TITRATION CALORIMETRIC STUDIES TO ELUCIDATE THE SPECIFICITY OF THE INTERACTIONS OF POLYMYXIN-B WITH LIPOPOLYSACCHARIDES AND LIPID-A

Lipopolysaccharide (LPS), the major cell wall constituent of Gram-nega tive bacteria, evokes a multitude of biological effects in mammals inc luding pyrogenicity and toxic shock syndrome. Polymyxin B (PmB), a pol ycationic cyclic peptide, is known to neutralize most of its activitie s. The nature of the interaction of PmB with LPS and lipid A was inves tigated by isothermal titration calorimetry. PmB binds to LPS as well as lipid A stoichiometrically and non-co-operatively with micromolar a ffinity. These interactions are driven primarily by a favourable chang e in entropy (Delta S) and are endothermic in nature. These positive c hanges in enthalpies decrease with increasing temperature, yielding a heat capacity change, Delta C-p, of -2385 J . mol(-1). degree(-1) for PmB-LPS interactions while the binding of PmB to lipid A displays a De lta C-p of -2259 J . mol(-1). degree-1. The negative heat capacity cha nges provide strong evidence for the role of hydrophobic interactions as the driving force for the association of PmB with LPS and lipid A. A correlation of the energetics of these interactions with analyses of the molecular models of PmB suggests that a cluster of solvent-expose d non-polar amino acid side-chains that line one surface of the molecu le, together with a ring of positively charged residues on its other s urface, are responsible for its strong and stoichiometric binding to L PS.