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. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.