OPTICAL SPECTROSCOPY OF BILAYER MEMBRANES

Abstract— The absorption and fluorescence spectroscopy of natural and model bilayer lipid membranes is reviewed. Basic structural features of biological membranes and the relative advantages of black lipid membranes (BLM) and of liposomes are discussed. Theoretical considerations show that the wavelengths of absorption maxima are affected by the refractive index and dielectric constant of the medium surrounding the chromophore. Techniques of obtaining photoelectric action spectra, direct absorption spectra, and reflection spectra of BLM are described. Polarized spectra can give information about the orientation of membrane constituents and show, for example, that the porphyrin ring of chlorophyll in BLM is tilted at 45 ± 5° to the membrane surface. Absorption maxima of chlorophyll in BLM are compared with solution spectra of various chlorophyll adducts and aggregates. It is concluded that chlorophyll in BLM exists largely as solvated monomer and dimer (or oligomer), depending on concentration, and is not coordinated with water. From the theory of fluorescence spectroscopy it follows that aggregation and the polarity of the environment affect the fluorescence yield and lifetime of a membrane component, and also the wavelength of its emission maximum. The microviscosity of the membrane matrix affects the anisotropy of fluorescence. Techniques of steady-state fluorescence spectroscopy and of fluorescence lifetime measurements are reviewed. Examples of the use of fluorescent probes in membrane studies are given. Certain probes such as anilinonaphthalene sulfonate (ANS) preferentially bind to membrane proteins. The location of a probe in a particular membrane region can be pinpointed from its fluorescence yield and emission maximum. The orientation of the hydrocarbon chains of membrane lipids has been found, from fluorescence polarization of certain probes, to be normal to the membrane surface as postulated a priori on the basis of the lipid bilayer model. Anisotropy of fluorescence shows that elongated probe molecules rotate rapidly about their long axes when surrounded by phospholipids but become immobilized when bound to proteins. Changes in intensity and anisotropy of fluorescence as function of temperature have demonstrated the existence of phase transitions and phase equilibria of membrane lipids. Excimer fluorescence has been used as a measure of the available lipid core volume of membranes. Mechanisms of energy transfer between membrane components are reviewed. The theoretical dependence of energy transfer on distance and orientation for several rigid and fluid membrane models is discussed in terms of the structural information it can provide. Fluorescence sensitization resulting from energy transfer within and across bilayer membranes has been demonstrated in various systems. Quantitative measurement of energy transfer efficiency in BLM has shown that such transfer is about five times more efficient than in solutions at comparable donor-acceptor distances. Lipid membranes can be viewed as structures which maintain their components at high concentrations, in a reactive state, and at favourable orientations.     

1,3,6,8-Tetraethynylpyrene and 1,3,6,8-tetrakis (trimethylsilylethynyl) pyrene: Photophysical properties in homogeneous media

The photophysical properties of two new tetra substituted derivatives of pyrene: 1,3,6,8-tetraethynylpyrene (TEP) and 1,3,6,8-tetrakis(trimethylsilylethynyl)pyrene (TEP-TMS) have been studied. Studies were done with respect to mirror image symmetry in the absorption and emission spectra and permissive or forbidden nature of S₀–S₁ transition, solvent sensitivity of the first and third vibronic bands and fluorescence anisotropy. Both the derivatives exhibited a strongly allowed S₀–S₁ transition, high fluorescence quantum yield, shorter fluorescence lifetime compared to pyrene and invariance of the vibronic band intensity ratio to solvent polarity. The behavior of the two pyrene derivatives validates the hypothesis “solvent polarity mediates vibronic coupling and therefore the emission band intensities, for forbidden S₀–S₁ transitions”. The trimethylsilyl derivative (TEP-TMS) was characterized by a strong fluorescence in solid state. The tetraethynyl derivative (TEP) showed high fluorescence anisotropy comparable to the well-known anisotropy probe DPH in glycerol at 0 °C. The fluorescence intensities of TEP and TEP-TMS did not show any significant change in the temperature ranger 0–40 °C for a low viscous solvent like ethanol and in the range 0–60 °C in glycerol. Unlike pyrene, no excimer emission was observed even up to 10⁻³ M for TEP and TEP-TMS.     

