Physicochemical properties of the lipopolysaccharide unit that activates B lymphocytes

We have examined the physical state of highly purified deep rough chemotype lipopolysaccharide (ReLPS) from Escherichia coli D31m4 as an aqueous suspension and as complexes with bovine serum albumin min (BSA). The ReLPS suspension showed large ellipsoidal particles 12-38 nm wide and 40-100 nm long. The solubility of this form of ReLPS was determined by equilibrium dialysis experiments to be 3.3 x 10(-8) M at 22 degrees C and 2.8 x 10(-8) M at 37 degrees C in 150 mM Tris-KCl, pH 7.5; 3.0 x 10(-8) M at 37 degrees C in 0.75 mM Tris-KCl, pH 7.5. The BSA-ReLPS complexes were fractionated on a Sephacryl S-200 column to yield peaks I and II with apparent masses of about 240 and 70 kDa, respectively. Peak II was a BSA monomer with estimated BSA:ReLPS molar ratios of 1:1-1:7. The ReLPS suspension and the two complexes were compared as antigens in enzyme-linked immunosorbent assays using three select monoclonal antibodies to lipopolysaccharide. The results were consistent with the high state of disaggregation of the ReLPS in both peaks I and II. Since the ReLPS in these complexes were not visible by electron microscopy, they did not contain vesicles or large particles. All forms of ReLPS tested were capable of stimulating 70Z/3, a lipopolysaccharide-responsive murine pre-B cell line. However, peak II was consistently more stimulatory at very low concentrations than the other preparations. The maximally stimulatory concentration of ReLPS for 70Z/3 cells was 40 ng/ml (1.6 x 10(-8) M) for peak II and 70 ng/ml (2.8 x 10(-8) M) for the ReLPS suspension. As expected, the above concentrations were at or below the solubility of the ReLPS. These results suggested that the highly disaggregated form of ReLPS (possibly the monomer) is the active unit that stimulates the cellular response in 70Z/3 cells.