Lateral organization of pyrene-labeled lipids in bilayers as determined from the deviation from equilibrium between pyrene monomers and excimers

In lipid bilayers, pyrene and pyrene-labeled lipids form excimers in a concentration-dependent manner. The aromatic amine N,N-diethylaniline (DEA), which has a high membrane-to-medium partition coefficient, quenches the monomers only, and therefore it is expected that under conditions in which the monomers are in equilibrium with the excimers due to the mass law, the Stern-Volmer coefficient (K(sv)) for monomers (M), defined as K(M), should be identical to that of the excimer (E), defined as K(E), and K(E)/K(M) = 1.0. This is indeed the case for pyrene and pyrene valerate in egg phosphatidylcholine small unilamellar vesicles. However, for pyrene decanoate and pyrene dodecanoate in these vesicles, and for N-[12-(1- pyrenyl)dodecanoyl]-sphingosylphosphocholine in a matrix of either N-stearoyl sphingosylphosphocholine or 1-palmitoyl-2-oleoyl phosphatidylcholine, K(E) < K(M). This can be explained either by the existence of (a) two subpopulations of excimers, one in fast equilibrium with the monomers and the other, related to ground-state protoaggregates of pyrene lipids; (b) two monomer subpopulations where part of M cannot be quenched by DEA; or (c) two monomer subpopulations, both quenched by DEA, but only one of which produces excimers. The good agreement between the photophysical processes determined by steady state and time-resolved measurements supports the third explanation for the bilayers containing pyrene phospholipids. It also suggests that the main factors determining the immiscibility of pyrene lipids in phospholipid bilayers are the temperature, the difference in the gel-to-liquid-crystalline phase transition temperature (ΔT(m)) between the matrix and the pyrene lipid, and the structural differences between the matrix lipid and the pyrene-labeled lipid. These results indicate that the K(E)/K(M) ratio can serve as a very sensitive tool to quantify isothermal microscopic immiscibility in membranes. This novel approach has the following advantages: applicability to fluid phase immiscibility, requirement of a relatively low mol fraction of pyrene lipids, and conceivably, applicability to biological membranes.