Stable vesicle formation through intra- and inter-chain aggregation of poly[sodium N-(11-acrylamidoundecanoyl)-l-valinate] in aqueous solution

The vesicle-forming surfactant, sodium N-(11-acrylamidoundecanoyl)-l-valinate was polymerized to obtain corresponding polysoap. Light scattering and fluorescence probe techniques were used to characterize the polysoap. Fluorescence probe studies suggested that the polymer forms intra-chain as well as inter-chain aggregates. The microenvironment of the aggregates was studied by fluorescence measurements using 1-anilinonaphthalene, pyrene, and 1,6-diphenylhexatriene (DPH) as probe molecules. Fluorescence anisotropy studies by use of DPH have indicated a high local viscosity of the aggregates formed by the polysoap in water. The pH-induced change of the aggregate structure has been studied. The phase transition temperature of the polysoap was determined from temperature dependence of fluorescence anisotropy of DPH. Dynamic light scattering measurements were performed to determine the mean size of the aggregates. Transmission electron micrographs revealed closed vesicles in water.     

Radiation damage to lymphocyte membranes

The changdes of some physical parameters of lymphocyte membranes following neutron irradiation from a²⁵²Cf sourcein vitro in range of very low doses were studied. We aimed our attention at fluorescence intensity, fluorescence polarisation, microviscosity and order parameter of lipid bilayer of lymphocyte membranes. The values of above mentioned parameters were obtained by means of fluorescent spectrometry using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a fluorescent probe. It was shown, that with increasing doses of radiation the values of all studied parameters have been decreasing. The microviscosity of membranes decreased to 37% of the control value.     

Effect ofcholinergic ligands on the lipids of acetylcholine receptor-rich membrane preparations from Torpedo californica.

Ion permeation, triggered by ligand-receptor interaction, is associated with the primary events of membrane depolarization at the neuromuscular junction and synaptic connections. To explore the possible sites of ion permeation, the long-lived fluorescent probe pyrene (fluorescence lifetime approximately 400 nsec) has been inserted into the lipid phase of acetylcholine receptor-rich membrane (AcChR-M) preparations from Torpedo californica. The pyrene probe is susceptible to both fluidity and permeability changes in the lipid bilayer. These changes are detected by variations in the rate of decay of the excited singlet state of pyrene after pulsation with a 10-nsec ruby laser flash. Variations of these lifetimes in the membrane preparations alone or in the presence of quenchers show that binding of cholinergic agonists and antagonists, neurotoxins, and local anesthetics to AcChR-M produces varying effects on the properties of the pyrene probe in the lipid phase. It is concluded that binding of cholinergic ligands to the receptor does not significantly alter the fluidity of permeability of the lipids in the bilayer contact with pyrene. On the other hand, local anesthetics do affect these properties.     

Fluorescent Probing of Urea-induced Chemical Unfolding of Bovine Serum Albumin by Intramolecular Charge Transfer Fluorescence Probe E-3-(4-Dimethylamino-Naphthalen-1-yl)-Acrylic Acid

Changes in polarity at the immediate binding site in protein bovine serum albumin (BSA) produces distinct changes in the solvent polarity-dependent emission band of fluorescence probe E-3-(4-dimethylamino-naphthalen-1-yl)-acrylic acid. Steady-state spectroscopy and time-resolved spectroscopy have been used to investigate this binding process. Attaching the probe to BSA and then monitoring its spectral changes with increasing urea concentration and raising temperature has also tracked the denaturation of BSA chemically and thermally. The polarity of the microenvironment was investigated employing the Reichardt E_T(30) scale. Fluorescence anisotropy, red edge excitation shifts and acrylamide-induced quenching of fluorescence have been exploited to gain better insight into this binding process.     

