Dioleoylphosphatidylcholine Monolayer Research Articles

Monitoring protein binding to phospholipid monolayers using electrochemical impedance spectroscopy

Biotin was incorporated into dioleoyl phosphatidylcholine (DOPC) monolayers as dioleoyl phosphatidylethanolamine (DOPE)–biotin, DOPE–caproyl–biotin and DOPE–polyethylene glycol (PEG)–biotin. Experiments were carried out in electrolyte KCl (0.1 mol dm −3) buffered with 0.001 mol dm −3 phosphate buffer to pH 6.9. Bovine serum albumin (BSA), avidin, biotinylated anti-haemoglobin (IgG) and haemoglobin were added to the solution. The frequency dependence of the complex impedance of the coated electrode surfaces was estimated between 65,000 and 0.1 Hz at potentials −0.4 V versus Ag/AgCl 3.5 mol dm −3 KCl. The capacitance of the monolayers was measured at 75 Hz between potentials −0.4 and −1.1 V. Both non-specific and specific binding of the soluble proteins to DOPC gave rise to the occurrence of low frequency relaxations in the impedance data. The non-specific binding of BSA to DOPC can be suppressed by the incorporation of DOPE–PEG into the DOPC monolayer. The nature of the effect of specific binding of neutravidin to biotin on the impedance data depended on the positioning of the biotin group in relation to the DOPC monolayer surface. Successive binding of proteins to the biotin and then to each other gave rise to an increase in the significance of low frequency relaxations in the impedance data respectively.

Penetration of amphotericin B into DOPC monolayers containing sterols of cellular membranes

In order to examine the interactions between amphotericin B (AmB) and cellular membrane components, the penetration of AmB into mixed Langmuir monolayers formed by dioleoyl phosphatidylcholine (DOPC)–ergosterol and DOPC–cholesterol of the composition corresponding to the membrane of fungi and mammalians, respectively, have been studied. The penetration of AmB into mixed monolayers of DOPC–sterols was found to be dependent both on the concentration of AmB in the subphase and on the kind of a sterol. In this study, two different concentrations of AmB have been applied: below and above its critical micelle concentration (c.m.c.) (6 × 10 −7 M). Our results indicate that when AmB is incorporated in the monomeric form, its penetration is more efficient into mammalians membranes. Under these conditions, the penetration of the antibiotic is higher into host cells (mammalians) versus fungi, which explains the high toxicity of AmB. However, when AmB is aggregated, its penetration is higher into fungi cells as compared to mammalians, which causes the fungicidal effect, and thus in such a form AmB is less toxic to host cells.

Incorporation of channel-forming peptides in a Hg-supported lipid bilayer

The channel-forming peptides gramicidin and alamethicin were incorporated in a mercury-supported lipid bilayer composed of a tethered thiolipid monolayer with a self-assembled dioleoylphosphatidylcholine monolayer on top of it. The thiolipid consists of a hexapeptide chain with a high tendency to form a 3 10-helical structure, which terminates at the N-terminus end with a sulfydryl group for anchoring to the metal while the C-terminus end is covalently linked to the polar head of dimyristolylphosphatidylethanolamine. The hexapeptide moiety has two triethyleneoxy side chains that impart a satisfactory hydrophilicity and are intended to keep the anchored thiolpeptide chains sufficiently apart, so as to accommodate water molecules and inorganic ions and to create a suitable environment for the incorporation of integral proteins. Changes in the conductance of this biomimetic membrane following the incorporation of gramicidin and alamethicin were detected by impedance spectroscopy. The surface dipole potential of the hexapeptide chain and the transmembrane potential of the lipid bilayer were estimated by using a simple electrostatic model of the mercury|solution interphase.

Interaction of gramicidin derivatives with phospholipid monolayers.

