Interaction of nitrophenols with lipids at the air/water interface

The uptake of ortho and para nitrophenol to charged and neutral lipid monolayers spread at the air/solution interface was studied by reflection spectroscopy. The adsorption characteristics of the two nitrophenols have been studied by measuring the surface pressure and surface potential as a function of molecular area of the different lipid monolayers in the presence of nitrophenols in the subphase. The results have been interpreted in terms of the electrostatic interaction between the negatively charged dissociated phenolate ions and the positively charged head group of dioctadecyldimethylammonium bromide monolayers.     

Organization of complex monolayers by matrix controlled adsorption

Complex monolayers can be organized at the air | water interface by adsorption of a water-soluble, negatively charged porphyrin dye from the aqueous subphase to a matrix monolayer containing positively charged head groups at the surface of the solution. The organization of the complex monolayer depends on the composition of the matrix monolayer which controls the molecular interactions with the dissolved porphyrin. Surface pressure versus area isotherms in combination with measurement of the enhanced light reflection from the interface in the presence of the porphyrin provides information on the packing and orientation of the chromophores. In an optimal situation, the surface density of the positively charged head groups matches the surface density of the negative charges in the densely packed porphyrins attached to the matrix monolayer, and neutral molecules in the monolayer occupy the free space to provide a dense packing of the matrix.     

Mixed DPPC/DPTAP monolayers at the air/water interface: influence of indolilo-3-acetic acid and selenate ions on the monolayer morphology

The interactions of mixed monolayers of two lipids, zwitterionic 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and positively charged 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), with phytohormone indolilo-3-acetic acid (IAA) and selenate anions in the aqueous subphase were studied. For this purpose, isotherms of the surface pressure versus the mean molecular area were recorded. Domain formation was investigated by using Brewster angle microscopy (BAM). The method of grazing incidence X-ray diffraction (GIXD) was also applied for the characterization of the organization of lipid molecules in condensed monolayers. It was found that selenate ions contribute to monolayer condensation by neutralizing the positive net charge of mixed monolayers whereas IAA molecules penetrated the lipid monolayer, causing its expansion/fluidization. When both solutes were introduced into the subphase, a competition between them for interaction with the positively charged lipids in the monolayer was observed.     

Amino-substituted amphiphilic calixarenes: self-assembly and interactions with DNA

The amphiphilic 5,11,17,23-tetramino-25,26,27,28-tetradodecyloxycalix[4]arene is shown to self-assemble as stable and well-defined Langmuir monolayers at the air-water interface. The effect of the presence of DNA in the subphase reveals interactions taking place at the interface between the positively charged surface and the negatively charged DNA, causing an expansion of the monolayers and a phase transition from a liquid-condensed to a liquid-expanded phase; a slight decrease in the stability of the monolayers is also observed. The title compound is shown to self-assemble, with the absence of a cosurfactant, as stable colloidal suspensions. Photon correlation spectroscopy, zeta-potential measurements, and atomic force microscopy reveal that these colloidal suspensions present a monodisperse size distribution and are composed of positively charged solid lipid nanoparticles (SLNs), with an average hydrodynamic diameter of 190 nm and a surface potential of +13.2 mV. The interaction of these SLNs with double-stranded DNA is demonstrated.     

Hepatitis A Synthetic Peptide VP3(110-121) Miscibility with Dipalmitoylphosphatidylcholine, Dipalmitoylphosphatidylglycerol, and Stearylamine Monolayers

To prepare liposomes containing a synthetic hepatitis A virus antigen (HAV) [VP3(110-121)] as a vaccine, the miscibility of this peptide (with negative net charge) with a neutral lipid [dipalmitoylphosphatidylcholine (DPPC)], a negatively charged lipid [dipalmitoylphosphatidylglycerol (DPPG)], and a positively charged lipid [Stearylamine (SA)] was studied through compression isotherms of monolayers. Mixtures with DPPC and SA showed a low degree of interaction with the peptide, the composition of the monolayer being stable through compression. For DPPG-containing monolayers larger positive deviations from ideality were found, and the peptide was squeezed out from the monolayer at a DPPG/VP3(110-121) mole fraction of 0.8/0.2. All this suggests that besides hydrophobic interactions between the peptide and the lipid, electrostatic forces also play a role; thus it seems that neutral and positively charged lipids would be more suitable for preparing stable liposomes with VP3(110-121). Copyright 2000 Academic Press.     