The styrylpyridine dye for the silane sol–gel transition studies by time-dependent fluorescence

A trans-4-(p-N,N-dimethylaminostyryl)-N-vinylbenzylpyridinium chloride (vbDMASP) fluorescence probe was optimized in ground and excited state as a function of change in the microenvironment polarity, using the Amsol HyperChem program package. In the calculations, protic and aprotic solvents were used. On this basis a change in the molecule geometry after excitation, depending on the surrounding solvent, was determined.Absorption and steady-state fluorescence spectra of vbDMASP in the solvent of different polarity and in the model water–glycerol solutions were also recorded. On the basis of Stokes’ shift change with the Onsager polarity scale a change in the dipole moment of the probe during transition from ground to excited state, in protic and aprotic solvents was determined.Since during the sol–gel transition of tetraethylorthosilane in the acidic environment both polarity and viscosity of the microenvironment change the vbDMASP probe was applied and fluorescence time-resolved measurements were done. On this basis the correlations between the results of time-resolved measurements for the multichromophoric probe applied in the gelation process and molecular optimization data are discussed.     

Effect of phenyltin compounds on lipid bilayer organization

Phenyltin compounds are known to be biologically active. Their chemical structure suggests that they are likely to interact with the lipid fraction of cell membranes. Using fluorescence and NMR techniques, the effect of phenyltin compounds on selected regions of model lipid bilayers formed from phosphatidylcholine was studied. The polarization of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) dipalmitoyl-L -phosphatidylethanolamine and desorption of praseodymium ions was used to probe the polar region, whereas the polarization of 1 - (4 - trimethylammoniumphenyl) - 6 - phenyl - 1,3,5-hexatriene p-toluenesulfonate measured the hydrophobic core of the membrane. In addition, changes in the N-(5-fluoresceinthiocarbanoly)dipalmitoyl - L - α - phosphatidyl - ethanolamine fluorescence intensity indicated the amount of charge introduced by organotin compounds to the membrane surface. There were no relevant changes of measured parameters when tetraphenyltin was introduced to the vesicle suspension. Diphenyltin chloride causes changes of the hydrophobic region, whereas the triphenyltin chloride seems to adsorb in the headgroup region of the lipid bilayer. When the hemolytic activity of phenyltin compounds was measured, triphenyltin chloride was the most effective whereas diphenyltin chloride was much less effective. Tetraphenyltin causes little damage. Based on the presented data, a correlation between activity of those compounds to hemolysis (and toxicity) and the location of the compound within the lipid bilayer could be proposed. In order to inflict damage on the plasma membrane, the compound has to penetrate the lipid bilayer. Tetraphenyltin does not partition into the lipid fraction; therefore its destructive effect is negligible. The partition of the compound into the lipid phase is not sufficient enough, by itself, to change the structure of the lipid bilayer to a biologically relevant degree. The hemolytic potency seems to be dependent on the location of the compound within the lipid bilayer. Triphenyltin chloride which adsorbs on the surface of the membrane, causes a high level of hemolysis, whereas diphenyltin chloride, which penetrates much deeper, seems to have only limited potency. © 1998 John Wiley & Sons, Ltd.     

Fluorescence studies of the interactions of ubiquinol-10 with liposomes

Ubiquinone-10 plays a central role in energy production and its reduced form, ubiquinol-10 is also capable of acting as a potent radical scavenging antioxidant against membrane lipid peroxidation. Efficiency of this protection depends mostly on its localization in lipid bilayer. The intrinsic fluorescence of ubiquinol-10 and of the exogenous probe, Laurdan, has been used to determine the location of ubiquinol-10 in unilamellar liposomes of egg phosphatidylcholine (EggPC) and dimyristoyl phosphatidylcholine. Laurdan fluorescence moiety is positioned at the hydrophilic-hydrophobic interface of the phospholipid bilayer and its parameters reflect the membrane polarity and microheterogeneity, which we have used to explore the coexistence of microdomains with distinct physical properties. In liquid-crystalline bilayers ubiquinol has a short fluorescence lifetime (0.4 ns) and a high steady-state anisotropy. In a concentration-dependent manner, ubiquinol-10 influences the Laurdan excitation, emission and generalized polarization measurements. In EggPC liposomes ubiquinol-10 induces a decrease in membrane water mobility near the probe, while in dimyristoyl liposomes a decrease in the membrane water content was found. Moreover the presence of ubiquinol results in the formation of coexisting phospholipid domains of gel and liquid-crystalline phases. The results indicate that ubiquinol-10 molecules are mainly located at the polar-lipid interface, inducing changes in the physico-chemical properties of the bilayer microenvironment.     

