1-Naphthol as an excited state proton transfer fluorescent probe for erythrocyte membranes

The microenvironmental dependence of excited state prototropism of 1-naphthol and the corresponding changes in its fluorescence emission is utilized to monitor the acyl chain melting phase transition behavior of liposome membrane made from human erythrocyte lipids. A sharp increase in the ratio of neutral/anionic form fluorescence intensity is noticed at the phase transition temperature (19 degrees C). This provides a convenient method for obtaining phase transition temperature in lipid membranes. The membrane modifying effect of cholesterol on the erythrocyte liposome is successfully sensed by 1-naphthol fluorescence.     

Effect of pressure on the Prodan fluorescence in bilayer membranes of phospholipids with varying acyl chain lengths

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) were observed as a function of pressure for the bilayer membrane systems of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). The wavelength of the emission maximum, lambdamax, was found to be 480, 430, and 500 nm for the liquid crystalline (Lalpha), ripple gel (P'beta), and pressure-induced interdigitated gel (LbetaI) phase, respectively. Since the lambdamax reflects the solvent property around the probe molecules, we could speculate on the location of the Prodan molecules in the bilayer membranes; in the Lalpha phase of the lipid bilayer, the Prodan molecules distribute around the phosphate of the lipids (i.e. the polar region). The Lalpha/P'beta phase transition caused the Prodan molecules to move into the less polar region near the glycerol backbone. The fluorescence intensity of the Prodan in the P'beta phase was dependent on the chain length of the lipids and on pressure; the shorter the chain length of the lipid, the stronger the fluorescence intensity of the Prodan. Moreover, for the DLPC bilayer membrane system, the fluorescence intensity at 430 nm increased with increasing pressure, indicating that the partition of Prodan into the DLPC bilayer membrane is promoted by applying pressure. In the case of the DPPC and DSPC bilayers, as the pressure increased further, the pressure-induced interdigitation caused the Prodan molecules to squeeze out of the glycerol backbone region and to move the hydrophilic region near the bilayer surface. The ratio of fluorescence intensity at 480 nm to that at 430 nm, F480/F430, showed a sharp change at the phase-transition pressure. In the case of the DPPC and DSPC bilayers, the values of F480/F430 showed an abrupt increase above a certain pressure higher than the Lalpha/P'beta transition pressure, which corresponds to the interdigitation from the P'beta to the LbetaI phase. The plot of F480/F430 versus pressure is available for recognition of the bilayer phase transitions, especially the bilayer interdigitation.     

Chain elongation of diacylphosphatidylcholine induces fully bilayer interdigitation under atmospheric pressure

The phase transitions of dibehenoylphosphatidylcholine (C22PC) bilayer membrane were observed by differential scanning calorimetry under atmospheric pressure and light-transmittance measurements under high pressure. The constructed temperature-pressure phase diagram suggests that the gel phase at low temperatures is the interdigitated gel phase. To confirm the phase state, we performed small-angle neutron scattering and fluorescence measurements using a polarity-sensitive probe Prodan for the C22PC bilayer membrane under atmospheric pressure. The peaks obtained in both measurements clearly showed the characteristic patterns of the fully interdigitated gel phase. Taking into account of previous studies on the gel phase for long-chain PC bilayers under atmospheric pressure and our studies on the pressure-induced bilayer interdigitaion of diacyl-PCs, it turned out that the interdigitation of diacyl-PC bilayer membranes occurs when the carbon number of acyl chain reaches at least 22. The present study revealed that the interdigitation of PC bilayer membranes occurs not only by weakening the attractive force of polar head groups but also by strengthening the cohesive force of acyl chains. When dominating the force of acyl chains, the interdigitation can be induced even in a diacyl-PC bilayer membrane by only hydration under atmospheric pressure.     

Carbazole as an excited state proton transfer fluorescent probe for lipid bilayers in alkaline medium

In our effort to look for novel excited state proton transfer (ESPT) fluorescent probes in alkaline pH range, we have examined carbazole as a possible candidate because of its high extinction coefficient, high quantum yield and a larger difference in ionization constant between the ground and excited state (pKa - pKa*). The photodissociation of carbazole was studied in liposome membrane by steady state fluorescence measurements at alkaline pH ranges. The neutral form and the anionic form of carbazole emit at 362 and 417 nm, respectively. This large shift in emission makes it convenient to monitor the physical properties of liposomes. The neutral form fluorescence intensity of carbazole is sensitive to phase changes in the membrane and also shows a maximum at phase transition temperature. This variation of intensity can be explained in terms of redistribution of probe between the surface and interior of the liposomes. Cholesterol induced phase changes of liposomes were also sensed by the ESPT of carbazole.     