A study of the interaction of gramicidin A (gA), tert-butyloxycarbonyl-gramicidin (g-BOC), and desformyl gramicidin (g-des) with dioleoyl phosphatidylcholine (DOPC) and DOPC/phosphatidylserine (PS) mixed monolayers on a mercury electrode is reported in this paper. Experiments were carried out in electrolytes KCl (0.1 mol dm(-3)) and Mg(NO3)2 (0.05 mol dm(-3)). The channel-forming properties of the gramicidins were studied by following the reduction of Tl(I) to Tl(Hg). The frequency dependence of the complex impedance of coated electrode surfaces in the presence and absence of the gramicidins was estimated between 65,000 and 0.1 Hz at potentials of -0.4 V versus Ag/AgCl with 3.5 mol dm(-3) KCl. Epifluorescence microscopy was used to qualitatively correlate the interaction of the gramicidin peptides with dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) at the air-water interface. gA was shown to form Tl+ conducting channels in a DOPC monolayer, while g-BOC and g-des did not. In DOPC-30% PS (DOPC-0.3PS) layers, there is a marked increase in channel activity of all three gramicidin derivatives. None of the peptides facilitate the permeability of the DOPC-0.3PS layer to Cd2+. All three peptides interact with the layer as shown by capacitance-potential curves and impedance spectroscopy indicated by penetration of the peptide into the dielectric, an increase in surface "roughness", and an increased significance of low-frequency relaxations. The order of interaction is gA > g-des > g-BOC. The epifluorescence study of DPPC and DPPG layers at the air-water interface shows a selective action of the different gramicidins.

Application of Electrochemical Impedance Spectroscopy to the Study of Dioleoyl Phosphatidylcholine Monolayers on Mercury

Electrochemical impedance spectroscopy has been applied to the analysis of the behavior of monolayers of dioleoyl phosphatidylcholine (DOPC) on a mercury electrode. Experiments were carried out in electrolytes KCl and NaCl (0.1 mol dm(-3)) and Mg(NO3)2 (0.05 mol dm(-3)), and the frequency dependence of the complex impedance was estimated between 65 000 and 0.1 Hz at potentials -0.4 to -1.5 V versus Ag/AgCl 3.5 mol dm(-3) KCl at uncoated and coated electrode surfaces. Experiments were also carried out in the presence of gramicidin A (gA). Between the potentials of -0.4 and -0.7 V, the DOPC monolayer behaves as an almost ideal capacitor with little frequency dispersion. At more negative potentials, the impedance data show the formation of defects (-0.7 to -0.85 V), ingression of electrolyte into the layer (capacitance peak approximately -0.935 V), reorientation of phospholipid-water structures (capacitance peak approximately -1.0 V), and initiation of phospholipid desorption (approximately -1.3 V). gA interaction with the phospholipid monolayer at -0.4 V is shown as an additional low-frequency element. A general "one capacitor model" in a RC series equivalent circuit is developed incorporating the frequency dispersion of the capacitance, distribution of the time constants of the dispersion, and a coefficient related to the interface between the solution and the coated electrode. This latter coefficient is the most robust and decreases at potentials approaching those coincident with the DOPC phase transitions.

How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy

Confocal fluorescence correlation spectroscopy (FCS) allows for the determination of lateral diffusion coefficients and surface densities in planar phospholipid systems. The determination of the vertical (z-) position of the laser focus relative to the phospholipid surface plane is of crucial importance for the accuracy of the confocal FCS experiment. In this work we determine for the first time this vertical (z-) position of the laser focus by a so-called “Z-scan”, which is based on the determination of diffusion times and particle numbers in 0.2 μm steps along the vertical (z-) axis. Experiments on supported phospholipid bilayers composed of dioleoylphosphatidylcholine (DOPC) and small amounts of Rhodamine Red-X 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Rhodamine Red-X DHPE) adsorbed onto atomically flat mica and borosilicate glass demonstrate that results obtained by the Z-scan approach are significantly more precise than those results obtained when the fluorescence intensity maximum is used as an indicator in the determination of the vertical (z-) position of the sample. In addition to this basic contribution for the investigation of planar bilayer systems by confocal FCS, the lateral diffusion coefficients of Rhodamine Red-X DHPE in supported phospholipid bilayers composed of DOPC and cholesterol as well as in DOPC or dipalmitoylphosphatidylcholine (DPPC) monolayers adsorbed at a liquid−liquid interface were determined.

