Study of plasma membrane heterogeneity using a phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene [2-(3-(diphenylhexatriene)propanoyl)-3-palmitoyl-l-α-phosphatidylcholine]

The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH derivatives have been used to characterize structural and physicochemical properties of specific membrane domains. Steady-state and fluorescence decay measurements of three probes, DPH (1,6-diphenyl-1,3,5-hexatriene), TMA-DPH [1-(4-trimethyl-ammonium-phenyl)-6-phenyl-1,3,5-hexatriene], and a phosphatidylcholine derivative of DPH, DPH-pPC [2-(3-(diphenylhexatriene)propanoyl)-3-pamitoyl-L--phosphatidyl choline], have been performed in erythrocyte membranes and in lymphocyte plasma membranes. The steady-state fluorescence polarization of the three probes showed a similar trend in both membranes. In fact either in erythrocyte or in lymphocyte plasma membranes the fluorescence polarization values of DPH-pPC and TMA-DPH were similar, but significantly higher with respect to DPH. A better characterization of erythrocyte and lymphocyte plasma membranes was possible by using fluorescence decay measurements. The data suggest the possible use of different DPH derivatives to characterize specific domains in biological membranes.     

Fluorescence evidence for cholesterol regular distribution in phosphatidylcholine and in sphingomyelin lipid bilayers

Our previous studies indicated that sterols (including cholesterol and dehydroergosterol) can be regularly distributed into hexagonal superlattices in the plane of liquid-crystalline phosphatidylcholine bilayers. It was suggested that regular and irregular regions coexist in the membrane. In the present study, we report supporting evidence for our sterol regular distribution model. We have examined the fractional concentration dependencies of dehydroergosterol (a naturally occurring cholesterol analogue) fluorescence intensity and lifetime in various phosphatidylcholine and sphingomyelin bilayers. Fluorescence intensity and lifetime dips have been observed at specific sterol mole fractions. At those mole fractions, the acrylamide quenching rate constant of dehydroergosterol fluorescence reaches a local maximum. Those mole fractions match the critical sterol mole fractions at which sterol molecules are expected to be regularly distributed into hexagonal superlattices. The results support the idea that the sterols in the regular region are embedded in the bilayer less deep than those in the irregular regions. We have also examined the fractional cholesterol concentration dependencies of diphenylhexatriene (DPH) fluorescence intensity, lifetime, and polarization in DMPC vesicles. DPH fluorescence intensity and polarization also exhibit distinct dips and peaks, respectively, at critical sterol mole fractions for hexagonal superlattices. However, DPH lifetime changes little with sterol mole fraction. As a comparison, the fluorescence properties of DHE and DPH behave differently in response to the formation of sterol regular distribution. Furthermore, finding evidence for sterol regular distribution in both phosphatidylcholine and sphingomyelin membranes raises the possibility that sterol regular distribution may occur within phospholipid/cholesterol enriched domains of real biological membranes.     

Investigation of the Dynamic Properties of the Lipid Layer of Intraplastid Membranes by Lipophilic Fluorescent Probes

The dynamic properties of the lipid layer of intraplastid membranes have been studied by analyzing the stationary and kinetic spectral polarization characteristics of the fluorescence of lipophilic probes of pyrene and diphenylhexatriene (DPH). Based on the data of the decay of the fluorescence anisotropy of pyrene, the value of the microviscosity of lipids in the membranes of prolamellar bodies (PLB) and protilakoids of etioplasts has been calculated. The pyrene molecules built into the membranes of etioplasts have a relatively high rotational mobility (stationary anisotropy r _s < 0.1). The DPH molecules rotate with difficulty in etioplast membranes (r _s > 0.3). After photoreduction of protochlorophyllide (Pd) in vivo, the rotation of the pyrene and DPH molecules in the membranes of prolamellar bodies becomes easier and this leads to a decrease in r _s. Illumination raised the degree of excimerization of the pyrene immersed into lipids (_exc = 337 nm), and the microsurrounding of the molecules of the probe in lipids became more hydropholic (the relationship between the vibronic maxima at 373 and 387 nm decrease). The set of data obtained points to a decrease in the microviscosity of the lipid layer of the membranes of prolamellar bodies as a result of illumination of sproutings.     