1-Naphthol as an excited state proton transfer fluorescent probe for sensing bound-water hydration of polyvinyl alcohol

When 1-naphthol incorporated polyvinyl alcohol (PVA) films are allowed to swell in water, there is a loss of fluorescence intensity of the neutral form with a concomitant increase of the anionic form fluorescence intensity. This fluorescence response due to the excited state prototropism (ESPT) of 1-naphthol is very sensitive to the initial stage of hydration of the PVA. Using an existing model of hydrogel swelling and DSC experiments, it was reasoned that 1-naphthol senses the bound-water component of PVA hydration. Thus, 1-naphthol is proposed as an ESPT fluorescent sensor for the specific sensing of bound-water hydration of PVA hydrogel.     

Probing the Interdigitated Phase of a DPPC Lipid Bilayer by Micropipette Aspiration

We used micropipette aspiration of giant unilamellar vesicles to directly measure the areal expansion of gel (L_β′) phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers induced by exposure to ethanol/water mixtures. Areal expansion began in 7 vol% ethanol and increased monotonically as the concentration of ethanol was increased to 15 vol% at which point areal expansion reached a plateau of 50%. This ethanol concentration range is in good agreement with that of the interdigitated phase (L_βI) of DPPC, therefore, we believe that this is the first direct measurement of the areal expansion accompanying interdigitation of gel-phase lipids. Our observations are consistent with the presence of coexisting L_βI and L_β′ phases in ethanol concentrations between 7% and 15 vol% and 100% L_βI phase in 15 vol% ethanol and higher. We observed a bimodal distribution of areal expansion (0% and 20%) induced by 7 vol% ethanol indicating that at the threshold concentration, interdigitation is induced in only a portion of DPPC vesicles. Areal expansion could not be easily reversed, consistent with kinetic trapping of the L_βI phase. DPPC vesicles exposed to butanol at the known threshold and plateau concentrations for the L_βI phase displayed areal expansion behavior consistent with our ethanol observations. However, the area expanded significantly faster for DPPC bilayers exposed to butanol vs. ethanol, which we attribute to enhanced partitioning of the longer-chained butanol into the lipid headgroups. Ethanol-induced areal expansion of DPPC bilayers was inhibited by inclusion of 10 mol% and 25 mol% cholesterol in the bilayer. However, areal expansion could be induced by application of tensions (∼8 mN/m) similar to the phenomena of interdigitation induced by high pressure. The presence of 20 vol% ethanol significantly decreased surface cohesion of DPPC bilayers containing 25 mol% cholesterol as evidenced by a decreased area compressibility modulus and lysis tension.     

Effect of submicellar concentrations of conjugated and unconjugated bile salts on the lipid bilayer membrane

The interaction of submicellar concentrations of various physiologically important unconjugated [sodium deoxycholate (NaDC), sodium cholate (NaC)] and conjugated [sodium glycodeoxycholate (NaGDC), sodium glycocholate (NaGC), sodium taurodeoxycholate (NaTDC), sodium taurocholate (NaTC)] bile salts with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles in solid gel (SG) and liquid crystalline (LC) phases was investigated using the excited-state prototropism of 1-naphthol. Steady-state and time-resolved fluorescence of the two excited-state prototropic forms of 1-naphthol indicate that submicellar bile salt concentration induces hydration of the lipid bilayer membrane into the core region. This hydration effect is a general phenomenon of the bile salts studied. The bilayer hydration efficiency of the bile salt follows the order NaDC > NaC > NaGDC > NaTDC > NaGC > NaTC for both DPPC and DMPC vesicles in their SG and LC phases.     