Pore formation by 6-ketocholestanol in phospholipid monolayers and its interpretation by a general nucleation-and-growth model accounting for the sigmoidal shape of voltage-clamp curves of ion channels.

6-Ketocholestanol (KC), a steroid that differs from cholesterol mainly by the presence of a carbonyl group, forms pores inside a dioleoylphosphatidylcholine monolayer self-assembled on mercury by a mechanism similar to that of channel-forming peptides and proteins. The potential steps responsible for pore formation by KC molecules give rise to potentiostatic charge vs time curves whose sigmoidal shape and potential dependence can be quantitatively interpreted on the basis of a mechanism of nucleation and growth of KC clusters. Pore formation by KC allows the penetration of thallous ions across the otherwise impermeable phosphatidylcholine monolayer, while pore disruption taking place at more negative potentials causes a drop in thallous ion permeation. Pore disruption is also accounted for by a mechanism of nucleation and growth of holes inside the KC clusters. The kinetic model of nucleation and growth is general, and accounts quantitatively for the sigmoidal shape and potential dependence of the classical Hodgkin-Huxley voltage-clamp curves of potassium channels in squid giant axon,(1) using a minimum number of free parameters.

Epi-fluorescence microscopic characterization of potential-induced changes in a DOPC monolayer on a Hg drop.

Characterization of the potential-induced changes of a lipid-coated Hg-0.1 M KCl interface through electrochemical techniques and newly developed in situ fluorescence microscopy is described. Fluorescence of a fluorophore-containing dioleoyl phosphatidylcholine (DOPC) layer deposited from the gas-solution interface was observed to be dependent upon the potential of the Hg surface. The largest changes occurred for potentials where the lipid layer was desorbed: the lipid moved away from the electrode surface, reducing the efficiency of metal-mediated quenching of the excited state resulting in an increase in fluorescence. Electric potential-induced changes in the morphology of the adsorbed or desorbed DOPC lipid monolayer were observed optically for the first time using this technique. The observed potential-dependent fluorescence was compared to previous studies on an octadecanol-coated Au(111) electrode. Fluorescence microscopy was also used to characterize the fusion of DOPC liposomes with a previously adsorbed DOPC layer. Large changes in fluorescence were observed for the DOPC layer after fusion with liposomes. The fusion was accomplished via potential-created defects in the adsorbed DOPC monolayer through which the liposomes interact. The integration of the liposomes into the adsorbed monolayer results in a hybrid layer in which some lipid exists further from the electrode surface, resulting in a large increase in fluorescence. Possibilities for the creation of a biomimetic adsorbed hybrid lipid layer on Hg are also discussed.

Effect of polar head groups on the activity of aspartyl protease adsorbed to lipid membranes

The proteolytic activity of an aspartyl protease of Mucor miehei was correlated with the adsorption of the protease to lipid vesicles. It was observed that the presence of phosphatidylethanolamines (PE's) in the membrane increased the enzyme activity in a 20% in the gel phase and 10% in the fluid phase. The effects of protease on the surface pressure of monolayers composed by dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dimyristoyl phosphatidylethanolamine (DMPE) were measured at constant temperature as a function of the surface pressure. At low surface pressures, the major changes were induced by protease on DOPC and DMPC monolayers. However, the effect were much lower when the monolayer was composed by DMPE. The low hydration and strong head–head interaction between the phosphates and the amine groups of adjacent PE's would result in an area per molecule much lower in PE than in phosphatidylcholine (PC) in concordance with the lower penetration in PE. Protease adsorption on PE membranes increases the proteolytic activity in which condition is less susceptible to inhibition by pepstatin. However, PC's do not alter the enzyme activity being the action of inhibitor unaffected.