Determination of the rate of rapid lipid transfer induced by poly(ethylene glycol) using the SLM Fourier transform phase and modulation spectrofluorometer

Rate constants were determined for the transfer of the fluorescent lipid probe 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] oxy]carbonyl]-3-sn-phosphatidylcholine (DPHpPC) between large, unilamellar extrusion vesicles composed either of dipalmitoyl phosphatidylcholine (DPPC) or of DPPC mixed with a small amount (0.5 mol%) of lyso phosphatidylcholine (Lyso PC). Transfer of the lipid probe in the presence of varying concentrations of poly(ethylene glycol) (PEG) was monitored using the SLM 48000-MHF Multi-Harmonic Fourier Transform phase and modulation spectrofluorometer to collect multifrequency phase and modulation fluorescence data sets on a subsecond time scale. The unique ability of this instrument to yield accurate fluorescence lifetime data on this time scale allowed transfer to be detected in terms of a time-dependent change in the fluorescent lifetime distribution associated with the lipid-like DPHpPC probe. This probe demonstrates two short fluoresence decay times (ca. 1.1–1.4 and 4.3–4.8 ns) in a probe-rich environment but a single long lifetime (ca. 7 ns) in a probe-poor environment. A simple two-state model for initial lipid transfer was used to analyze the multifrequency data sets collected over a 4-s time frame to obtain the time rate of change of the concentrations of donor and acceptor probe populations following rapid mixing of vesicles with PEG. The ability to measure fluorescence lifetimes on this time scale has allowed us to show that the of rate of lipid transfer increased dramatically at 35% PEG in both fusing and nonfusing vesicle systems. These results are interpreted in terms of a distinct interbilayer structure associated with intimate bilayer contact induced by high and potentially fusogenic concentrations of PEG.     

Diphenylhexatriene-phosphatidylcholine fluorescence in POPC vesicles: Comparison of the exponential-series and the maximum-entropy methods

We have investigated the time-resolved fluorescence of diphenylhexatriene (DPH) covalently linked to phosphatidylcholine (PC) in palmitoyloleoylglycerophosphocholine (POPC) vesicles with special consideration of the comparison of two methods for distributional lifetime analysis: the exponential-series method (ESM) and the maximum-entropy method (MEM). Generally, both methods were found to reveal equivalent results on high-quality data. Different are the shapes of the recovered distributions (symmetry and width) as well as the time effort for the numerical analysis.     

The Susceptibility of Non-UV Fluorescent Membrane Dyes to Dynamical Properties of Lipid Membranes