High pressure effect on phase transition behavior of lipid bilayers

Phase behavior of lipid bilayers at high pressure is critical to biological processes. Using coarse grained molecular dynamic simulations, we report critical characteristics of dipalmitoylphosphatidylcholine bilayers with applied high pressure, and also show their phase transition by cooling bilayer patches. Our results indicate that the phase transition temperature of dipalmitoylphosphatidylcholine bilayers obviously shifts with pressure increasing in the rate of 37 °C kbar(-1), which are in agreement with experimental data. Moreover, the main phase transition is revealed to be strongly dependent on lipid area. A critical lipid area of ~0.57 nm(2) is found on the main phase transition boundary. Similar structures of acyl chains lead to the same sensitivity of phase transition temperature of different lipids to the pressure. Based on the lateral density and pressure profiles, we also discuss the different effects on bilayer structure induced by high temperature and high pressure, e.g., increasing temperature induces higher degree of interdigitation of lipid tails and thinner bilayers, and increasing pressure maintains the degree of interdigitation and bilayer thickness.     

Unusual fluorescence spectral response of 1-(4-N,N-dimethylaminophenylethynyl)pyrene towards the thermotropic phase change in lipid bilayer membranes

The applicability of 1-(4-N,N-dimethylaminophenylethynyl)pyrene (DMAPEPy), a pyrene derivative showing intramolecular charge transfer, as a prospective probe for lipid bilayer membranes has been evaluated. High sensitivity of DMAPEPy to solvent polarity and viscosity makes it to act both as a polarity-sensitive probe and as a fluorescence anisotropy probe. The molecule shows high partition efficiency towards bilayer membranes in both solid gel as well as in the liquid crystalline phases. The emission spectrum, quenching experiment and lifetime data suggest bimodal distribution of DMAPEPy in the bilayer. Using the solvent polarity scales the polarity parameters of the two locations in lipid bilayer have been estimated. In the bilayer environment it exhibits remarkable spectral changes with temperature. The thermotropic phase change of the bilayer is sensitively monitored by fluorescence intensity as well as fluorescence anisotropy parameters. DMAPEPy is also capable of sensing the changes induced by membrane modifiers like cholesterol.     

Adsorption of phenyltin compounds onto phosphatidylcholine / cholesterol bilayers

Phenyltin compounds are known to be biologically active and, whan widely spread, are potentially hazardous. As their chemical structure suggests, they interact with the lipid fraction of the cell membrane. Their effect on the model phosphatidylcholine/cholesterol bilayer has been studied using fluorescence and ¹H NMR techniques. The change in the fluorescein‐PE fluorescence intensity indicates the amount of charge added by phenyltin compounds to the membrane surface. Although the presence of cholesterol alone does not alter membrane interface properties measured with fluorescein‐PE, ¹H NMR measurements show that lipid mobility is altered throughout the hydrophobic core of the membrane. Cholesterol in the phosphatidylcholine bilayer does not alter tetraphenyltin interaction with the membrane, though the effect of diphenyltin dichloride, penetrating deeply into the hydrophobic core of the membrane, is reduced when the amount of cholesterol in the membrane is increased, suggesting decreased compound adsorption. Triphenyltin chloride has a qualitatively different effect on the lipid bilayer, when observed using this fluorescence technique. The adsorption of triphenyltin onto the phosphatidylcholine/cholesterol membrane induces a lateral phase separation of membrane components. Since triphenyltin chloride is known to be adsorbed onto the interface of the lipid bilayer, this separation mechanism must originate in this region and does not seem to be electrostatic in origin. ¹H NMR measurements have confirmed the observation that these two active phenyltin compounds interact with the phosphatidylcholine/cholesterol membrane differently, disrupting different regions of the bilayer to a different degree. Copyright © 2000 John Wiley & Sons, Ltd.     

Prodan fluorescence detects the bilayer packing of asymmetric phospholipids

The bilayer phase behavior of asymmetric phospholipids, palmitoylstearoylphosphatidylcholine (PSPC) and stearoylpalmitoylphosphatidylcholine (SPPC), with different vesicle sizes (large multilamellar vesicle (LMV) and giant multilamellar vesicle (GMV)) was investigated by fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan under high pressure. The results were compared with those of a symmetric phospholipid, diheptadecanoyl PC (C17PC). The difference in phase transitions of the PSPC and SPPC bilayers and in thermodynamic quantities of the transitions was hardly observed between LMV and GMV as the case of the C17PC bilayer. On the other hand, the Prodan fluorescence showed clear differences between LMV and GMV of the asymmetric PC bilayers. From the second derivative of Prodan fluorescence spectra, the three dimensional image plots in which we can clearly see the location of Prodan in the bilayer membrane as blue valleys were constructed for LMV and GMV under high pressure. We revealed from the plots that the bilayer packing is significantly dependent on not only the vesicle size but also the acyl-chain asymmetry of PC molecule in addition to the phase states. It was found that the packing of the gel phases of the asymmetric PC bilayers is weaker than that of the symmetric PC bilayer, and the size of vesicle affects the packing of the interdigitated gel phase the most markedly among three gel phases. This study suggests that the Prodan molecules can detect the effect of vesicle size on the phase states for the asymmetric PC bilayers, and they become a useful indicator for various membrane properties, especially bilayer interdigitation.     

