Keller Sandro, Böthe Matthias, Bienert Michael, Dathe Margitta, Blume Alfred
Leibniz Institute of Molecular Pharmacology FMP, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Chembiochem. 2007 Mar 26;8(5):546-52. doi: 10.1002/cbic.200600553.
We have established a combination of fluorescence-spectroscopic uptake, release, and dilution experiments as a powerful tool for studying the translocation of fluorescent compounds across lipid membranes, demonstrating this through intrinsic tryptophan fluorescence for the interaction of the cell-penetrating peptide penetratin with phospholipid membranes, for which conflicting results have been reported. We found that penetratin is not membrane-permeant under the conditions used here. To confirm this finding and to validate the approach, we also employed an established titration-calorimetric method, the results of which were in excellent agreement with a thermodynamic analysis of the fluorescence-spectroscopic experiments. Further support was provided by a comparison with published data obtained under similar conditions by using a variety of techniques. Unlike these methods, however, the new approach allows consistent and simultaneous assessment of membrane binding and transbilayer movement without depending on extrinsic labels attached to the molecule of interest or on reporter moieties inserted into the lipid membrane.
我们已经建立了一套荧光光谱摄取、释放和稀释实验的组合方法，作为研究荧光化合物跨脂质膜转运的有力工具。通过内在色氨酸荧光，我们展示了细胞穿透肽穿膜肽与磷脂膜的相互作用，此前关于这一相互作用的报道结果相互矛盾。我们发现，在此处使用的条件下，穿膜肽不能透过膜。为了证实这一发现并验证该方法，我们还采用了一种既定的滴定量热法，其结果与荧光光谱实验的热力学分析结果高度一致。通过与使用各种技术在类似条件下获得的已发表数据进行比较，进一步提供了支持。然而，与这些方法不同的是，新方法能够一致且同时地评估膜结合和跨双层运动，而无需依赖附着于目标分子的外在标记或插入脂质膜的报告基团。