Hybridization of DNA at the surface of phospholipid monolayers. Effect of orientation of oligonucleotide chains

We studied the properties of lipid monolayers formed at the air–water interface composed of dioleoylphosphatidylcholine (DOPC) with incorporated short (19-mer) oligonucleotides. These oligonucleotides were modified by oleylamine at both (3′ and 5′) terminals or only at one (3′) terminal. Interaction of single-stranded (19-mer) oligonucleotides without oleylamine with DOPC monolayers resulted only in slight increase of surface pressure and the area per phospholipid molecule, while more substantial and significant increase of these values were observed following incorporation of oligonucelotides modified by oleylamine. This influence is similar for both types of oligonucleotide modifications. However, considerable differences in changes of monolayer properties took place after hybridization with complementary oligonucleotides. The hybridization of oligonucleotides with the DNA modified by oleic acid at both 3′ and 5′ terminals at the surface of lipid monolayer resulted in further increase of surface pressure and in the increase of the area per phospholipid molecule, while decrease of both the surface pressure and the area per phospholipid molecules were observed for hybridization with DNA modified by oleic acid at 3′ terminal. It is possible that in latter case, the hybridization caused the loss of hybridized molecules from monolayers. Interaction of noncomplementary chains with DOPC monolayers with incorporated oleyl acid-modified DNA also influenced the properties of monolayers, but the effect was weaker in comparison with that observed for complementary chains.

Detection of Tl(I) Transport through a Gramicidin−Dioleoylphosphatidylcholine Monolayer Using the Substrate Generation−Tip Collection Mode of Scanning Electrochemical Microscopy

We report the use of scanning electrochemical microscopy (SECM) to control the generation of Tl(I) at a mercury substrate and detect this species at a mercury-coated tip to study the transport of these ions through ion channels. The transport of Tl(I) across gramicidin D half-channels imbedded in a dioleoylphosphatidylcholine (DOPC) monolayer supported on a Tl amalgam hanging mercury drop electrode (HMDE) was studied using the substrate generation−tip collection mode of SECM. A Hg/Pt “submarine” electrode, used as the SECM tip, was made through simple contact of the Pt ultramicroelectrode with the HMDE. The tip transient response for the collection of generated Tl(I) at the amalgam HMDE was recorded for several tip to substrate distances. This collection−generation experiment was repeated with a DOPC-modified Tl/HMDE and a gramicidin−DOPC-modified Tl/HMDE. An apparent heterogeneous rate constant (khet = 2.8 (±0.1) × 10-4 cm/s) for the transport of Tl(I) through the gramicidin to the tip was extracted.

Electroreduction of Diphenyl Disulfide on a Self-Assembled Lipid Monolayer on Mercury

In the present work, a voltammetric study of permeability and redox behavior of diphenyl disulfide (Ph2S2) through a self-assembled monolayer of dioleoylphosphatidylcholine adsorbed on mercury is carried out; the results are compared with those obtained on a bare electrode; in this case, the reduction of Ph2S2 proceeds in a reversible way with the formation of a mercurial compound. The presence of the monolayer of phospholipid provokes an increase in the process irreversibility. Different mechanisms based on Ph2S2 adsorption either directly on a mercury drop or on phospholipids heads are proposed. The charge associated with the adsorbed Ph2S2 electroreduction was employed to calculate its solubility, and the method is compared with semiempirical expressions proposed in the literature for obtaining approximate values of solubility.

Photoelectric response of purple membrane fragments adsorbed on a lipid monolayer supported by mercury and characterization of the resulting interphase

Purple membrane (PM) fragments were adsorbed on a dioleoylphosphatidylcholine (DOPC) monolayer supported by mercury to investigate the kinetics of light-driven proton transport by bacteriorhodopsin (bR). PM fragments were also adsorbed on a mercury-supported triethyleneoxythiol (TET) monolayer. On both monolayers, the light-on current exhibits a finite, potential dependent stationary component that decreases linearly with a positive shift in the applied potential. The light-on and light-off capacitive photocurrents were interpreted on the basis of a simple equivalent circuit, which accounts for the potential dependence of the stationary light-on current. The potential of zero stationary current is about equal to +0.010 V vs. saturated calomel electrode (SCE) on DOPC-coated mercury. The absolute potential difference across the PM fragments adsorbed at this applied potential was estimated on the basis of extrathermodynamic considerations and amounts to about +260 mV; it compares favorably with the value, +250 mV, of the transmembrane potential of zero stationary current across an oocyte plasma membrane incorporating bR [Biophys. J. 74 (1998) 403.]. The effect of the proton pumping activity of photoexcited PM fragments on the electroreduction kinetics of ubiquinone-10 incorporated in the DOPC monolayer underlying the PM fragments was investigated.

