Determination of the rate of rapid lipid transfer induced by poly(ethylene glycol) using the SLM Fourier transform phase and modulation spectrofluorometer

Abstract

Rate constants were determined for the transfer of the fluorescent lipid probe 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] oxy]carbonyl]-3-sn-phosphatidylcholine (DPHpPC) between large, unilamellar extrusion vesicles composed either of dipalmitoyl phosphatidylcholine (DPPC) or of DPPC mixed with a small amount (0.5 mol%) of lyso phosphatidylcholine (Lyso PC). Transfer of the lipid probe in the presence of varying concentrations of poly(ethylene glycol) (PEG) was monitored using the SLM 48000-MHF Multi-Harmonic Fourier Transform phase and modulation spectrofluorometer to collect multifrequency phase and modulation fluorescence data sets on a subsecond time scale. The unique ability of this instrument to yield accurate fluorescence lifetime data on this time scale allowed transfer to be detected in terms of a time-dependent change in the fluorescent lifetime distribution associated with the lipid-like DPHpPC probe. This probe demonstrates two short fluoresence decay times (ca. 1.1-1.4 and 4.3-4.8 ns) in a probe-rich environment but a single long lifetime (ca. 7 ns) in a probe-poor environment. A simple two-state model for initial lipid transfer was used to analyze the multifrequency data sets collected over a 4-s time frame to obtain the time rate of change of the concentrations of donor and acceptor probe populations following rapid mixing of vesicles with PEG. The ability to measure fluorescence lifetimes on this time scale has allowed us to show that the of rate of lipid transfer increased dramatically at 35% PEG in both fusing and nonfusing vesicle systems. These results are interpreted in terms of a distinct interbilayer structure associated with intimate bilayer contact induced by high and potentially fusogenic concentrations of PEG.