Ferrer-Montiel A V, Gonzalez-Ros J M, Ferragut J A
Department of Neurochemistry (School of Medicine), University of Alicante, Spain.
Biochim Biophys Acta. 1988 Jan 22;937(2):379-86. doi: 10.1016/0005-2736(88)90260-x.
1,6-Diphenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene are fluorophores used to explore different hydrophobic domains of membrane bilayers (Andrich, M.P. and Vanderkooi, J.M. (1976) Biochemistry 15, 1257-1265; Prendergast, F.G., Haugland, R.P. and Callahan, P.J. (1981) Biochemistry 20, 7333-7338). Fluorescence resonance energy transfer between these fluorophores, acting as energy donors, and the anthracycline, daunomycin, as the acceptor, was used to analyze the interaction of the drug with natural membranes, and its relative location within the membrane bilayer. The transfer process was demonstrated by: (1) emission fluorescence of the acceptor when the samples were excited at the excitation maximum of the donor (360 nm); and (2) progressive quenching of the energy donor (at 428 nm) when in the presence of increasing acceptor concentration. Also, the disruption of the energy transfer by solubilization of the membrane with Triton X-100 evidences a role for the membrane in providing the appropriate site(s) for energy transfer to occur. At moderately low daunomycin/membrane lipid ratios, the different efficiencies of resonance energy transfer between the two donors and daunomycin predicts a preferential, but not exclusive, location of the drug at membrane 'surface' domains, i.e., those regions of the bilayer explored by the 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probe. In support of this observation, a large fraction (approx. 75%) of membrane-associated daunomycin was rapidly sequestered away from the membrane upon addition of excess DNA, which forms high-affinity complexes with daunomycin (Chaires, J.B., Dattagupta, n. and Crothers, D.M. (1982) Biochemistry 21, 3927-3932), thus acting as a drug 'sink'. Also, a large fraction of drug was accessible to fluorescence quenching by iodide, a collisional water-soluble quencher. On the other hand, a smaller population of the membrane-associated daunomycin was characterized by slow sequestering by the added DNA and inaccessibility to quenching by iodide. We conclude that the daunomycin, which is only slowly sequestered, is located deep within the hydrophobic domains of the bilayer, likely to be those probed by 1,6-diphenyl-1,3,5-hexatriene.
1,6 - 二苯基 - 1,3,5 - 己三烯和1 -（4 - 三甲基铵苯基） - 6 - 苯基 - 1,3,5 - 己三烯是用于探索膜双层不同疏水区域的荧光团（安德里奇，M.P.和范德库伊，J.M.（1976年）《生物化学》15卷，第1257 - 1265页；普伦德加斯特，F.G.，豪格兰德，R.P.和卡拉汉，P.J.（1981年）《生物化学》20卷，第7333 - 7338页）。这些作为能量供体的荧光团与作为受体的蒽环类药物柔红霉素之间的荧光共振能量转移，被用于分析该药物与天然膜的相互作用及其在膜双层中的相对位置。转移过程通过以下方式得到证明：（1）当样品在供体的最大激发波长（360 nm）处激发时受体发出荧光；（2）当存在浓度不断增加的受体时，能量供体（在428 nm处）的荧光逐渐猝灭。此外，用Triton X - 100溶解膜导致能量转移中断，这证明了膜在提供能量转移发生的合适位点方面的作用。在柔红霉素/膜脂比例适度较低时，两个供体与柔红霉素之间共振能量转移的不同效率预示着药物在膜“表面”区域存在优先但不唯一的定位，即1 -（4 - 三甲基铵苯基） - 6 - 苯基 - 1,3,5 - 己三烯探针所探索的双层区域。为支持这一观察结果，加入过量DNA后，很大一部分（约75%）与膜结合的柔红霉素迅速从膜中被隔离出来，DNA与柔红霉素形成高亲和力复合物（蔡尔斯，J.B.，达塔古普塔，N.和克罗瑟斯，D.M.（1982年）《生物化学》21卷，第3927 - 3932页），因此起到了药物“汇”的作用。此外，很大一部分药物可被碘离子（一种碰撞性水溶性猝灭剂）猝灭荧光。另一方面，一小部分与膜结合的柔红霉素的特征是被添加的DNA缓慢隔离且对碘离子猝灭不敏感。我们得出结论，仅被缓慢隔离的柔红霉素位于双层的疏水区域深处，可能是1,6 - 二苯基 - 1,3,5 - 己三烯所探测的区域。