Some bioelectrochemical applications of self-assembled films on mercury

The homogeneous, defect-free surface of a hanging mercury drop electrode (HMDE) is used to self-assemble films apt to the investigation of problems of biological interest. The electrochemical behavior in the dark of a film of chlorophyll (Chl) molecules self-assembled on mercury and the peculiar features of the photocurrents recorded upon illuminating this film at different applied potentials are discussed. The limiting flux of hydrated Tl + ions through the channel-forming polypeptide gramicidin (GR), incorporated in a dioleoylphosphatidylcholine (DOPC) monolayer self-assembled on mercury, is shown to be controlled both by diffusion and by a dehydration step, while the limiting flux of dehydrated Tl + ions stemming from Tl amalgam electrooxidation is exclusively controlled by the diffusion of thallium atoms within the amalgam. The photocurrents of purple membrane (PM) fragments adsorbed on a DOPC monolayer and on a mixed alkanethiol/DOPC bilayer self-assembled on mercury are examined to investigate the kinetics of light-driven proton transport by bacteriorhodopsin (bR). The photocurrents on the mixed bilayer are interpreted on the basis of a simple equivalent circuit and their pH dependence is analyzed to estimate the p K a values of the transitions releasing protons to, and taking up protons from the solution.

Amphotericin B interactions with a DOPC monolayer. Electrochemical investigations

A model lipid membrane consisting of a monolayer of dioleoyl phosphatidylcholine (DOPC) adsorbed onto a Hg electrode has been used to study the interaction between the lipid and different formulations of Amphotericin B (AmB) [Fungizone® (FZ), Heated Fungizone (HFZ), and Abelcet®]. The lipid organizational order was measured by electrochemical methods [capacitance and metal ion (Tl +) reduction], characterizing the change in lipid order due to interaction with the drug. The mean size and number density of pores formed in the monolayer were estimated by fitting the reduction current transients to a random array of microelectrode model. This method was shown sensitive for investigation of the interaction of drugs with the DOPC monolayer. Abelcet was found to have a smaller disruptive effect on lipid order than FZ and HFZ. The formulations used to solubilize the AmB were also studied. Sodium deoxycholate used as a solubilizer in FZ displayed significant influence on lipid order similar to that observed for Abelcet. The lipid complex, used in Abelcet, did not significantly perturb the DOPC monolayer order. The lipid complex used in Abelcet may have an annealing or healing effect that buffers the disruption possible due to AmB.

DC photoelectric signals from bacteriorhodopsin adsorbed on lipid monolayers and thiol/lipid bilayers supported by mercury

Purple membrane (PM) fragments were adsorbed on a dioleoylphosphatidylcholine (DOPC) monolayer and on a mixed alkanethiol/DOPC bilayer supported by mercury to investigate the kinetics of light-driven proton transport by bacteriorhodopsin (bR). The light-on and light-off capacitive currents on an alkanethiol/DOPC bilayer at pH 6.4 were interpreted on the basis of a simple equivalent circuit. The pH dependence of the biphasic decay kinetics of the light-on currents was analyzed to estimate the pK a values for the transitions releasing protons to, and taking up protons from, the solution. The linear dependence of the stationary light-on current of bR on a DOPC monolayer self-assembled on mercury upon the applied potential was interpreted on the basis of an equivalent circuit.

Electrochemistry of Langmuir–Blodgett and self-organized monolayers of an azocrown ether, both pure and mixed with a phospholipid