Antioxidant activity of daidzein, a natural antioxidant, and its spectroscopic properties in organic solvents and phosphatidylcholine liposomes

Daidzein, one of major isoflavones found in soybeans, has a wide spectrum of physiological and pharmacological functions. The observed biological effects involve its interactions with lipid bilayers, usually detected by indirect methods. In this study we use the native fluorescence of daidzein to report changes observed during its interactions with organic solvents and in a phosphatidylcholine membrane.We have investigated interactions of daidzein with lipid bilayers of egg phosphatidylcholine (PC) by absorption and fluorescence methods. The data obtained indicate emission arises from the conjugate anion in excited singlet state. The fluorescence is found to increase with the basicity of the solution and the polarity of the solvent. An increase in fluorescence anisotropy in the presence of membranes suggests partial incorporation of daidzein molecules into the bilayer. Two fluorescence lifetime components, 1.5 ns and 3.5 ns, reflects the partition of daidzein between aqueous and membrane environments, respectively. On the basis of the obtained spectroscopic data we conclude that up to 15% of daidzein is located in hydrophilic region of the membrane whereas the rest is distributed in aqueous bulk and aqueous/membrane interface.For studying the antioxidant activity of daidzein against lipid peroxidation initiated by AAPH the molecule of C11-BODIPY581/591 has been used as a fluorescent oxidation indicator. The results show that the presence of daidzein anions in the membrane interface increases the inhibitory effect on lipid peroxidation compared to the neutral form of daidzein.     

Effect of leucinyl-phenylalanyl-valine on DMPC liposome membrane

Leucinyl-phenylalanyl-valine (LFV) is a hydrophobic tripeptide with a flat egg shaped structure with the long axis dimension of about 12 A. The effect of LFV on dimyristoylphosphatidylcholine (DMPC) liposome membrane has been studied by differential scanning calorimetry (DSC) and fluorescence spectroscopy. Calorimetric studies shows that incorporation of LFV completely abolishes the pretransition temperature with broadening of main transition temperature. Four conceptually different fluorescence probes, 1-naphthol (1-ROH) an excited state proton transfer probe, 8-anilino-1-naphthalenesulphonate (ANS) a solvent polarity probe, 1-6-diphenylhexatriene (DPH) an anisotropy probe and pyrene an excimer-forming probe have been used for fluorescence spectroscopic studies. For 1-ROH, ANS and DPH, a decreased partitioning with increasing mol.% of LFV was observed. Increasing LFV mol.% caused a decrease in the neutral form emission of 1-ROH, and a decrease in fluorescence intensity with red shift in ANS. The excimer formation ability of pyrene also decreased. The phase transition behavior of DMPC membrane in the presence of LFV was similar to the known effect of cholesterol on lipid bilayers. These results suggest that LFV cause an increased compactness of membrane.     

INTRAMOLECULAR EXCIMER FLUORESCENCE: A NEW PROBE OF PHASE TRANSITIONS IN SYNTHETIC PHOSPHOLIPID MEMBRANES

Abstract— The molecule (1,1'-dipyrenyl)-methyl ether [dipyme] shows intramolecular excimer fluorescence in competition with fluorescence from the locally excited pyrene chromophore. This intensity ratio I_c/I_m is sensitive to solvent viscosity. The molecule is soluble in synthetic phospholipid membranes. ¹H NMR studies suggest that the molecule is localized in the hydrocarbon region of the membrane. Fluorescence measurements at various temperatures of dipyme dissolved in these membranes show that I_c/I_m is exceedingly sensitive to fluidity changes accompanying both the pretransitions and the gel to liquid crystalline transitions of the membrane. These studies can be carried out at a mole ratio of probe to lipid 10²-10³ smaller than that necessary to observe bimolecular pyrene excimer formation.     

