Membrane Translocation Assayed by Fluorescence Spectroscopy
Assessing the ability of biomolecules or drugs to overcome lipid membranes in a receptor-independent way is of great importance in both basic research and applications involving the use of liposomes. A combination of uptake, release, and dilution experiments performed by steady-state fluorescence spectroscopy provides a powerful, straightforward, and inexpensive way of monitoring membrane translocation of fluorescent compounds. This is particularly true for peptides and proteins carrying intrinsic tryptophan residues, which eliminates the need for attaching extrinsic labeling moieties to the compound of interest. The approach encompasses three different kinds of fluorescence titrations and some simple calculations that can be carried out in a spreadsheet program. A complete set of experiments and data analyses can typically be completed within two days.
Key wordsMembrane binding Membrane permeability Membrane permeation Flip-flop Transbilayer movement Uptake Release Dilution Tryptophan fluorescence Vesicles
We thank Heike Nikolenko (FMP) and Matthias Böthe (Robert Koch Institute, Berlin, Germany) for excellent technical assistance and Sebastian Fiedler (FMP) for helpful comments on the manuscript. We are indebted to Dr. Michael Beyermann, Dagmar Krause, and Bernhard Schmikale for synthesis and purification and to Drs. Eberhard Krause and Michael Schümann (all FMP) for mass-spectrometric characterization of penetratin peptide. This work was supported by the European Commission with grant No. QLK3-CT-2002-01989 to S.K.
- 4.Hagen V, Dekowski B, Nache V, Schmidt R, Geissler D, Lorenz D, Eichhorst J, Keller S, Kaneko H, Benndorf K, Wiesner B (2005) Coumarinylmethyl esters for ultrafast release of high concentrations of cyclic nucleotides upon one- and two-photon photolysis. Angew Chem Int Ed 44:7887-7891CrossRefPubMedGoogle Scholar