Lipopolysaccharide-protein interactions: determination of dissociation constants by affinity electrophoresis.
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'-methylenebisacrylamide 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.
Borneleit
P
P
Sektion Biowissenschaften, Karl-Marx-Universität, Leipzig, German Democratic Republic.
Blechschmidt
B
B
Kleber
H P
HP
D000091:Acetobacter / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008070:Lipopolysaccharides / Q000032:analysis*
D001425:Bacterial Outer Membrane Proteins; D008070:Lipopolysaccharides