Analysis of the binding of polymyxin B to endotoxic lipid A and core glycolipid using a fluorescent displacement probe

Abstract

Dansylcadaverine, a cationic fluorescent probe binds to bacterial lipopolysaccharide and lipid A, and is displaced competitively by other compounds which possess affinity toward endotoxins. The binding parameters of dansylcadaverine for lipid A were determined by Scatchard analysis to be two apparently equivalent sites with apparent dissociation constants (K_d) ranging between 16 μM to 26 μM, while that obtained for core glycolipid from Salmonella minnesota Re595 yielded a K_d of 22 μM to 28 μM with three binding sites. The K_d of polymyxin B for lipid A was computed from dansylcadaverine displacement by the method of Horovitz and Levitzki. The applicability of this method for analyzing fluorescence data was validated by comparing the K_ds of melittin for lipid A obtained by direct Scatchard analysis, and by the Horovitz-Levitzki method. The displacement of dansylcadaverine from lipid A by polymyxin B was distinctly biphasic with K_ds for polymyxin B-lipid A interactions corresponding to 0.4 μM and 1.5 μM, probably resulting as a consequence of lipid A being a mixture of mono- and di-phosphoryl species. This was not observed with core glycolipid, for which the K_d for polymyxin was estimated to range from 1.1 μM to 5.8 μM. The use of dansylcadaverine as a displacement probe offers a novel and convenient method of quantitating the interactions of a wide variety of substances with lipid A.