Fluorescence spectroscopy and microscopy are powerful techniques to detect dynamic properties in artificial and natural lipid membrane systems. Unfortunately, most fluorescent dyes that sense dynamically relevant membrane parameters are UV sensitive. Their major disadvantage is a high susceptibility to fluorescence bleaching. Additionally, the risk for hazardous damages in biological components generally increases with decreasing excitation wavelength. Therefore the use of non-UV–sensitive membrane dyes would provide significant advantage, particularly for applications in fluorescence microscopy, which usually implies high local excitation intensities. We applied steady-state fluorescence spectroscopy techniques to several UV and non-UV membrane dyes to detect and compare dynamically relevant excitation and emission characteristics. Small unilamellar liposomes (composed of egg yolk phosphatidylcholine) served as a model system for biological membranes. The dynamic properties of the membranes were varied by two independent parameters: the intrinsic cholesterol content (0–50 mol%) and temperature (10–50°C). We tested four non-UV–sensitive membrane dyes: 9-diethylamino-5H-benzophenoxazine-5-one (Nile Red), 4-(dicyanovinyl)julolidine (DCVJ), N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM 4-64), and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiIC₁₈). We also tested three derivatives of DiIC₁₈: DiIC₁₆ and DiIC₁₂ differ in acyl chain length and Fast-DiIC₁₈ provides double bonds between hydrocarbon atoms. The spectral results were compared to established fluorescence characteristics of four UV membrane dyes: the anisotropy of 1-6-phenyl-1,3,5,-hexatrien (DPH), two derivatives of DPH (TMA-DPH and COO^–-DHP), and the generalized polarization of 6-dodecanoyl-2-dimethyl-aminonaphthalene (Laurdan). Our results indicate that the tested non-UV dyes do not reveal dynamically relevant membrane parameters in a direct manner. However, spectral characteristics make DiIC₁₈, Nile Red, and DCVJ promising probes for the microscopic detection of lateral lipid organization, an indirect indicator of membrane dynamics. In particular, DiIC₁₈ showed very selective shifts in the emission spectra at defined temperatures and cholesterol contents that have not been reported elsewhere.     

Combined Fluorescence and NMR Studies of Reversed HPLC Stationary Phases with Different Ligand Chain Lengths

Alkyl chain bonded reversed HPLC phases consisting of 6 to 30 carbon atoms are investigated by fluorescence spectroscopy, steady-state and time-resolved fluorescence anisotropy, and solid-state NMR spectroscopy. The structure and dynamics of the interphase formed by alkyl chains and liquid phase penetrating each other are studied as a function of alkyl chain length. Increasing alkyl chain lengths lead to enhanced partitioning of the fluorescent probe diphenylhexatriene (DPH) into the interphase, as monitored by fluorescence decay curves. The concomitant spectral red shift of DPH fluorescence excitation maxima is evidence of increased interphase polarizability. Time-resolved fluorescence anisotropy measurements reveal that the motion of the probe molecule in the interphase is wobble in cone-like. Cone angles and rotational correlation times _R change from = 63° and _R = 0.75 ns in C₆ phases to = 42° and _R = 1.50 ns in C₃₀ phases, thus indicating decreasing probe mobility with increasing ligand length. This interpretation is supported by ¹³C CP/MAS NMR spectra, which show reduced contributions of alkyl chain gauche conformations, i.e., enhanced interphase order, in phases with long alkyl chains and high surface coverage. A concomitant increase in the line-widths of ¹H MAS NMR peaks indicates reduced mobility of the longer chains. The spectroscopic observations are consistent with the results of HPLC separations, where enhanced shape selectivity is found with increasing ligand length, rod-shaped molecules like DPH showing the greatest increase in retention time.     

Spectral-Kinetic Characteristics of Pyrene Fluorescence in Model Systems and Membranes of Etioplasts

The decay kinetics parameters of monomer and excimer fluorescence of pyrene in artificial galactolipid protein-free micelles and membranes of prolamellar bodies (PLB) of etioplasts have been determined. A complex law of probe fluorescence decay in artificial and native membranes has been discovered. It has been found that the addition of protochlorophyllide (Pd) to lipid micelles led to a considerable reduction in the lifetime of the long-lived component of the fluorescence-decay kinetics of the monomer form of pyrene (₂) and to the appearance of luminescence in the region of 640 nm in the stationary spectra of fluorescence at excitation of the pyrene molecules present in the bilayer. In native membranes of etioplasts, the monomer duration ₂ did not depend on the pigment content and the Pd fluorescence upon excitation through pyrene was absent. The membranes of etioplasts and the pigment-containing artificial micelles did not differ in the ₂ value of the excimer. They only differed in the contribution of the long-lived component to the total fluorescence (low in native membranes). The pigment-protein interaction in the etioplast membranes and the absence from them of pigment directly associated with lipids are discussed.     

