Steady-state fluorescence polarization in planar lipid membranes. Experimental and theoretical analysis of the fluorophores 8-anilino-1-naphthalenesulfonate, 1,6-Diphenyl-1,3,5-hexatriene, dansyllysine-valinomycin and n-(9-anthroyloxy) fatty acids.

In continuation of earlier work, the steady-state fluorescence polarization in a globally oriented system of planar lipid membranes was analyzed experimentally and theoretically for the fluorophores 8-anilino-1-naphthalenesulfonate, 1,6-diphenyl-1,3, 5-hexatriene, dansyllysine-valinomycin and n-(9-anthroyloxy) fatty acids. The theoretical analyses of experiments were mainly done in terms of the mean orientation of transition moments with respect to the membrane normal, an angle describing the region of hindered rotational diffusion and the coefficients of rotational diffusion perpendicular to the membrane and around the membrane normal. The nonvanishing angle between the moments of absorption and emission was taken into account. In the case of n-(9-anthroyloxy) fatty acids it was found that the orientational disorder increases significantly with the depth of the fluorophore within the membrane. In order to compare with recent results from time-dependent fluorescent polarization in globally isotropic membrane suspensions and with 2H-NMR experiments, the second moment ('order parameter') of the steady-state orientational distribution of absorption dipoles was calculated. For all fluorophores the theoretical analysis indicates a preferred orientation of absorption moments within the membrane plane.

Kinetics of proton-hydroxyl transport across lecithin vesicle membranes as measured with a lipoid pH-indicator.

When unilamellar vesicles are prepared in the presence of 3-Palmitoyl-7-ocy-coumarin abbreviation 3P-UBF) this lipoid pH-Indicator is anchored by its fatty acid chain to the membrane and can be used to measure pH-changes at the outer and inner membrane surface (ranging from pH to pH 9.5). By rapidly changing the pH of the outer aqueous phase a pH-gradient is set up across the vesicle membrane. The rate of the subsequent H+ or OH- influx into the vesicles can be measured as a change of the 3P-UBF absorbance at 424 nm. This was done with a stopped-flow -spectrophotometer at temperatures between 10 degree C and 50 degree C. Suspensions of vesicles prepared from egg-lecithin or L-dipalmitoyl-lecithin were investigated in buffered salt solutions. The influence of Na+, K+, Cl-, SO2-(4) and valinomycin on the rate of absorbance changes was studied at different temperatures. It was found that the rate of the pH-equilibration between the aqueous phase outside and inside the vesicles depends on the direction of the pH-gradient. This new result together with a high H+/OH- permeability of vesicle membranes found in recent studies from other laboratories and confirmed by this investigation is interpreted to indicated a higher permeability of the vesicle membrance to OH--ions compared to H+-ions. (Calculated values are: POH = 1.4 x 10(-4) cm/s at pH9 and PH = 8.3 x 10 (-7) cm/s at pH 5 and 20 degree C.) All data described in the literature in detail agree with this suggestion but a pH-dependence of POH and PH cannot be excluded.

The specificity of ionophore A23187 in cation transport across lipid membranes. Studies with lecithin vesicles.

Lecithin vesicles containing different cations were prepared by sonication and characterized. When the concentration of the divalent cations in the buffer was increased from 1 mM to about 100 mM the cation-concentration inside of the vesicles did not increase proportional to the outside concentration, but showed a saturation behaviour. The efflux of various cations mediated by ionophore A23187 was measured and relative transport-rates were determined. The following sequence was obtained: Zn2+ > Ca2+ > Mg2+ > Sr2+ > Ba2+ approximately Li+ > Na+. The efflux of Ca2+ increased proportional to the square of the A23187-concentration. To lecithin-vesicles containing ethylenediamine tetraacetate (EDTA) inside different cations were added on the outside. In the presence of A23187 divalent cations are transported into the vesicles and bound there by EDTA. During the influx of Me2+-ions into vesicles a H+-efflux was observed. The resulting pH-decrease was measured. The rate of the pH-change depended on the Me2+-ion used. The sequence was: Cd2+ > Zn2+ > Ca2+ > Mn2+ > MG2+ > Sr2+, Ba2+.

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187.

The antibiotic A23187 carries Ca2+ across Müller-Rudin membranes made from 1,2-dierucoyl-sn-glycero-3-phosphocholine and n-decane. The conductance of the membranes is not increased by the Ca2+-transport. The flux depends linearly on Ca2+ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4-5 between pH 6 and pH 8. Maximal Ca2+-fluxes of about 10(-10) mol-cm-2-s-1 were found. A counter transport of H+ could not be detected. The complex formation between A23187 and Ca2+ in egg phosphotidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2:1 complex. Optical absorption measurements on single phophatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.

Properties of bilayer membranes in the presence of dipicrylamine. A comparative study by optical absorption and electrical relaxation measurements.

In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes larger than or equal to 4-10(-5). A minimal concentration of about 6-10(11) dipicrylamine ions per cm2 of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method Ntopt is compared with the concentrations Ntel obtained from simultaneous electrical relaxation meausurements. Ntopt and Ntel agreed at low dipicrylamine concentrations (10(-8)--10(-7) M in the aqueous phase) and showed saturation at higher concentrations (up to 5-10(-6) M). In the saturation range Ntopt was maximally four times higher than Ntel. The significance of this difference is discussed together with general aspects of the saturation phenomenon.

The use of fluorescent probes for studying the interaction of proteins with black lipid membranes.

The number of 1-anilino-8-naphthalene sulphonate (ANS) molecules bound to black phosphatidylcholine (PC) and phosphatidylinositol (PI)-membranes was calculated. The fluorescences change of membrane bound ANS was measured after the additon of positively charged proteins to the same side as ANS. Cytochrome c caused a fluorescence decrease, lysozyme and protamine an increase. These effects were completely reversible in the case of cytochrome c and lysozyme and only partly reversible in the case of protamine by increasing the ionic strength. The fluorescence polarization of membrane bound ANS was not significantly changed by protein addition. The results are discussed with respect to the binding of proteins to black lipid membranes and the use of ANS as a probe compared with other fluorescent probes.