Bonding in Protein and Polypeptide Monolayers

The logarithm of the surface viscosity of protein and polyamino acid monolayers was found to be a linear function of the surface pressure in agreement with the Moore-Eyring theory. The area of the flow unit was similar for all proteins and corresponded to a segment of the molecule of about seven or eight amino acid residues. The free energies of activation for flow at pH 5.5 were fairly constant, falling between 15.6 and 16.6 kcal mole^?1 of flow unit. Evidence was obtained for ascribing a large part of the free energy of activation to the breaking of intermolecular keto-imido hydrogen bonds. The interactions of protein monolayers with mercuric ion in the subphase and with lipid incorporated in the monolayer are discussed.     

Langmuir monolayers as models to study processes at membrane surfaces

The use of new sophisticated and highly surface sensitive techniques as synchrotron based X-ray scattering techniques and in-house infrared reflection absorption spectroscopy (IRRAS) has revolutionized the monolayer research. Not only the determination of monolayer structures but also interactions between amphiphilic monolayers at the soft air/liquid interface and molecules dissolved in the subphase are important for many areas in material and life sciences. Monolayers are convenient quasi-two-dimensional model systems. This review focuses on interactions between amphiphilic molecules in binary and ternary mixtures as well as on interfacial interactions with interesting biomolecules dissolved in the subphase. The phase state of monolayers can be easily triggered at constant temperature by increasing the packing density of the lipids by compression. Simultaneously the monolayer structure changes are followed in situ by grazing incidence X-ray diffraction or IRRAS. The interactions can be indirectly determined by the observed structure changes. Additionally, the yield of enzymatic reaction can be quantitatively determined, secondary structures of peptides and proteins can be measured and compared with those observed in bulk. In this way, the influence of a confinement on the structural properties of biomolecules can be determined. The adsorption of DNA can be quantified as well as the competing adsorption of ions at charged interfaces. The influence of modified nanoparticles on model membranes can be clearly determined. In this review, the relevance and utility of Langmuir monolayers as suitable models to study physical and chemical interactions at membrane surfaces are clearly demonstrated.     

PORPHYRIN-LIPID INTERACTIONS AT THE AIR-WATER INTERFACE IN SPREAD MONOLAYERS. A FLUORESCENCE STUDY.

In membrane systems, carboxylic porphyrins may interact with both the lipid pseudophase and the adjacent aqueous environment through their hydrophobic core and their polar acid chains, respectively. These interactions are monitored in model membrane systems, i.e. spread monolayers of dioleoylphosphatidylcholine as functions of lipid organization and pH of the aqueous subphase using steady state and time resolved fluorescence techniques. In all cases contact between porphyrin and aqueous subphase, as indicated through quenching by I^-, is observed at low surface pressure. This contact decreases and becomes almost insignificant as the monolayer approaches maximum organization through compression. On deprotonation of the monocarboxylic porphyrin, methylpyrroporphyrin, increased contact with water is observed in liquid compressed monolayers. In liquid expanded layers, however, it appears that organization of lipid molecules surrounding this dissymmetric charged form affords some isolation from water. The effect of esterification of carboxylic chains is also examined.     

Dynamic Behaviors and Morphology Change of Anionic Phospholipid DPPG Monolayer Caused by Bovine Serum Albumin at Air-Water Interface

The interaction between bovine serum albumin (BSA) and the anionic 1.2-dipalmitoyl-snglycero- 3-(phospho-rac-(1-glycerol)) (sodium salt) (DPPG) phospholipid at different subphase pH values was investigated at air-water interface through surface pressure measurements and atomic force microscopy (AFM) observation. By analyzing surface pressure-mean molecular area (π-A) isotherms, the limiting molecular area in the closed packing state-the concentration of BSA (A_lim-[BSA]) curves, the compressibility coefficient-surface pressure (C_S^-1-π) curves and the difference value of mean molecular area-the concentration of BSA (ΔA-[BSA]) curves, we obtained that the mean molecular area of DPPG monolayer became much larger when the concentration of BSA in the subphase increased at pH=3 and 5. But the isotherms had no significant change at different amount of BSA at pH=10. In addition, the amount of BSA molecules adsorbed onto the lipid monolayer reached a threshold value when [BSA]>5×10^-8 mol/L for all pHs. From the surface pressure-time (π-t) data, we obtained that desorption and adsorption processes occurred at pH=3, however, there was only desorption process occurring at pH=5 and 10. These results showed that the interaction mechanism between DPPG and BSA molecules was affected by the pH of subphase. BSA molecules were adsorbed onto the DPPG monolayers mainly through the hydrophobic interaction at pH=3 and 5, and the strength of hydrophobic interaction at pH=3 was stronger than the case of pH=5. At pH=10, a weaker hydrophobic interaction and a stronger electrostatic repulsion existed between DPPG and BSA molecules. AFM images revealed that the pH of subphase and [BSA] could affect the morphology features of the monolayers, which was consistent with these curves. The study provides an important experimental basis and theoretical support to understand the interaction between lipid and BSA at the air-water interface.     

