A photophysical study of the polyene antibiotic filipin. Self-aggregation and filipin--ergosterol interaction.

Filipin, a macrolide polyene antibiotic, is known to interact selectively with ergosterol, a constituent of fungi membranes. In this work, the fluorescence resonance energy transfer (FRET) between a fluorescent analog of ergosterol, dehydroergosterol (DHE), and filipin was measured in small unilamellar vesicles of dipalmitoylphosphatidylcholine at 25 degrees C. The time-resolved FRET results were rationalized in the framework of the mean concentration model, and were complemented with steady-state fluorescence intensity, anisotropy and absorption measurements. The results point to the formation of both DHE--filipin aggregates (evidence from static quenching of DHE fluorescence by filipin) and filipin--filipin aggregates (evidence from: (i) the FRET acceptor concentration distributions; (ii) spectral changes of filipin absorption in the vesicles, the excitonic interaction suggesting a stack arrangement; (iii) filipin fluorescence self-quenching), even in presence of DHE and low antibiotic mole fractions (<1 mol%). These results point out that apparently contradictory biochemical models for the action of filipin (some based on the presence of sterols, others not) can be equally valid. Moreover, since results (ii) and (iii) are also observed when a sterol is present, both models of action can actually coexist in membranes with a low sterol content.