Distribution of Hydrophobic Probe Molecules in Lipid Bilayers. 2. Time-Resolved Fluorescence Anisotropy Study of Perylene in Vesicles

The orientational distribution and rotational dynamics of planar perylene molecules incorporated into spherical vesicles of palmitoyl-δ-9-oleoyl(16:0,18:1)phophatidylcholine (POPC) was studied using time-resolved fluorescence anisotropy. The experimental anisotropy decay curves were analyzed using a global target approach. Here, anisotropy curves obtained at six temperatures above the gel-lamellar phase transition of the vesicles as well as the two combinations of excitation and emission wavelengths, (256, 470 nm) and (410, 470 nm), were fitted simultaneously. We have utilized two sets of orientational distributions of perylene in the bilayer. One set contains various one-population orientational distributions, while the other consists of models in which the probes are distributed over two distinct orientational populations. We find that in general the models yield statistically equivalant solutions, though the two-population models need a substantially smaller number of fit parameters. The existence of two distinct orientational populations of perylene in the lipid vesicle bilayer is in agreement with the results of Monte Carlo dynamics simulations in the preceding paper, but we argue that additional independent information is needed in order to remove the remaining ambiguities. We conclude that time-resolved anisotropy experiments on macroscopically unoriented samples do not provide sufficient information in order to fully characterize the orientational distribution of probe molecules in the bilayers.