Small,
unilamellar vesicles (SUV) or large,
unilamellar vesicles (LUV) containing a small amount of N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (
NBD-PE) or the corresponding
phosphatidylserine (
NBD-PS) were made asymmetric in labeled
lipid by reduction of outer leaflet probe with externally added
sodium dithionite. Following removal of
dithionite, transbilayer
lipid redistribution (presumably due to
lipid flip-flop) was indicated by a loss of fluorescence intensity upon readdition of
dithionite. Vesicle
rupture and fusion in the presence of PEG were measured by changes in the fluorescence of trapped Tb3+ complexed with
dipicolinic acid (DPA) or by the increase of fluorescence from 8-aminonaphthalene-1,3,6-trisulfonic
acid (ANTS) coencapsulated with a quenching agent.
NBD-PE redistributed slowly (approximately 2%/h) in all symmetrically labeled vesicles examined, while
NBD-PS did not.
NBD-PE redistribution was not accelerated by treatment of vesicles with PEG below concentrations that induced vesicle
rupture or fusion, but was enhanced at or above these PEG concentrations. SUV prepared from hen egg yolk
phosphatidylcholine (egg PC) or from
dioleoylphosphatidylcholine (
DOPC)/dilinolenoylphosphatidylcholine (
diC(18:3)PC) (85/15) mixtures were shown to fuse without rupturing in the presence of appropriate concentrations of PEG. Matching the osmolalities inside and outside the vesicle 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 systems, 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 probe to total probe. Both probes detected inwardly directed redistribution of
lipid mass under conditions that induced fusion of SUV. We conclude that inward mass redistribution must accompany PEG-mediated SUV fusion, but that
lipid flip-flop is not mechanistically related to the fusion process.