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

Abstract 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 .