Continuous measurement of rapid transbilayer movement of a pyrene-labeled phospholipid analogue

The excimer forming capacity of the fluorescent moiety pyrene is employed to measure continuously the transbilayer (re)distribution of a pyrene-labeled phosphatidylcholine analogue (pyPC) in liposomal membranes. pyPC with a lauroyl residue ( sn-1 position) and a short (butyroyl) fatty acid chain ( sn-2 position) bearing the pyrene moiety incorporates rapidly into the outer leaflet of liposomes. The fluorescence intensities of excimers ( I E) and of monomers ( I M) of pyPC depend on the concentration of the analogue in a membrane leaflet. Therefore, the redistribution of pyPC from the outer to the inner leaflet can be followed by changes of the ratio I E/ I M. The transbilayer movement of pyPC in pure phospholipid vesicles is very slow indicated by a constant I E/ I M. However, addition of membrane active peptides (melittin, magainin 2 amide or a mutant of magainin 2 amide) induced a rapid translocation of pyPC from the outer to the inner leaflet. An approach is presented which allows estimating the transbilayer distribution of pyPC from the measured ratio I E/ I M.