Abstract
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 (Ksv) for monomers (M), defined as KM, should be identical to that of the excimer (E), defined as KE, and KE/KM = 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, KE < KM. 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 (deltaTm) 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 KE/KM 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.
Highlights
In lipid bilayers, pyrene and pyrene-labeled lipids form excimers in a concentration-dependent manner
At the level of resolution of the wavelength of visible light (ϳ0.5 m), studies based on determination of the immobile fraction in a fluorescence recovery after photobleaching experiment indicate that biological membranes are laterally heterogeneous and have in-plane domains distributed in a homogeneous lipid continuum (4 – 6)
The model systems to be used to characterize lateral organization at the submicron range should be applicable to both lipid bilayers and biological membranes, which is the case for the fluorescence recovery after photobleaching approach used for the micron range organization (5, 7, 8, 11)
Summary
Pyrene and pyrene-labeled lipids form excimers in a concentration-dependent manner. We evaluate a novel approach based on the ability to detect and quantify the lateral organization of pyrene-labeled lipids present in lipid bilayers of defined compositions This method can be applied to biological membranes that can be labeled by introducing a pyrene fluorophore either metabolically (for review, see Ref. 16), or enzymatically by phospholipid exchange proteins (17) using liposomes as donors. The most common use of pyrene and pyrene lipid is based on the ability of one excited pyrene moiety, together with a nonexcited pyrene moiety, to form an excited-state dimer (excimer) This reaction was studied extensively in organic solvents and proved to be a diffusion-controlled bimolecular reaction (19, 20). Chong and co-workers (26, 27), using pyrene lipids, showed that the fluorescent lipids were organized into hexagonal super-lattices in the bilayer plane, information that could not be obtained by other methods
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