Polarized Fluorescence of Diphenylhexatriene-Incorporated Poly(Styrene) Film at Room Temperature

Poly(styrene) is a highly viscous, and cross-linked polymer at room temperature. This makes it ideal to use as a molecular fixer. The polarized fluorescence of a diphenylhexatriene (DPH)-incorporated poly(styrene) film has been studied. The excitation and emission wavelength dependence of the anisotropy of fluorescence of a luminophore-incorporated poly(styrene) film reveals that with decreasing excitation wavelength the anisotropy changes remarkably but is independent of emission wavelength. The investigation estimates an angle of no more than 7.4° between the absorption and the emission transition dipole moment for DPH, suggesting poly(styrene) as a suitable medium to evaluate the mutual orientation of absorption and the emission transition dipole moments at room temperature.     

Hemolysis of Erythrocytes and Erythrocyte Membrane Fluidity Changes by New Lysosomotropic Compounds

This work contains the results of studies on the influence of newly synthesized lysosomotropic substances (lysosomotropes) on human erythrocytes. Six homologous series of the compounds differing in the alkyl chain length and counterions were studied. They were found to hemolyse erythrocytes and to change their osmotic resistance. The observed hemolytic effects were dependent both on the compounds structure (polar head dimension and alkyl chain length of compound) and its form (the kind of the counterion). In parallel, the influence of lysosomotropes on fluidity of the erythrocyte membrane was studied. Three different fluorescent probes were used; 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, p-toluenesulfonate (TMA-DPH) and 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan). Their anisotropy (DPH and TMA-DPH) or general polarization (laurdan) values after incorporation into ghost erythrocyte membranes were measured. The results obtained show that fluidity changes accompanied the effects observed in hemolytic experiments both quantitatively and qualitatively.     

Fluorescence and ESR studies on membrane oxidative damage by gamma radiation

Oxidative damage to cellular membranes critically controls the manifestation of cellular response to ionizing radiation. To gain further insight into the damaging mechanisms, we have investigated the effects of γ-radiation-generated free-radical-mediated peroxidative damage in egg yolk lecithin unilamellar liposomal membranes by employing 1,6-diphenyl-1,3,5-hexatriene (DPH). Alterations in lipid bilayer fluidity and malondialdehyde (MDA) formation were measured in irradiated liposomal membranes as a function of radiation dose (0.1-1 kGy). A relationship seems to exist between the degree of radiation-induced peroxidative damage and the magnitude of DPH fluorescence decay in irradiated membranes. Radiation-induced membrane rigidization and MDA formation were significantly reduced when α-tocopherol, a natural membrane antioxidant, was present in the liposomes suggesting an involvement of lipid free radicals in the mechanism of the damage process. The results of the present study have been compared with those obtained by the electron spin resonance (ESR) technique on human erythrocyte ghost membranes with spin-labeled phospholipids having the unique capability to sensitively report on the dynamic state of the lipid environment inside the bilayer membrane. Iodoacetamide and N-ethylmaleimide spin labels were used to investigate alterations in membrane proteins. These results have contributed to our understanding of mechanisms involved in radiation membrane oxidative damage in terms of lipid peroxidation, fluidity changes and involvement of -SH groups of membrane proteins. Combined use of fluorescence and ESR spin-label techniques is of potential interest in probing the deeper molecular mechanisms of radiation injury in cellular membranes for developing strategies to modify the radiation damage to cells.     

Wavelength-Selective Fluorescence of a Model Transmembrane Peptide: Constrained Dynamics of Interfacial Tryptophan Anchors

WALPs are prototypical, α-helical transmembrane peptides that represent a consensus sequence for transmembrane segments of integral membrane proteins and serve as excellent models for exploring peptide-lipid interactions and hydrophobic mismatch in membranes. Importantly, the WALP peptides are in direct contact with the lipids. They consist of a central stretch of alternating hydrophobic alanine and leucine residues capped at both ends by tryptophans. In this work, we employ wavelength-selective fluorescence approaches to explore the intrinsic fluorescence of tryptophan residues in WALP23 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. Our results show that the four tryptophan residues in WALP23 exhibit an average red edge excitation shift (REES) of 6 nm, implying their localization at the membrane interface, characterized by a restricted microenvironment. This result is supported by fluorescence anisotropy and lifetime measurements as a function of wavelength displayed by WALP23 tryptophans in POPC membranes. These results provide a new approach based on intrinsic fluorescence of interfacial tryptophans to address protein-lipid interaction and hydrophobic mismatch.     

