F. W. Schneider

Binding and relaxation behaviour of prodan and patman in phospholipid vesicles: a fluorescence and 1H NMR study

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.

Solvent relaxation behaviour of n-anthroyloxy fatty acids in PC-vesicles and paraffin oil: a time-resolved emission spectra study.

Time-resolved fluorescence measurements were performed for a set of n-anthroyloxy fatty acids (n-AS; n = 2, 3, 6, 9, 12, 16) in both solvent and vesicle systems. The Stokes shifts and the mean relaxation times calculated from the time-resolved emission spectra (TRES) are shown to be strongly dependent on the position of the fluorophore in small unilamellar vesicles (SUV) composed of phosphatidylcholine (PC), while they are essentially independent of the fluorophore position in isotropic paraffin oil. The concept of an intramolecular relaxation process which had been suggested to explain the wavelength dependence of the emission behaviour of the n-AS dyes in viscous solvents is supported by semiempirical calculations showing that a more planar conformation is favoured in the excited compared to the ground state. However, in order to explain the results in vesicle systems, the concept of intramolecular relaxation is not sufficient. Rather, we show that intermolecular solvent relaxation processes play the dominant role for the wavelength dependent emission behaviour in polar, viscous environments.

Influence of vesicle curvature on fluorescence relaxation kinetics of fluorophores.

The effect of membrane curvature on the fluorescence decay of 2-p-toluidinyl-naphthalene-6-sulfonic acid (TNS), 2-(9-anthroyloxy) stearic acid (2-AS) and 12-(9-anthroyloxy)-stearic acid (12-AS) was investigated for egg lecithin vesicles of average diameter dm = 22 nm and 250 nm. The biexponential fluorescence decay of TNS at the red edge of the emission spectrum was analysed according to the model of Gonzalo and Montoro [1]. Over the entire temperature range (1-40 degrees C) the small TNS labelled vesicles showed significantly shorter solvent relaxation times tau(r) than their larger counterparts (e.g. 1.3 ns compared with 2.1 ns at 5 degrees C), indicating a higher mobility of the hydrated headgroups in the highly curved, small vesicles. The fluorescence decay of both AS derivatives is also biexponential. While the shorter decay times (1-3 ns) are practically identical for small and large vesicles, the longer decay times (5-14 ns) are identical only for 12-AS but not for 2-AS. This indicates that the microenvironment is similar in small and large vesicles deep in the membrane in spite of the differences in curvature.

Binding of prothrombin and its fragment 1 to phospholipid membranes studied by the solvent relaxation technique

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.

Resonance multiplex cars of fluorescing acridines

Abstract Resonance enhanced coherent anti-Stokes Raman scattering (CARS) spectra have been obtained for the highly fluorescing acridine dyes, acridine orange and proflavine, in dilute methanol solutions at submillimolar concentrations. Spectra have also been taken in the multiplex mode by the use of a broad-band Stokes laser and a Vidicon OMA detection system. Several Raman bands are observed in the 1100–1600 cm −1 region originating from the acridine ring modes. Upon decreasing the beam crossing angle a continuous transition from the normal CARS spectrum to a negative spectrum in the nonresonant background is observed.

Time-resolved emission spectra and anisotropy profiles for symmetric diacyl- and dietherphosphatidylcholines

The solvent relaxation behavior of Patman (6-palmitoyl-2-[[2-(trimethylammonium) ethyl]methylamino]naphthalene chloride) was investigated in small unilamellar vesicles composed of symmetric diacyl( 1,2-dipalmitoylphosphatidylcholine; DPPC) and diether lipids (l,2-dihexadecylphosphatidylcholine; DHPC), calculating time-resolved emission spectra (TRES) and correlation functions. Both the steady-state spectra as a function of temperature and excitation wavelength and the TRES of Patman in DPPC are blue-shifted compared to those in DHPC. The solvent relaxation at three temperatures above and below the phase transition is considerably faster in DHPC than in DPPC. As the steady-state anisotropies of Patman and TMA-DPH [l-(4-trimethylammoniumphenyl)-6-phenyl-l,3,5-hexatriene] are similar in both lipids as a function of both temperature and emission wavelength, we conclude that the introduction of ether linkages allows more efficient water penetration in the glycerol region, leading to a more polar environment and therefore faster solvent relaxation of the incorporated dyes. Using a series ofn-(9-anthroyloxy) fatty acids (n = 2, 3, 6, 9, 12; 16-AP), we show that anisotropy profiles can be used to distinguish between noninterdigitated (DPPC) and fully interdigitated (DHPC) gel-phase structures. 16-(9Antroyloxy) palmitic acid (16-AP) is an especially useful probe exhibiting pronounced differences in the steady-state anisotropies in non- and fully interdigitated gel phases.

