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) by LPS. This process is presently not adequately understood. Using the equilibrium dialysis apparatus and a highly purified and well-characterized radiolabeled deep rough chemotype LPS ([C-14]ReLPS) from Escherichia 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]ReLPS suspended in 50 mM Tris-HCl-100 mM KCl buffer (or 50 mM MES-100 mM 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, respectively. These experimental data satisfactorily fitted the curve generated by the solubility equation C(T)=S0(1+K5/[H+])/([H+]/K4'+1), where S0 is the concentration of the tetraanionic ReLPS, K5 is the dissociation constant of the tetraanionic ReLPS in solution, and K4' is the dissociation constant of the trianionic ReLPS at the surface of the solid particles in suspension. The increase in solubility of ReLPS with increase in pH from 7.00 to 8.20 is primarily caused by the formation of the pentaanionic form from the tetraanions. The pK5 (primarily the second 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 (monosaccharide monophosphates where pK = 6.1 for the second dissociation) because of electrostatic effects caused by the other phosphate and carboxylate groups of two 2-keto-3-deoxyoctonate (Kdo) moieties of ReLPS. Using the same theoretical arguments, pK6 was calculated to be much higher, 10.8. In the absence of Kdo groups, as is the case of 1,4'-diphosphoryl lipid A, the same theoretical approach showed that the pK values of the second dissociations of the two phosphate groups are lower and 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. This 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, pK values, ionic states, and charge distribution of a model, toxic LPS dissolved in aqueous media. Such information is necessary to understand the molecular basis for the biological activities of LPS.
