Lateral diffusion in the hydrophobic region of membranes: use of pyrene excimers as optical probes

Abstract

A new (optical) method of diffusion measurement is described which allows the determination of the coefficient of lateral diffusion, D diff, of aromatic molecules in the hydrophobic region of lipid bilayers. In the present work pyrene is used as a fluorescence probe. The method is based on the finding that the formation of excited pyrene dimers (excimers) in fluid membranes is a diffusion controlled process. The value of D diff is obtained from the second order rate constant of excimer formation which is determined from the ratio of the excimer to the monomer fluorescence quantum yields The method has been applied to dipalmitoyllecithin membranes and mixed dipalmitoyllecithin-cholesterol bilayers. The diffusion coefficient for pyrene in dipalmitoyllecithin membranes at 50 °C (that is above the lipid phase transition) is D diff = 1.4 · 10 −7 cm 2/s. The activation energy for the pyrene diffusion is ΔE = 8.8 kcal/mole. Below the lipid phase transition pyrene aggregates into small clusters embedded in the lipid matrix. For bilayers of unsonicated lipid dispersions the cluster formation is observed at very low pyrene concentration of 0.1 mole %, showing that the lipid matrix forms a rather regular crystal structure. Above the lipid phase transition cholesterol reduces the lateral mobility of pyrene considerably (at 30 mole cholesterol: D diff = 0.64 · 10 −7 cm 2/s). Below the phase transition cholesterol suppresses the cluster formation. The excimer formation of pyrene is also sensitive to the pretransition of the lipid matrix occurring about 10 °C below the main lipid phase transition at T t = 41 °C.