INTERACTION OF DIVALENT-CATIONS AND POLYMYXIN-B WITH LIPOPOLYSACCHARIDE

Cation binding to the lipopolysaccharide of Salmonella typhimurium was investigated by equilibrium dialysis and fluorometric titration of lipopolysaccharides derivatized with dimethylaminonaphthalenesulfonyl chloride (dansyl chloride). In the presence of Tris buffer (50 mM), addition of Ca2+ or Mg2+ resulted in a blue shift in the emission maximum and an increase in the relative fluorescence of dansylated lipopolysaccharides obtained from the rough mutants G30 (galactose deficient, Rc chemotype) and G30A (heptose deficient, Re chemotype). These effects were completely reversed by EDTA. Fluorometric titrations revealed two types of binding sites with markedly different affinities for divalent cations in both lipopolysaccharides. The first class, of relatively low affinity, gave Kd values of 0.2 and 1-2 mM with G30A and G30, respectively, and was attributed to pyrophosphoryl and/or phosphodiester groups of the 3-deoxy-mannoo-octulosonate (KDO)-lipid A region of the molecule. The second class, of higher affinity, yielded Kd values for Ca2+ and Mg2+ of 6 and 15 μM, respectively, with both lipopolysaccharides. Titrations with polymyxin B showed only high-affinity binding with Kd values of 0.3 μM for G30A and 0.5 μM for G30. The nature of the high-affinity Ca2+ and Mg2+ binding site was further investigated by equilibrium dialysis of lipopolysaccharides and related products with 45Ca2+. All lipopolysaccharides tested (wild type, G30, and G30A) bound 1 mol of Ca2+ per mol of lipopolysaccharide monomer with a Kd of 12-13 μM. In contrast, lipid A (obtained by mild acid hydrolysis of lipopolysaccharide) and an incomplete biosynthetic precursor of lipid A lacking 3-deoxy-d-manno-octulosonate yielded Kd values of 100 and 56 μM and a reduction in the stoichiometry of binding to 0.5 and 0.3 mol of Ca2+ per mol, respectively. The results suggest that the branched 3-deoxy-d-manno-octulosonate trisaccharide unit of lipopolysaccharide may afford a specific high-affinity site for interaction with divalent cations required for assembly and maintenance of the normal structural organization of the outer membrane. © 1979, American Chemical Society. All rights reserved.