholipid by weight. When compared with lipopolysaccharide obtained by phenol extraction of control cells, this preparation is more immunogenic, and at least as lethal in mice. It yields two fractions on ultracentrifugation. One fraction, containing 40 to 60% of the lipopolysaccharide and most of the protein and lipid, is heterodisperse on ultracentrifugation but changes to a single symmetrical peak with a sedimentation constant of approximately 11 S when the phospholipid is extracted. Its composition is the same as that of control lipopolysaccharide obtained by phenol extraction of normal cells of this strain. The second fraction contains 30 to 40% of the released lipopolysaccharide and yields on ultracentrifugation a hyper-sharp peak with a sedimentation constant of approximately 5.5 S at infinite dilution. Per mole of heptose this fraction is twice as rich in glucose and galactose, and 4 times as rich in colitose, as control lipopolysaccharide. EDTA treatment also releases lipopolysaccharide from two other strains of E. coli and two species of Salmonella.