Fluorescence of the Protein of the Prolamellar Bodies of Etioplasts

In the high-purity membranes of the prolamellar bodies (PLB) of etioplasts, the main protein component of which is protochlorophyllide oxidoreductase (POR), protein fluorescence in the 320–350-nm region with a maximum in the excitation spectrum at 278–280 nm has been revealed. The microsurrounding of protein fluorophors in an acidic medium (pH 4.0) is the most hydrophobic. Transfer of energy from the protein to pyrene present in the lipid of the PLB membranes is observed, but fluorescence of protochlorophyllide (Pd) is absent when pyrene is excited via protein. A change in the content of Pd and carotenoids in the membranes of PLB influences the degree of excimerization of the acceptor molecules of pyrene (inverse dependence). The character of interaction of POR with a membrane with allowance for the data on its primary and secondary structures is discussed.     

Specific molecular properties of organic solvents determine the fluorescence depolarization of DPH and TMA-DPH in membranes

In pig liver microsomes and protein-free egg PC liposomes the effects of organic solvent molecules on the fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)phenyl]-6-phenyl-hexa-3,5-triene (TMA-DPH) were investigated. Aromaticity, alkyl chain length, and stereometry of the solvent molecules are shown to determine the changes of fluorescence depolarization. A concentration-dependent decrease in the fluorescence anisotropy is obtained with aromatic molecules but not with aliphatic molecules. The decrease correlates with the increasing side chain length of alkylbenzenes for both fluorophors and both membrane systems. Benzene in microsomes deviates characteristically from this trend. Measurements of TMA-DPH reveal smaller changes of anisotropy but yield the same qualitative results. Moreover, it is possible to distinguish the effects of the different stereometric properties of the three xylene isomers on the fluorescence anisotropy of DPH. According to their alkyl chain length and their specific stereometry, they exert the strongest fluidizing effect of all solvents investigated.     

Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance

Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of hemifusion is observed and quantified. In this state, the outer leaflets of both vesicles bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.Received: 26 May 2004, Published online: 3 August 2004PACS: 87.16.Dg Subcellular structure and processes: Membranes, bilayers, and vesicles - 87.15.Vv Biomolecules: structure and physical properties: Diffusion - 64.70.Nd Structural transitions in nanoscale materials     

Pressure Effects on Submicrosecond Phospholipid Dynamics Using a Long-Lived Fluorescence Probe

The effects of applied external hydrostatic pressure on submicrosecond lipid motions in DPPC⁴ bilayers have been examined using coronene (a long-lived planar fluorescent molecule) and DPH. Steady-state fluorescence emission anisotropy (EA) values () obtained for probe-labeled DPPC SUVs measured at different fixed temperatures above T _c as a function of increasing hydrostatic pressure reveal pressure-induced lipid phase transition profiles. For coronene-labeled samples, the observed lipid melt profiles are broad and shifted to higher midpoint EA pressure values (P _1/2) compared with corresponding DPH-labeled SUVs at the same temperature. The data suggest lipid motions occurring on the submicrosecond time scale, detected only by using a long-lived fluorescence probe, which occur well above the normally reported fluid–gel lipid phase transition. Slopes of the pressure-to-temperature equivalence plots (dP _1/2/dT = 39 bar/K) obtained for DPH-or coronene-labeled DPPC SUVs are identical within experimental error and reflect probe independence. For DPH, the slope of the P _1/2(T) plot provides the expected phase transition phospholipid volume change. However, intercept values (at P _1/2 = 1 bar) or apparent phase transition temperatures obtained from the equivalence plots for the two probes are not equal. Differences appear to arise due to the very disparate fluorescence lifetime values of the two probes, which result in rotational sensitivity of coronene to gel lipid volume fluctuations occurring during the extended time window provided by coronene fluorescence.     

