Lipopolysaccharide‐protein interactions: Determination of dissociation constants by affinity electrophoresis

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Abstract
An affinity electrophoresis system is described to allow determination of dissociation constants of lipopolysaccharide (LPS)-protein complexes. The LPS ligand is incorporated into polyacrylamide gels by addition to the polyacrylamide-N, N′-methyl-enebisacrylamide polymerization mixture. Quantitative evaluation revealed formation of immobile protein-ligand complexes. The method was applied both to R- and S- form LPS from Acinetobacter calcoaceticus. For a heat-modifiable outer membrane protein with Mr 18 000 from strain 69V the dissociation constant was determined to be 0.5 mM (EDTA-salt extracted R-LPS) and 0.3 mM (phenol-chloroform-petrolether extracted R-LPS). In comparison, for another A. calcoaceticus strain, CCM 5593, a higher dissociation constant of 1.0 mM (phenol-chloroform-petrolether extracted R-LPS) - indicative of lower affinity - was obtained. When S-LPS from A. calcoaceticus 69V was incorporated into the affinity gels, a dissociation constant of 0.02 mM was determined which indicates much stronger interactions than those exerted by R-LPS forms.