Kachel K, Asuncion-Punzalan E, London E
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, 11794-5215, USA.
Biochim Biophys Acta. 1998 Sep 23;1374(1-2):63-76. doi: 10.1016/s0005-2736(98)00126-6.
The location of commonly used charged fluorescent membrane probes in membranes was determined in order to: (1) investigate the relationship between the structure of hydrophobic molecules and their depth within membranes; and (2) aid interpretation of experiments in which these fluorescent probes are used to examine membrane structure. Membrane depth was calculated using parallax analysis, a method in which the quenching induced by lipids carrying a nitroxide group at different locations in the membrane is compared. Shallow locations were found for xanthene dyes (fluorescein, eosin, Texas Red and rhodamine) both in free form and when attached either to the headgroup of phospholipids or long hydrocarbon chains. The exact structure of the xanthene and the nature of its linkage to lipid had only a modest effect on membrane location, which ranged between 19 and 24 A from the center of the bilayer in a charged state. Thus, the location of these fluorophores largely reflects their intrinsic properties rather than the nature of the groups to which they are attached. Furthermore, cationic and anionic xanthene derivatives had similar depths, indicating the type of charge does not have a large effect on depth. Consistent with this conclusion, shallow locations were also found for other hydrocarbon chain-linked cationic (acridine orange and styrylpyridinium) and anionic (coumarin, anilinonaphthalenesulfonic acid (ANS), and toluidinylnaphthalenesulfonic acid (TNS)) charged probes. These all located at 16-18 A from the bilayer center. We conclude that both anionic and cationic molecules that are otherwise hydrophobic predominantly occupy shallow locations within the polar headgroup region of the bilayer no matter how hydrophobic the molecule to which they are linked. This depth is significantly shallower than that occupied by most previously studied uncharged polar molecules that locate near the membrane surface. Consistent with this conclusion, a 2-4 A deeper location was found for xanthene probes with no net charge. In other experiments, methods to avoid chemical reactions that can distort the measurement of depth by fluorescence quenching were developed.
确定了常用的带电荷荧光膜探针在膜中的位置，目的是：（1）研究疏水分子的结构与其在膜内深度之间的关系；（2）辅助解释使用这些荧光探针检测膜结构的实验。使用视差分析计算膜深度，该方法是比较膜中不同位置带有氮氧化物基团的脂质所诱导的猝灭。无论是游离形式还是连接到磷脂头基团或长烃链上，都发现呫吨染料（荧光素、伊红、德克萨斯红和罗丹明）处于较浅位置。呫吨的确切结构及其与脂质的连接性质对膜位置的影响较小，在带电状态下，其距离双层中心的范围为19至24埃。因此，这些荧光团的位置很大程度上反映了它们的固有性质，而不是它们所连接基团的性质。此外，阳离子和阴离子呫吨衍生物具有相似的深度，表明电荷类型对深度没有很大影响。与这一结论一致，对于其他烃链连接的阳离子（吖啶橙和苯乙烯基吡啶鎓）和阴离子（香豆素、苯胺基萘磺酸（ANS）和甲苯胺基萘磺酸（TNS））带电探针也发现了较浅位置。这些探针都位于距离双层中心16 - 18埃处。我们得出结论，无论与之连接的分子疏水性如何，原本疏水的阴离子和阳离子分子主要占据双层极性头基团区域内的浅位置。这个深度明显比大多数先前研究的位于膜表面附近的不带电极性分子所占据的深度要浅。与这一结论一致，发现不带净电荷的呫吨探针位置要深2 - 4埃。在其他实验中，开发了避免化学反应的方法，这些反应会因荧光猝灭而扭曲深度测量。
