Polymyxin B-phosphatidylglycerol interactions. A monolayer (pi, delta V) study.

Through a monolayer investigation (pi, delta V), it is shown that the cationic antibiotic polymyxin B (or E) strongly interacts with films of acidic lipids, namely the didodecanoyl- and dihexadecanoylphosphatidylglycerol. The zwitterionic dihexadecanoylphosphatidylcholine was an unsuitable substrate. Interactions occurred at and above a polymyxin B concentration in the subphase of 2.5 . 10(-7) M, bringing about a considerable increase of both pi and delta V. These interactions proceeded in two steps, as revealed by a biphasic change of delta V with time. They were independent of the film molecular packing (fluid or gel states) and of the initial film pressure. Since it was possible to monitor the relative number of polymyxin B and didodecanoyl- or dihexadecanoylphosphatidylglycerol molecules in the monolayer, it is demonstrated that, at saturation, one polymyxin B molecule is bound to five phosphatidylglycerol molecules, a result which accounts for an exact neutralization of the charges. From competition experiments, it is shown that Na+ is ineffective in removing polymyxin B from the interface. Ca2+ appeared to be a stronger competitor but no complete antibiotic desorption was observed even at a Ca2+ concentration of 100 mM. As a working hypothesis, the antibiotic/lipid (1/5) system was assumed to constitute by itself one molecular species. The mixing of the polymyxin B/didodecanoylphosphatidylglycerol (1/5) system with an excess of lipid molecules in the monolayer was found to be ideal both in terms of pi and delta V. With dihexadecanoylphosphatidylglycerol, a small condensing effect could be detected only at intermediate surface pressures, in a region where the lipid phase transition occurred. The molecular area of polymyxin B interacting with didodecanoylphosphatidylglycerol can be calculated to be 1.23 +/- 0.05 nm2. It is proposed that the whole antibiotic molecule penetrates the film, the five bound lipid molecules being distributed aroung the peptide structure, at given positions imposed by the five 2,4-diaminobutyric acid residues.