Preferential Solvation in Aqueous–Organic Mixed Solvents Using Solvatochromic Indicators

Solvatochromism of the twisted intramolecular charge-transfer (TICT) fluorescence of 4-(N,N-dimethylamino)benzonitrile (DMABN) in pure water, methanol, ethanol, 1-propanol, 2-propanol, acetone, acetonitrile, and in the corresponding aqueous–organic binary mixed solvents was systematically studied and an empirical solvent polarity scale (F _B) based on the DMABN solvatochromism was defined. The F _B parameters of the explored binary mixed solvents as a function of solvent composition were analyzed by a stepwise solvent-exchange (SSE) model to clarify the preferential solvation (PS) of the probe dye in these binary mixed solvents. Solvation diagrams toward DMABN in the mixed solvents, i.e., the local solvent composition in the solvation shell of DMABN molecules was depicted as a function of bulk solvent composition to visualize the PS in these mixed solvents. For comparison, a similar PS analysis was applied to the solvatochromism of 2,6-diphenyl-4-(2,4,6-triphenylpyridinium-1- yl)phenolate (ET-30) and pyrene (Py) in these mixed solvents; the responsive PS pattern of the mixed solvents toward the specific indicator dye of DMABN, ET-30, and Py was then discussed in terms of the chemical properties of the probe dye, the properties of the mixed solvents, and the solute–solvent and solvent–solvent interactions.     

Interrogating interfacial organization in planar bilayer structures

The field of biomimetic planar lipid membranes is finding increased importance as the need to devise sensing systems for biologically important species increases. We approach this area with an eye toward understanding how to interrogate local organization in these complex media. Our primary tools for this purpose are spectroscopy and electrochemistry, where imbedded reporter molecules serve as the information transducers. We use Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) methods to construct planar lipid membranes on hydrophilic solid substrates (Au for electrochemistry, SiO x for spectroscopy). Pyrene tethered to the substrate acts as our probe and AC voltammetry was used to evaluate its redox kinetics, showing slow, distance independent electron transfer between the pyrene moieties and the electrode for both monolayer and bilayer systems. Time-resolved fluorescence data indicate that tethered pyrene resides in a highly rigid environment and that the addition of the top lipid leaflet improves the organization of the bottom lipid leaflet. These data point to the cooperative effect of the bilayer leaflets in creating a system that is comparatively rigid on short length scales, and capable of mediating motion and accessibility of imbedded species.     

Effect of chlorogenic acid on the phase transition in phospholipid and phospholipid/cholesterol membranes

Chlorogenic acid (CGA) is present in many plants, especially in green coffee, dry plums, and bilberries. It is an important bioactive polyphenol. Studies showed that CGA has an antioxidative, bacteriostatic, anticancer, antiviral, and anti-inflammatory activity. Despite great interest in this compound, its interaction with the lipid model membrane has not yet been investigated. To better understand the relationship between the biological activity of CGA and its interaction with biological membranes, the thermotropic behavior of model lipid membranes was investigated. The effect of CGA on the model lipid membrane, specifically on the lipid bilayer phase transitions, was examined by the combined methods: differential scanning calorimetry and fluorescence spectroscopy. In particular, the degree of packing order of the hydrophilic phase of the lipid bilayer was determined using the fluorimetric method with Laurdan and Prodan probes, while the fluorescence anisotropy of the hydrophobic phase with the DPH and TMA-DPH probes. The results of the study show that CGA incorporates mainly into the hydrophilic part of membrane, changing the packing order of the polar heads of lipids. No significant changes were recorded in membrane fluidity of the hydrophobic membrane region, for the fluorescence anisotropy practically did not change. One can thus infer that CGA does not penetrate deep into the hydrophobic area of the membrane.     

