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, que nches the monomers only, and therefore it is expected that under condi tions in which the monomers are in equilibrium with the excimers due t o 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), define d as K-E, and K-E/K-M = 1.0. This is indeed the case for pyrene and py rene valerate in egg phosphatidylcholine small unilamellar vesicles. H owever, for pyrene decanoate and pyrene dodecanoate in these vesicles, and for [12-(1-pyrenyl)dodecanoyl]sphingosylphosphocholine in a matri x of either N-stearoyl sphingosylphosphocholine or 1-palmitoyl -2-oleo yl phosphatidylcholine, K-E < K-M. This can be explained either by the existence of (a) two subpopulations of excimers, one in fast equilibr ium with the monomers and the other, related to ground-state protoaggr egates of pyrene lipids; (b) two monomer subpopulations where part of M cannot be quenched by DEA; or (c) two monomer subpopulations, both q uenched by DEA, but only one of which produces excimers, The good agre ement between the photophysical processes determined by steady state a nd time-resolved measurements supports the third explanation for the b ilayers containing pyrene phospholipids. It also suggests that the mai n factors determining the immiscibility of pyrene lipids in phospholip id bilayers are the temperature, the difference in the gel-to-liquid-c rystalline phase transition temperature (Delta T-m) between the matrix and the pyrene lipid, and the structural differences between the matr ix 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 isotherm al microscopic immiscibility in membranes, This novel approach has the following advantages: applicability to fluid phase immiscibility, req uirement of a relatively low mol fraction of pyrene lipids, and concei vably, applicability to biological membranes.