R. Hutterer

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 of Prodan and Patman: A Useful Tool for the Determination of Polarity and Rigidity Changes in Membranes

The time dependence of the solvent relaxation behavior of two polarity-sensitive dyes called Prodan and Patman has been investigated in detail in artificial membrane systems of different compositions. The works provides a comparison of the solvent relaxation behavior of the mentioned dyes, using steady-state and time-resolved fluorescence methods, and exemplifies their complementary use for membrane studies.

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.

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.

Probing Ethanol-Induced Phospholipid Phase Transitions by the Polarity Sensitive Fluorescence Probes Prodan and Patman

The emission behaviour of the two polarity sensitive probes Prodan and Patman in phospholipid vesicles was studied as a function of the concentration of ethanol. Comparing the spectral shifts in both the symmetric lipid 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) showing a phase transition from a normal to a fully interdigitated gel phase and the strongly asymmetric lipid 1-stearoyl-2-lauroyl-sn-glycero-3-phosphatidylcholine (C(18):C(12)-PC) favouring a mixed interdigitated gel phase we show that the huge red shifts of Prodan in presence of higher ethanol concentrations cannot be easily attributed to a specific lipid phase transition. Rather, probe relocation and a pronounced increase in solvent relaxation (SR) as monitored by time-resolved emission spectra (TRES) in presence of ethanol contribute to the large shifts observable in both lipid systems in case of Prodan. While Patman exhibits a red shift caused by increased SR due to the ethanol induced formation of a fully interdigitated phase in DPPC, hardly any shift occurs in C(18):C(12)-PC, which is supposed not to undergo an ethanol-induced phase transition.

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.

Dynamics in Diether Lipid Bilayers and Interdigitated Bilayer Structures Studied by Time-Resolved Emission Spectra, Decay Time and Anisotropy Profiles

We present a comparative fluorescence spectroscopic investigation of diacyl and diether phosphatidylcholine vesicles using different probes with well-defined localization within either the hydrophilic headgroup region or the hydrophobic part of the bilayer. Time-resolved emission spectra have been used to characterize the solvent relaxation behavior in both symmetric and asymmetric diether and diacyl phosphatidylcholines. It is shown that time-resolved emission spectra of Prodan (6-propionyl-2-(dimethylamino)-naphthalene) and its long-alkyl chain derivative Patman (6-palmitoyl-2-[[trimethylammoniumethyl]methylamino]-naphthalene chloride) are a sensitive tool for the detection of differences in the micropolarities and viscosities at the hydrophobic/hydrophilic membrane interface of diether and diacyl lipids, respectively. Moreover, a new approach for the detection of interdigitated bilayers is discussed. It relies on the construction of anisotropy and decay time profiles for the set of n-anthroyloxy fatty acids and is compared with an older fluorescence assay based on intensity measurements only. The shape of plots of the fluorescence steady-state anisotropy versus the position of the chromophore (anthracene-9-carboxylic acid) combined with fluorescence lifetime measurements can be used to differentiate among non-fully, and mixed interdigitated gel phase structures and to predict structures for new lipid species.

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.

Influence of lipid composition and membrane curvature on fluorescence and solvent relaxation kinetics in unilamellar vesicles.

Time-resolved fluorescence on unilamellar vesicles shows that increasing amounts of anionic, natural lipid lead to a larger increase in polarity close to the headgroups than in the hydrophobic core of the bilayer. The region close to the headgroups is less polar in vesicles containing phosphatic acid rather than phosphatidylserine. A greater membrane curvature increases the mobility of the hydrated headgroups.