ANALYSIS OF THE BINDING OF POLYMYXIN-B TO ENDOTOXIC LIPID-A AND CORE GLYCOLIPID USING A FLUORESCENT DISPLACEMENT PROBE

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 muM to 26 muM, while that obtained for core glycolipid from Salmonella minnesota Re595 yielded a K(d) of 22 muM to 28 muM 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 (Horovitz, A., and Levitzki, A. (1987) Proc. Natl. Acad. Sci. USA 84, 6654-6658). The applicability of this method for analyzing fluorescence data was validated by comparing the K(d)s 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(d)S for polymyxin B-lipid A interactions corresponding to 0.4 muM and 1.5 muM, 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 muM to 5.8 muM. 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.