Polymyxin interaction with negatively charged lipid bilayer membranes and the competitive effect of Ca2+.

ABSTRACT

The binding of cationic polymyxin-B to negatively charged phosphatidic acid and phosphatidylglycerol membranes has been investigated by fluorescence polarization study. Competition experiments with Ca2+ were performed. 1. Binding of polymyxin-B to mixed dipalmitoylphosphatidic acid/distearoylphosphatidylcholine membranes leads to a phase separation. Domains of polymyxin-bound phosphatidic acid are formed. 2. Ca2+ is found to be a strong competitor in displacing polymyxin from the complex in the mixed membrane system. Complete displacement is obtained at pH 9.0. With decreasing pH value, Ca2+ becomes a less strong competitor and is ineffective at pH 5.0. 3. Binding of polymyxin to dipalmitoylphosphatidylglycerol membranes is observed. Incorporation of polymyxin lowers the lipid phase transition by 10 degrees C. One polymyxin is found to bind five phosphatidylglycerol molecules. The binding curve is determined and in contrast to phosphatidic acid membranes, a noncooperative binding could be established. 4. Addition of Ca2+ decreases the amount of phosphatidylglycerol bound to polymyxin by about 20%. No complete displacement is achieved even at 10-fold excess of Ca2+ with respect to phosphatidylglycerol.