Amphiphilic Fluorescence Resonance Energy-Transfer Dyes: Synthesis,Fluorescence, and Aggregation Behavior in Water

Amphiphilic pyrene/perylene bis-chromophore dyes were synthesized from unsymmetrically substituted perylene bisimide dyes, which were obtained through three synthetic methods. The optical and aggregation behaviors of these functional dyes were studied by means of UV/Vis absorption and fluorescence spectroscopy, dynamic light scattering, and TEM. These dyes are highly fluorescent and cover the whole visible-light region. A donor/acceptor dye displays intramolecular fluorescence resonance energy transfer (FRET), with a high efficiency of up to 96.4 % from pyrene to perylene bisimide chromophores, which leads to a high fluorescence color sensitivity to environmental polarity. Under a λ=365 nm UV lamp, the light-emitting colors of the donor/acceptor dye change from green to yellow with increasing solvent polarity, which demonstrates application potential as a new class of FERT probes. The donor/acceptor dye in water was assembled into hollow vesicles with a narrow size distribution. The bilayer structure of the vesicular wall was directly observed by means of TEM. These vesicular aggregates in water are fluorescent at λ=650–850 nm within the near-infrared region.     

Synthesis of environmentally sensitive 2′-deoxyguanosine containing solvatochromic pyrene fluorophore

The synthesis and photophysical property of novel solvatochromic pyrene derivative, Apa5 and fluorescent guanine ^ApaG8 were described. These newly synthesized fluorescent pyrene derivatives exhibited solvent polarity dependent fluorescence at longer wavelengths. Such environmentally sensitive pyrene derivatives can be used as a reporter probe that is sensitive to the changes in the microenvironment around DNA either in vitro or in vivo.     

Role of bound water in biological membrane structure: fluorescence and infrared studies.

Bound water is a major component of biological membranes and is required for the structural stability of the lipid bilayer. It has also been postulated that it is involved in water transport, membrane fusion, and mobility of membrane proteins and lipids. We have measured the fluorescence emission of membrane-bound 1-anilino-8-naphthalenesulfonate (ANS) and the infrared spectra of membranes, both as a function of hydration. ANS fluorescence is sensitive to polarity and fluidity of the membrane-aqueous interface, while infrared absorption is sensitive to the hydrogen bonding and vibrational motion of water and membrane proteins and lipids. The fluorescence results provide evidence of increasing rigidity and/or decreasing polarity of the membrane-aqueous interface with removal of water. The membrane infrared spectra show prominent hydration-dependent changes in a number of bands with possible assignments to cholesterol (vinyl CH bend, OH stretch), protein (amide A, II, V), and bound water (OH stretch). Further characterization of the bound water should allow its incorporation into current models of membrane structure and give insight into the role of membrane hydration in cell surface function.     

Thermodynamics and micellar properties of tetradecyltrimethylammonium bromide in formamide-water mixtures

The effect of formamide on the micellization of tetradecyltrimethylammonium bromide has been investigated by conductance and fluorescence probe experiments. The critical micelle concentration and the degree of counterion dissociation of micelles were obtained from conductance measurements in the temperature range of 20 to 40 degrees C. It was found that these two parameters increase with both temperature and formamide content in the solvent system. The thermodynamic parameters of micellization were estimated using the equilibrium model of micelle formation. The standard free energy of micellization was found to be negative in all cases and becomes less negative as the formamide content in the mixed solvent increases, but it is roughly independent of temperature. Although the entropic contribution was found to be larger than the enthalpy one, in particular at lower temperatures, an enthalpy-entropy compensation effect was observed for all systems. Micellar aggregation numbers were determined by the static quenching method, using pyrene as a probe and cetylpyridinium chloride as a quencher. The observed decrease in the micelle aggregation number, which is controlled by the increase in the surface area per headgroup, was attributed to an enhanced solvation in formamide rich solvent mixtures. Changes in the pyrene 1:3 ratio index, indicating a more polar environment, are consistent with an increased micellar solvation. Fluorescence polarization of both coumarin 6 and fluorescein are indicative of a decrease in microviscosity with cosolvent addition. The data on fluorescence anisotropy of coumarin 6 were analyzed using the wobbling in cone model. Data indicated the formation of micelles with a less ordered structure as the formamide increases in the solvent system.