TRANSBILAYER LIPID REDISTRIBUTION ACCOMPANIES POLY(ETHYLENE GLYCOL) TREATMENT OF MODEL MEMBRANES BUT IS NOT INDUCED BY FUSION

Small, unilamellar vesicles (SW) or large, unilamellar vesicles (LUV) containing a small amount of -2,1,3-benzoxadiazol-4-yl)phosphatidyleth anolamine (NBD-PE) or the corresponding phosphatidylserine (NBD-PS) we re made asymmetric in labeled lipid by reduction of outer leaflet prob e with externally added sodium dithionite. Following removal of dithio nite, transbilayer lipid redistribution (presumably due to lipid flip- flop) was indicated by a loss of fluorescence intensity upon readditio n of dithionite. Vesicle rupture and fusion in the presence of PEG wer e measured by changes in the fluorescence of trapped Tb3+ complexed wi th dipicolinic acid (DPA) or by the increase of fluorescence from 8-am inonaphthalene-1,3,6-trisulfonic acid (ANTS) coencapsulated with a que nching agent. NBD-PE redistributed slowly (similar to 2%/h) in all sym metrically labeled vesicles examined, while NBD-PS did not NBD-PE redi stribution was not accelerated by treatment of vesicles with PEG below concentrations chat induced vesicle rupture or fusion, but was enhanc ed at or above these PEG concentrations. SW prepared from hen egg yolk phosphatidylcholine (egg PC) or from dioleoylphosphatidylcholine (DOP C)/dilinolenoylphosphatidylcholine (diC(18:3)PC) (85/15) mixtures were shown to fuse without rupturing in the presence of appropriate concen trations of PEG. Matching the osmolalities inside and outside the vesi cle mitigated against rupture but did not prevent fusion. Under these conditions, NBD-PE flip-flop was proportional to the amount of fusion, but with different proportionality constants for the two lipid system s, while NBD-PS flip-flop did not occur. Redistribution of total mass from the outer to the inner leaflet during the fusion process could be detected in terms of a change in the ratio of dithionite-reducible pr obe to total probe. Both probes detected inwardly directed redistribut ion of lipid mass under conditions that induced fusion of SUV. We conc lude that inward mass redistribution must accompany PEG-mediated SW fu sion, but that lipid flip-flop is not mechanistically related to the f usion process.