Resonance cars spectroscopy of bacteriorhodopsin

Abstract Resonance-enhanced coherent anti-Stokes Raman scattering (CARS) spectra are reported for light and dark adapted bacteriorhodopsin in aqueous solution in the nanosecond time range. Spectra have been obtained in the scanning as well as in the multiplex mode. Minor differences between the spectra obtained recently by the conventional resonance Raman and the present resonance CARS method are discussed.

The localization of the local anesthetic tetracaine in phospholipid vesicles: A fluorescence quenching and resonance energy transfer study

Abstract Fluorescence quenching and resonance energy transfer have been used to determine the localization of the local anesthetic tetracaine in vesicles composed of 1,2-dimyristoyl- sn -glycero-3-phosphatidylcholine (DMPC) as a function of both temperature and ionic strength. The fluorescence behaviour of tetracaine in vesicles can be attributed to its different partition coefficients in acid and basic solution, in gel phase and fluid phase vesicles, respectively. Using both steady-state and time-resolved fluorescence measurements we show that a saturable binding rather than a partitioning model holds for the interaction of tetracaine with gel phase bilayers. The relative quenching efficiencies of the series of n -AS dyes depend on the phase state of the bilayer and suggest a deeper incorporation of tetracaine in fluid phase than in gel phase membranes. Resonance energy transfer measurements support the view that tetracaine is incorporated predominantly in the region of the 9-AS chromophore in DMPC-bilayers.

ANISOTROPY AND LIFETIME PROFILES FOR N-ANTHROYLOXY FATTY ACIDS : A FLUORESCENCE METHOD FOR THE DETECTION OF BILAYER INTERDIGITATION

Abstract A new fluorescence assay for the detection of interdigitated bilayer phases using the set of n -anthroyloxy stearic acids ( n -AS, where n = 2,3,6,9,12) and 16-anthroyloxy palmitic acid (16-AP) is presented. Anisotropy measurements were performed for the n -AS dyes in multilamellar vesicles (MLV) composed of either 1,2-dimyristoyl- sn -glycero-3-phosphatidylcholine (DMPC), 1,2-dipalmitoyl- sn -glycero-3-phosphatidylcholine (DPPC), 1,2-dihexadecyl- sn -glycero-3-phosphatidylcholine (DHPC) or 1-stearoyl-2-lauroyl- sn -glycero-3-phosphatidylcholine (C18/12-diacy1PC) with known gel phase structures. Plots of the steady-state anisotropy versus the position of the chromophore were obtained with characteristic shapes for non-interdigitated, mixed or fully interdigitated gel phases. Lifetime measurements revealed a somewhat smaller polarity gradient for the fully interdigitated DHPC compared to DPPC and extraordinarily long lifetimes for 16-AP in the mixed interdigitated C18/12-diacy1PC which can be of diagnostic value for this specific gel phase structure. The assay was used for the investigation of the new asymmetric 1-stearyl-2-lauryl- sn -glycero-3-phosphatidylcholine (C18/12-dietherPC), providing evidence for a mixed interdigitated structure similar to that described previously for the corresponding C18/12-diacy1PC.

The phases of small networks of chemical reactors and neurons

We present an experimental study of the phase relationships observed in small reactor networks consisting of two and three continuous flow stirred tank reactors. In the three-reactor network one chemical oscillator is coupled to two other reactors in parallel in analogy to a small neural net. Each reactor contains an identical reaction mixture of the excitable Belousov-Zhabotinsky reaction which is characterized by its bifurcation diagram, where the electrical current is the bifurcation parameter. Coupling between the reactors is electrical via Pt-working electrodes and it can be either repulsive (inhibitory) or attractive (excitatory). An external electrical stimulus is applied to all three reactors in the form of an asymmetric electrical current pulse which sweeps across the bifurcation diagram. As a consequence, all three reactors oscillate with characteristic oscillation patterns or remain silent in analogy to the firing of neurons. The observed phase behavior depends on the type of coupling in a complex way. This situation is analogous to the in vivo measurements on single neurons (local neurons and projection neurons) performed by G. Laurent and co-workers on the olfactory system of the locust. We propose a simple neural network similar to the reactor network using the Hodgkin-Huxley model to simulate the action potentials of the coupled single neurons. Analogies between the reactor network and the neural network are discussed.