patman and prodan

The subject of this report was to investigate headgroup hydration and mobility of two types of mixed lipid vesicles, containing nonionic surfactants; straight chain Brij 98, and polysorbat Tween 80, with the same number of oxyethylene units as Brij, but attached via a sorbitan ring to oleic acid. We used the fluorescence solvent relaxation (SR) approach for the purpose and revealed differences between the two systems. Fluorescent solvent relaxation probes (Prodan, Laurdan, Patman) were found to be localized in mixed lipid vesicles similarly as in pure phospholipid bilayers. The SR parameters (i.e. dynamic Stokes shift, Deltanu, and the time course of the correlation function, C(t)) of such labels are in the same range in both kinds of systems. Each type of the tested surfactants has its own impact on water organization in the bilayer headgroup region probed by Patman. Brij 98 does not modify the solvation characteristics of the dye. In contrast, Tween 80 apparently dehydrates the headgroup and decreases its mobility. The SR data measured in lipid bilayers in presence of Interferon alfa-2b reveal that this protein, a candidate for non-invasive delivery, affects the bilayer in a different way than the peptide melittin. Interferon alfa-2b binds to mixed lipid bilayers peripherally, whereas melittin is deeply inserted into lipid membranes and affects their headgroup hydration and mobility measurably.

Topics: 2-Naphthylamine; Animals; Chemistry Techniques, Analytical; Fluorescent Dyes; Laurates; Lipid Bilayers; Melitten; Palmitic Acids; Phosphatidylcholines; Plant Oils; Polyethylene Glycols; Polysorbates; Protein Binding; Solvents; Spectrometry, Fluorescence; Surface-Active Agents; Time Factors; Water

Topics: 2-Naphthylamine; Fluorescent Dyes; Lipid Bilayers; Palmitic Acids; Phosphatidylcholines; Phospholipids; Solvents; Stearic Acids

The phospholipid headgroup mobility of small unilamellar vesicles composed of different mixtures of phosphatidyl-L-serine (PS) and phosphatidylcholine is characterized by the solvent relaxation behavior of the polarity sensitive dyes 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and 6-palmitoyl-2-[trimethylammoniumethyl]-methylamino]naphthalene chloride (Patman). If the PS content exceeds 10%, the addition of calcium leads to a substantial deceleration of the solvent relaxation of both dyes, indicating the formation of Ca(PS)2 complexes. Addition of prothrombin and its fragment 1 leads to a further decrease of the headgroup mobility, as explained by the binding of more than two PS-molecules by a single protein molecule. Prodan monitors the outermost region of the bilayer and it clearly distinguishes between the binding of prothrombin and its fragment 1. The deeper incalated Patman does not distinguish between both proteins. The validity of the solvent relaxation technique for the investigation of the membrane binding of peripheral proteins is demonstrated by the studies of prothrombin induced changes in the steady-state fluorescence anisotropies of 1,6-diphenyl-1,3, 5-hexatriene.

Topics: 2-Naphthylamine; Calcium; Fluorescent Dyes; Lipid Bilayers; Palmitic Acids; Peptide Fragments; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Protein Precursors; Prothrombin; Solvents; Time Factors

The peptide toxin thionin from Pyrularia pubera binds to dipalmitoylphosphatidylglycerol (DPPG) large unilamellar vesicles as shown by an increase in the intensity and blue-shift of the fluorescence emission spectrum of the single tryptophan residue of the protein. The magnitude of these fluorescence changes increased with temperature near the thermotropic phase transition of DPPG (about 40 degrees C). Fluorescent probes sensitive to the structure and dynamics of the membrane were used to assess the effect of thionin binding on bilayer properties. The fluorescence emission spectra of Prodan, Patman, and Laurdan all showed spectral changes consistent with an increase in bilayer polarity at temperatures below the DPPG phase transition but a decrease in polarity at higher temperatures. Fluorescence polarization experiments and the ratio of monomer-to-excimer fluorescence of the probe 1,3-bis(1-pyrene)propane suggested that thionin increases the bilayer order above the transition temperature. Differential scanning calorimetry revealed that thionin broadens the transition and either increases or decreases the melting temperature depending on the concentration of the peptide. Taken together, the data are consistent with at least three distinct interactions of thionin with the bilayer: (1) thionin bound electrostatically to the bilayer surface; (2) tryptophan of the bound thionin inserted into the bilayer; (3) high-order aggregates of thionin-bound vesicles.

Topics: 2-Naphthylamine; Antimicrobial Cationic Peptides; Calorimetry, Differential Scanning; Fluorescence Polarization; Fluorescent Dyes; Laurates; Lipid Bilayers; Liposomes; Palmitic Acids; Phosphatidylglycerols; Plant Proteins; Plants, Toxic; Protein Binding; Pyrenes; Spectrometry, Fluorescence; Temperature; Tryptophan

The relative location, binding behaviour and the solvent relaxation behaviour of the polarity sensitive membrane probes 6-propionyl-2-(dimethylamino)naphthalene and 6-palmitoyl-2-[[trimethylammoniumethyl]methylamino]naphthalene chloride in vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine or egg yolk lecithin have been compared using steady-state and time-resolved fluorescence as well as high resolution NMR measurements. The reconstructed time-resolved emission spectra show unambiguously that the observed spectral shifts in vesicle systems have to be assigned to time-dependent solvent relaxation processes rather than to a probe relocation mechanism. All fluorescence as well as the NMR relaxation data suggest a deeper localization of Patman in the membrane, sensing a less polar and/or more restricted probe environment.

Topics: 2-Naphthylamine; Binding Sites; Fluorescent Dyes; Magnetic Resonance Spectroscopy; Palmitic Acids; Phospholipids; Protons; Spectrometry, Fluorescence