EFFECT OF PH ON SOLUBILITY AND IONIC STATE OF LIPOPOLYSACCHARIDE OBTAINED FROM THE DEEP ROUGH MUTANT OF ESCHERICHIA-COLI

The dissociation of the highly aggregated form of lipopolysaccharide ( LPS) from Gram-negative bacteria to the monomeric (or soluble) form is though to be the initial step in the activation of responding cells ( macrophages, B-cells, neutrophils, monocytes, and endothelial cells) b y LPS. This process is presently not adequately understood. Using the equilibrium dialysis apparatus and a highly purified and well-characte rized radiolabeled deep rough chemotype LPS ([C-14]ReLPS) from Escheri chia coli D31m4, we have examined the effect of pH on its solubility ( CT) and ionic states in aqueous media. The solubility range of [C-14]R eLPS suspended in 50 mM Tris-HCl-100 mM KCl buffer (or 50 mM MES-100 m M KCl buffer at pH 6.5) was determined to be from (2.91 +/- 0.01) X 10 -8 to (4.55 +/- 0.07) X 10(-8) M over a pH range of 6.50-8.20, respect ively. These experimental data satisfactorily fitted the curve generat ed by the solubility equation C(T)=S0(1+K5/[H+])/([H+]/K4'+1), where S 0 is the concentration of the tetraanionic ReLPS, K5 is the dissociati on constant of the tetraanionic ReLPS in solution, and K4' is the diss ociation constant of the trianionic ReLPS at the surface of the solid particles in suspension. The increase in solubility of ReLPS with incr ease in pH from 7.00 to 8.20 is primarily caused by the formation of t he pentaanionic form from the tetraanions. The pK5 (primarily the seco nd dissociation of the l-phosphate) of ReLPS was determined to be 8.58 from experimental data. Theoretical arguments were presented to show that this value is higher than that for simple model compounds (monosa ccharide monophosphates where pK = 6.1 for the second dissociation) be cause of electrostatic effects caused by the other phosphate and carbo xylate groups of two 2-keto-3-deoxyoctonate (Kdo) moieties of ReLPS. U sing the same theoretical arguments, pK6 was calculated to be much hig her, 10.8. In the absence of Kdo groups, as is the case of 1,4'-diphos phoryl lipid A, the same theoretical approach showed that the pK value s of the second dissociations of the two phosphate groups are lower an d are separated by smaller numbers, giving calculated pK values of 6.9 and 7.8. The presence of nearby Kdo units in ReLPS gives the molecule fewer negative charges on the phosphate groups compared to lipid A. T his may contribute to better binding of ReLPS to the LPS receptors and may explain its higher biological activity when compared to lipid A. From these results, we can now provide the monomeric concentrations, p K values, ionic states, and charge distribution of a model, toxic LPS dissolved in aqueous media. Such information is necessary to understan d the molecular basis for the biological activities of LPS.