The electrochemical behavior of monolayers of an azocrown ether (L16), both pure and mixed with dioleoylphosphatidylcholine (DOPC) was investigated using a mercury electrode. The monolayers formed at the air ∣ water interface were transferred onto the electrode using the Langmuir–Blodgett approach. The reversibility of the electrode processes depends on the surface pressure during the transfer of the monolayer. The reduction mechanism of the azo to the hydrazo group was studied in acidic medium by cyclic voltammetry and potential-step chronocoulometry. The dependence of the faradaic charge due to azo group reduction at constant pH upon scan rate (for voltammetry) or upon electrolysis time (for chronocoulometry) was examined on the basis of a general kinetic approach. The same approach was used to interpret the dependence of the faradaic charge upon pH at constant scan rate or electrolysis time. The reduction of the azo to the hydrazo group takes place via the reversible uptake of one electron, followed by the rate determining protonation of the resulting radical anion. When L16 is in the form of a pure monolayer its electroreduction is accompanied by a 2D phase transition involving the passage from a liquid-like to a solid-like structure. No such phase transition is observed in mixed L16–DOPC monolayers. At intermediate compositions of this mixture, strong attractive interactions between L16 and DOPC molecules decrease the mean area per molecule with respect to the ideal behavior at the air ∣ water interface, and prevent complete electroreduction of the L16 molecules in the monolayer.

Voltammetry of L-cysteine and 2-mercaptopyridine on a self-assembled phospholipid monolayer

The redox behaviour of 2-mercaptopyridine and the aminoacid L-cysteine was studied through a self-assembled monolayer of dioleoylphosphatidylcholine adsorbed on mercury by using cyclic voltammetry. 2-Mercaptopyridine penetrates into monolayer in the zone of stability of the phospholipid layer and shows an quasi-reversible behaviour while reversibility is observed in the absence of a monolayer. This fact is reflected in the occurrence of voltammetric peaks in the above mentioned region of potentials. Conversely, cysteine was found not to penetrate in the lipid layer as long as the latter behaves like a half-membrane. Voltammetric signal of cysteine was only obtained when the potential was scanned to values positive to -0.2 V. Beyond this potential the cyclic voltammograms show a series of anodic peaks, due to a rearrangement of the lipid film and to the formation of Hg(RS)2 , followed by three cathodic peaks when the scan is reversal. The behaviour of these peaks was analized.

Electrochemical Impedance Study of Tl+ Reduction through Gramicidin Channels in Self-Assembled Gramicidin-Modified Dioleoylphosphatidylcholine Monolayers on Mercury Electrodes

Impedance measurements for the reduction of Tl+ on gramicidin-modified dioleoylphosphatidylcholine-coated mercury electrodes have been performed. The frequency dependence of the admittance data fits well to a Randles circuit, and the Warburg coefficient, σ, and the irreversibility coefficient, p‘, can be obtained at every dc potential from the frequency analysis conforming to this circuit. However, the potential dependence of the Warburg coefficient is different from the one expected for a simple electron transfer. Instead, the σ−E data can be analyzed conforming to a mechanism including preceding and following homogeneous chemical steps to the electron transfer (CEC mechanism). In addition, from the irreversibility coefficient, p‘, a value of the standard rate constant for the electron transfer of 0.035 cm s-1 and a potential independent value for the transfer coefficient, α, of close to 0.5 are obtained. The possibility that the CEC mechanism originates partly from nonlinear diffusion is considered, and the results are discussed in comparison with those given in the literature for Tl+ reduction on pure mercury.

Voltammetry of 6,6'-dithiodinicotinic acid on a self-assembled phospholipid monolayer prive

This paper reports a voltammetric study of 6,6'-dithiodinicotinic acid (CPDS) across a biomimetic membrane system consisting of a monolayer of dioleoylphosphatidylcholine, deposited on mercury. Because of the low solubility of this compound and its potential decomposition in alkaline media, estimation of pK values for the carboxyl and amino groups of the pyridine ring of the CPDS entailed using the Hammett equation. UV spectra seem to confirm the presence of the dianionic form of CPDS above pH=3-4. Differential capacity and cyclic voltammetry measurements were made in order to characterize the voltammetric behavior directly on mercury and through a monolayer of dioleoylphosphatidylcholine. Estimation of the CPDS hydrophobicity degree from the partition coefficient in octanol/ water suggests no penetration of the dianion into the monolayer and supports the fact that the named dianion undergoes protonation at the phospholipid/solution interface to give a neutral specie which penetration into the phospholipid region, favored by its higher hydrophobicity, is followed by electrochemical reduction at the mercury surface.