Filipin fluorescence quenching by spin-labeled probes: studies in aqueous solution and in a membrane model system.
A detailed photophysical study of the fluorescence quenching (transient and steady state) of the macrolide antibiotic filipin by nitroxide-substituted fatty acids and a cholesterol derivative was carried out, aimed at determining its transverse position in a model system of membranes (multilamellar vesicles of dipalmitoylphosphatidylcholine). Filipin partitions efficiently into membranes (Kp = (5.0 +/- 1.0).10(3), 20 degrees C) and it was concluded that the antibiotic is buried in the membrane, away from the lipid-water interface. In addition, information on the organization of the quenchers was also obtained. The 5-nitroxide derivative of the fatty acid is essentially randomly distributed, while the 16-nitroxide is aggregated at concentrations higher than approximately 5% molar. For the cholesterol compound the results point to a phase separation at concentrations higher than 3% molar (below this limit concentration filipin associates with the derivatized sterol with KA = 20 M-1, assuming a 1:1 interaction). We propose that this phase separation and the aggregation state of filipin in the aqueous solution may be key processes in the antibiotic mode of action. A systematic and general approach to fluorescence quenching data analysis in complex (e.g., biochemical) systems is also presented.
Castanho
M
M
Centro de Química-Física Molecular, Instituto Superior Técnico, Lisboa, Portugal.
Prieto
M
M
D015060:1,2-Dipalmitoylphosphatidylcholine; D000975:Antioxidants; D003497:Cyclic N-Oxides; D005372:Filipin / Q000737:chemistry*; D007700:Kinetics; D008051:Lipid Bilayers; D008433:Mathematics; D008567:Membranes, Artificial; D008954:Models, Biological; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013113:Spin Labels
D000975:Antioxidants; D003497:Cyclic N-Oxides; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D012996:Solutions; D013113:Spin Labels; D015060:1,2-Dipalmitoylphosphatidylcholine; D005372:Filipin; C001803:tempol