Spectral-Luminescent Properties of Deposited Rhodamine 6G Films

We have investigated the absorption and fluorescence spectra of Rhodamine 6G thin (20–100 nm) films vacuum-deposited on solid dielectric substrates: quartz and borosilicate glasses and polymer films. By the absorption spectra several molecular absorption centers — monomers, dimers, and complex associates — have been revealed. For the first time luminescence of vacuum-deposited Rhodamine 6G films has been obtained and investigated. We classified it as fluorescence of plane dimers and dimers of the house-of-cards type and also as luminescence of complex associates. The dependence of the spectral composition of the films on the excitation wavelength in the 293–353 K temperature range has been studied. It has been proposed that amorphous Rhodamine 6G films obtained by the vacuum deposition method contain crystalline zones and microclusters of adsorbed water molecules.     

Transient excited singlet state absorption in Rhodamine 6G

Transient excited singlet state absorption (ESSA) has been studied in Rhodamine 6G in ethanol using a nitrogen laser and nitrogen laser-pumped dye laser. Broad absorption with several submaxima and possible shoulders, which represent the vibrational structure, has been observed in Rhodamine 6G in the region, 4175–4640 Å. The position of the lowest vibrational level of the first excited singlet stateS ₁ has been determined from the crossing point of the long and short wavelength spectral wings of absorption and fluorescence respectively. The energy level scheme of the molecule has been obtained with the help of the absorption and fluorescence spectra recorded. The observed structure in ESSA has been tentatively interpreted to be due to transitions from the different vibrational levels ofS ₁ to one or more vibrational levels of the upper singlet electronic stateS ₄.     

Triphenylbenzene Sensor for Selective Detection of Picric Acid

A C ₃-symmetric triphenylbenzene based photoluminescent compound, 1,3,5-tris(4′-(N-methylamino)phenyl) benzene ([NHMe]₃TAPB), has been synthesized by mono-N-methylation of 1,3,5-tris(4′-aminophenyl) benzene (TAPB) and structurally characterized. [NHMe]₃TAPB acts as a selective fluorescent sensor for picric acid (PA) with a detection limit as low as 2.25 ppm at a signal to noise ratio of 3. Other related analytes (i.e. TNT, DNT and DNB) show very little effect on the fluorescence intensity of [NHMe]₃TAPB. The selectivity is triggered by proton transfer from picric acid to the fluorophore and ground-state complex formation between the protonated fluorophore and picrate anion through hydrogen bonding interactions. The fluorescence lifetime measurements reveal static nature of fluorescence quenching.     

The Fluorescence Properties of Three Rhodamine Dye Analogues: Acridine Red,Pyronin Y and Pyronin B

The fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime of Acridine Red (AR), Pyronin Y (PYY), and Pyronin B (PYB) in aqueous and organic solvents were measured by steady state fluorescence, time-correlated single photon counting, and electronic absorption methods. The rate constants of radiation and non radiation process (k_f and k_ic) were calculated to elucidate the structural effect on the fluorescence mechanism. The data for each compound are compared with that of the corresponding rhodamine dye. AR showed significant longer lifetime and higher quantum yield than PYY and PYB, because the alkyls on N atom enhance the internal conversion (IC), the longer the alkyl the faster the IC. However, the structural variation does not alter the rate constant of radiation process (k_f) but does change k_ic significantly. The phenyl in Rhodamine B or Rhodamine 6G shows only a slight effect on the fluorescence properties. Ethanol is the solvent in which all five compounds exhibit longest lifetime and highest fluorescence quantum yield.