Interaction of polycationic antibiotics with Pseudomonas aeruginosa lipopolysaccharide and lipid A studied by using dansyl-polymyxin.
A fluorescent derivative of polymyxin B (dansyl-polymyxin) was used to study the interaction of polycations with lipopolysaccharide (LPS) and lipid A from Pseudomonas aeruginosa. Dansyl-polymyxin became bound to LPS and lipid A sites, including Mg2+-binding sites, resulting in a 20-fold enhancement of fluorescence. A Hill plot of the binding data showed that the binding of dansyl-polymyxin to LPS was cooperative (n = 1.98) and of high affinity (S0.5 = 0.38 microM). The maximal binding capacity of LPS was approximately four molecules of dansyl-polymyxin per mol of LPS. The dansyl-polymyxin interaction with lipid A displayed similar kinetics (n = 2.26; S0.5 = 0.38 microM), and the maximal binding capacity was approximately 2 mol of dansyl-polymyxin per mol of lipid A. A variety of polycationic compounds, including gentamicin, streptomycin, and polymyxin B, as well as Mg2+, were able to displace dansyl-polymyxin bound to LPS or to lipid A. Marked differences both in terms of the degree of displacement and in terms of the amount of competing polycation required to displace a given amount of dansyl-polymyxin were observed. Addition of excess polymyxin B resulted in displacement of all of the dansyl-polymyxin, demonstrating that only polymyxin-binding sites were being probed. Our data demonstrate that polymyxin B binds to multiple sites on LPS, including sites which bind aminoglycoside antibiotics and other polycationic compounds.
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001665:Binding Sites; D005456:Fluorescent Dyes; D007700:Kinetics; D008050:Lipid A / Q000032:analysis*; D008070:Lipopolysaccharides / Q000032:analysis*; D008274:Magnesium / Q000378:metabolism; D011112:Polymyxin B; D011113:Polymyxins; D011550:Pseudomonas aeruginosa / Q000187:drug effects*; D013050:Spectrometry, Fluorescence
D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D008050:Lipid A; D008070:Lipopolysaccharides; D011113:Polymyxins; D008274:Magnesium; D011112:Polymyxin B