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.     

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.     

Fluorescence properties of 1-naphthol, 2-naphthol and 1,2,3,4-tetrahydronaphthol in aqueous alcohol solvents with and without beta-cyclodextrin

The fluorescence properties of 1-naphthol, 2-naphthol and 1,2,3,4-tetrahydronaphthol were obtained in binary aqueous-alcohol solvents with and without beta-cyclodextrin. The fluorescence of both the molecular and anionic forms of 1-naphthol and 2-naphthol were observed in the binary solvents without beta-cyclodextrin. Only the fluorescence of the molecular form of 2-naphthol appeared in the binary solvents with beta-cyclodextrin present, and its fluorescence was quenched with increasing amounts of beta-cyclodextrin. However, the fluorescence intensity of the molecular form of 1-naphthol increased with an increasing amount of beta-cyclodextrin in the binary solvents. The fluorescence intensity of 1,2,3,4-tetrahydronaphthol decreased with an increase in the amount of beta-cyclodextrin. The fluorescence results were interpreted with the Stern-Volmer equation and a modified Stern-Volmer equation.     

Partitioning of dual-lipidated peptides into membrane microdomains: lipid sorting vs peptide aggregation

The lateral membrane organization and phase behavior of the lipid mixture DMPC(di-C(14))/DSPC(di-C(18))/cholesterol (0-33 mol %) with and without an incorporated fluorescence-labeled palmitoyl/farnesyl dual-lipidated peptide, BODIPY-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe, which represents a membrane recognition model system for Ras proteins, was studied by two-photon excitation fluorescence microscopy. Measurements were performed on giant unilamellar vesicles (GUVs) over a large temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (all-gel, fluid/gel, liquid-ordered, all-fluid). At temperatures where the fluid-gel coexistence region of the pure binary phospholipid system occurs, large-scale concentration fluctuations appear. Incorporation of cholesterol levels up to 33 mol % leads to a significant increase of conformational order in the membrane system and a reduction of large domain structures. Adding the peptide leads to dramatic changes in the lateral organization of the membrane. With cholesterol present, a phase separation is induced by a lipid sorting mechanism owing to the high affinity of the lipidated peptide to a fluid, DMPC-rich environment. This phase separation leads to the formation of peptide-containing domains with high fluorescence intensity that become progressively smaller with decreasing temperature. As a result, the local concentration of the peptide increases steadily within the confines of the shrinking domains. At the lowest temperatures, where the acyl-chain order parameter of the membrane has already drastically increased and the membrane achieves a liquid-ordered character, an efficient lipid sorting mechanism is no longer supported and aggregation of the peptide into small clusters prevails. We can conclude that palmitoyl/farnesyl dual-lipidated peptides do not associate with liquid-ordered or gel-like domains in phase-separated bilayer membranes. In particular, the study shows the interesting ability of the peptide to induce formation of fluid microdomains at physiologically relevant cholesterol concentrations, and this effect very much depends on the concentration of fluid vs ordered lipid molecules.     

Fluidity modulation of phospholipid bilayers by electrolyte ions: insights from fluorescence microscopy and microslit electrokinetic experiments

Fluidity and charging of supported bilayer lipid membranes (sBLMs) prepared from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) were studied by fluorescence recovery after photobleaching (FRAP) and microslit electrokinetic measurements at varying pH and ionic composition of the electrolyte. Measurements in neutral electrolytes (KCl, NaCl) revealed a strong correlation between the membrane fluidity and the membrane charging due to unsymmetrical water ion adsorption (OH(-) ? H(3)O(+)). The membrane fluidity significantly decreased below the isoelectric point of 3.9, suggesting a phase transition in the bilayer. The interactions of both chaotropic anions and strongly kosmotropic cations with the zwitterionic lipids were found to be related with nearly unhindered lipid mobility in the acidic pH range. While for the chaotropic anions the observed effect correlates with the increased negative net charge at low pH, no correlation was found between the changes in the membrane fluidity and charge in the presence of kosmotropic cations. We discuss the observed phenomena with respect to the interaction of the electrolyte ions with the lipid headgroup and the influence of this process on the headgroup orientation and hydration as well as on the lipid packaging.     

