A method for quantitative interpretation of fluorescence detection of poly(ethylene glycol)-mediated 1-palmitoyl-2-[[[2-[4-(phenyl-trans-1,3,5-hexatrienyl) phenyl]ethyl]oxyl]carbonyl]3-sn-phosphatidylcholine (DPHpPC) transfer and fusion between phospholipid vesicles in the dehydrated state.
A method has been developed for calculating the expected fluorescence lifetime of the DPH p PC probe distributed between different membrane environments. We show how this method can be used to distinguish between lipid transfer and fusion between large unilamellar vesicles occurring in the presence of poly(ethylene glycol) (PEG). This application of the calculation took into consideration the heterogeneity of microenvironments experienced by the probe in a sample containing vesicle aggregates of different sizes. Assuming that the aggregate size distribution was a delta function of the aggregate size, comparison of the calculated and observed lifetimes yielded an estimate of the vesicle aggregate size. For vesicles of varying compositions in the presence of dehydrating concentrations of PEG, this method suggested that only small aggreggates formed. For vesicles that could be demonstrated by other means not to have fused, the data were consistent with lipid transfer occurring only between the outer leaflets of two to four vesicles, even at high PEG concentrations. For vesicles that could be demonstrated to fuse by contents mixing and size changes, the fluorescence lifetime data were consistent with lipid transfer between both the inner and the outer leaflets of two to four fused vesicles. At very high PEG concentrations, where extensive rupture and large, multilamellar products were previously observed, the lifetime data were consistent with much more extensive lipid transfer within larger aggregates. The agreement of predictions made on the basis of lifetime measurements with other observations attests to the validity of the fluorescence lifetime method. In addition, the model and data presented here provide evidence that fusion occurs between small numbers of PEG-aggregated vesicles before the removal of PEG.
Department of Biochemistry & Biophysics, University of North Carolina, 27599-7260, Chapel Hill, North Carolina.
J R
JR
Wu
B R
BR
Lentz