Lipid fluidity and membrane protein monitoring using 1,6-diphenyl-1,3,5-hexatriene

Experiments have been designed to challenge the use of stedy-state fluorescence polarizaiton with 1,6-diphenyl-1,3,5-hexatriene as an evaluator of the fluidity of cell plasma membranes. We used paraffinic systems, of defined structure and composition (liquid paraffin, soap bilayers and phospholipid liposomes--with and without incorporated proteins), to demonstrate that corresponding polarizaiton values cannot be interpreted in terms of the overall fluidity of the labeled medium. In homogeneous structured paraffinic media (lipid bilayers), knowledge of the location of the probe is essential for a consistent interpretation of the observed fluorescence polarization. Due to the highly polarizable electronic structure of the diphenylhexatriene molecule, the presence of heterogeneities with potential sites for interaction (e.g., C18-coated Si particles, albumin molecules, etc.) can lead to relatively high polarization values, even in isotropic media. In cellular systems, translocation experiments from labeled cells to added proteins show a rather localized peripheral distribution of the probe as well as its high affinity for hydrophobic sites of proteins. This and other arguments presented here suggest that although cellular polarization values represent an intricate average over all labeled hydrophobic regions of the cell (phospholipid bilayers, membrane proteins, etc.), these values might reflect, to a large extent, interactions of the probe with proteins from the inner periphery of the cell.