脂双层膜表面结构与稳定性的原子力显微镜研究

用原子力显微镜研究了1,2－二油酸甘油－3－磷酸－1甘油（DOPG）脂双层膜 的表面结构与稳定性。实验结果表明,原子力显微镜的探针与脂双层膜的相互作用 导致脂双层膜表面产生一个永久的损伤。静电相互作用对脂双层膜结构和稳定性的 影响表明,在NaCl溶液中制成的脂质体,随着NaCl浓度的增加,它们的双层膜更稳 定。在低的NaCl浓度则经常被损伤,在1 mol/L NaCl溶液中制备的指双层变得更稳 定。在KCl溶液中结果恰好相反。在高的KCl浓度中经常被损伤,随着KCl浓度的降 低,它们的双层膜更稳定。葡萄糖和蔗糖对脂双层膜结构有稳定作用。     

Atomistic simulation of lipid and DiI dynamics in membrane bilayers under tension

Membrane tension modulates cellular processes by initiating changes in the dynamics of its molecular constituents. To quantify the precise relationship between tension, structural properties of the membrane, and the dynamics of lipids and a lipophilic reporter dye, we performed atomistic molecular dynamics (MD) simulations of DiI-labeled dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under physiological lateral tensions ranging from -2.6 mN m(-1) to 15.9 mN m(-1). Simulations showed that the bilayer thickness decreased linearly with tension consistent with volume-incompressibility, and this thinning was facilitated by a significant increase in acyl chain interdigitation at the bilayer midplane and spreading of the acyl chains. Tension caused a significant drop in the bilayer's peak electrostatic potential, which correlated with the strong reordering of water and lipid dipoles. For the low tension regime, the DPPC lateral diffusion coefficient increased with increasing tension in accordance with free-area theory. For larger tensions, free area theory broke down due to tension-induced changes in molecular shape and friction. Simulated DiI rotational and lateral diffusion coefficients were lower than those of DPPC but increased with tension in a manner similar to DPPC. Direct correlation of membrane order and viscosity near the DiI chromophore, which was just under the DPPC headgroup, indicated that measured DiI fluorescence lifetime, which is reported to decrease with decreasing lipid order, is likely to be a good reporter of tension-induced decreases in lipid headgroup viscosity. Together, these results offer new molecular-level insights into membrane tension-related mechanotransduction and into the utility of DiI in characterizing tension-induced changes in lipid packing.     

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.     

In situ formation and characterization of poly(ethylene glycol)-supported lipid bilayers on gold surfaces

Inclusion of a polymer cushion between a lipid bilayer membrane and a solid surface has been suggested as a means to provide a soft, deformable layer that will allow for transmembrane protein insertion and mobility. In this study, mobile, tethered lipid bilayers were formed on a poly(ethylene glycol) (PEG) support via a two-step adsorption process. The PEG films were prepared by coadsorbing a heterofunctional, telechelic PEG lipopolymer (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-(pyridyldithio)propionate]) (DSPE-PEG-PDP) and a nonlipid functionalized PEG-PDP from an ethanol/water mixture, as described in a previous paper (Munro, J. C.; Frank, C. W. Langmuir 2004, 20, 3339-3349). Then a two-step lipid adsorption strategy was used. First, lipids were adsorbed onto the PEG support from a hexane solution. Second, vesicles were adsorbed and fused on the surface to create a bilayer in an aqueous environment. Fluorescence recovery after photobleaching experiments show that this process results in mobile bilayers with diffusion coefficients on the order of 2 microm2/s. The mobility of the bilayers is decreased slightly by increasing the density of tethered lipids. The formation of bilayers, and not multilayer structures, is also confirmed by surface plasmon resonance, which was used to determine in situ film thickness, and by fluorimetry, which was used to determine quantitatively the fluorescence intensity for each 18 by 18 mm sample. Unfortunately, fluorescence microscopy also shows that there are large defects on the samples, which limits the utility of this system.