Barenholz Y, Cohen T, Haas E, Ottolenghi M
Department of Biochemistry, Hebrew University Hadassah Medical School, Jerusalem, Israel.
J Biol Chem. 1996 Feb 9;271(6):3085-90. doi: 10.1074/jbc.271.6.3085.
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.
在脂质双分子层中，芘及芘标记的脂质以浓度依赖的方式形成激基缔合物。芳香胺N，N - 二乙苯胺（DEA）具有较高的膜/介质分配系数，仅淬灭单体，因此可以预期，在质量作用定律导致单体与激基缔合物处于平衡的条件下，单体（M）的斯特恩 - 沃尔默系数（Ksv）（定义为KM）应与激基缔合物（E）的相同（定义为KE），且KE/KM = 1.0。对于芘和芘戊酸酯在卵磷脂小单层囊泡中的情况确实如此。然而，对于这些囊泡中的芘癸酸酯和芘十二酸酯，以及在N - 硬脂酰鞘氨醇磷脂酰胆碱或1 - 棕榈酰 - 2 - 油酰磷脂酰胆碱基质中的N - [12 - （1 - 芘基）十二烷酰基]鞘氨醇磷脂酰胆碱，KE < KM。这可以通过以下几种情况来解释：（a）激基缔合物存在两个亚群，一个与单体快速平衡，另一个与芘脂质的基态原聚集体有关；（b）两个单体亚群，其中部分M不能被DEA淬灭；或者（c）两个单体亚群，都能被DEA淬灭，但只有其中一个产生激基缔合物。稳态和时间分辨测量所确定的光物理过程之间的良好一致性支持了对含芘磷脂双层的第三种解释。这也表明，决定芘脂质在磷脂双层中不混溶性的主要因素是温度、基质与芘脂质之间凝胶 - 液晶相转变温度的差异（ΔTm）以及基质脂质与芘标记脂质之间的结构差异。这些结果表明，KE/KM比值可作为定量膜中等温微观不混溶性的非常灵敏的工具。这种新方法具有以下优点：适用于液相不混溶性，对芘脂质的摩尔分数要求相对较低，并且可以想象，适用于生物膜。