Study of adsorption and penetration of E2(279-298) peptide into Langmuir phospholipid monolayers

The peptide corresponding to the sequence (279-298) of the Hepatitis G virus (HGV/GBV-C) E2 protein was synthesized, and surface activity measurements, pi-A compression isotherms, and penetration of E2(279-298) into phospholipid monolayers spread at the air-water interface were carried out on water and phosphate buffer subphases. The results obtained indicated that the pure E2(279-298) Langmuir monolayer exhibited a looser packing on saline-buffered than on pure water subphase and suggest that the increase in subphase ionic strength stabilizes the peptide monolayer. To better understand the topography of the monolayer, Brewster angle microscopy (BAM) images of pure peptide monolayers were obtained. Penetration of the peptide into the pure lipid monolayers of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) and into mixtures of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) at various initial surface pressures was investigated to determine the ability of these lipid monolayers to host the peptide. The higher penetration of peptide into phospholipids is attained when the monolayers are in the liquid expanded state, and the greater interaction is observed with DMPC. Furthermore, the penetration of the peptide dissolved in the subphase into these various lipid monolayers was investigated to understand the interactions between the peptide and the lipid at the air-water interface. The results obtained showed that the lipid acyl chain length is an important parameter to be taken into consideration in the study of peptide-lipid interactions.     

Studies of monolayer/substrate adhesion as function of the monolayer headgroup charge: DMPE and DMPA

The variation of the work of adhesion between lipid monolayers and a plane silicon oxide surface in a typical LB-configuration is measured as function of the subphase pH. The adhesion energy is deduced via fluorescence microscopy from the equilibrium meniscus height. With increasing pH the negative headgroup charge of both, dimyristoylphosphatidylethanolamine (DMPE) and dimyristoylphosphatidic acid (DMPA) monolayers increases. The increasing charge of DMPE is reflected in a measured decrease of the work of adhesion at higher pH. The DMPA/SiO₂ interaction is not affected by increasing headgroup charges. These results are qualitatively understood in terms of an electrostatic double layer interaction between charged surfaces. It predicts decreasing adhesion for increasing, but low surface charge densities (DMPE). whereas the adhesion is constant for high surface charge densities (DMPA).     

Protein-lipid interactions at the air/water interface

Surface pressure measurements and external reflection FTIR spectroscopy have been used to probe protein-lipid interactions at the air/water interface. Spread monomolecular layers of stearic acid and phosphocholine were prepared and held at different compressed phase states prior to the introduction of protein to the buffered subphase. Contrasting interfacial behaviour of the proteins, albumin and lysozyme, was observed and revealed the role of both electrostatic and hydrophobic interactions in protein adsorption. The rate of adsorption of lysozyme to the air/water interface increased dramatically in the presence of stearic acid, due to strong electrostatic interactions between the negatively charged stearic acid head group and lysozyme, whose net charge at pH 7 is positive. Introduction of albumin to the subphase resulted in solubilisation of the stearic acid via the formation of an albumin-stearic acid complex and subsequent adsorption of albumin. This observation held for both human and bovine serum albumin. Protein adsorption to a PC layer held at low surface pressure revealed adsorption rates similar to adsorption to the bare air/water interface and suggested very little interaction between the protein and the lipid. For PC layers in their compressed phase state some adsorption of protein occurred after long adsorption times. Structural changes of both lysozyme and albumin were observed during adsorption, but these were dramatically reduced in the presence of a lipid layer compared to that of adsorption to the pure air/water interface.     

Properties of Spreaded Collagen I Monolayers on the Surface of Aqueous tert-Butanol and n-Hexanol Solutions

Properties of the monolayers of collagen isolated from the sclera of pig's eye are studied at the air–water interface with increasing tert-butanol or n-hexanol concentrations in a subphase. In the case of aqueous n-hexanol solutions, its adsorption on the subphase surface results in the formation of mixed monolayer whose properties depend on n-hexanol concentration in the subphase and the ratio between the number of alcohol and collagen molecules in the monolayer. At higher n-hexanol surface concentration, the phase separation of the monolayer into the domains of the condensed phase of alcohol and fibrous collagen occurs. A decrease in water activity in the presence of tert-butanol leads to a drastic reduction of collagen surface activity. This effect can be explained by both the constrained collagen spreading on the surface of tert-BuOH solutions and adsorption of alcohol molecules on collagen resulting in macromolecule hydrophilization. Alcohol critical concentrations are disclosed above which collagen monolayers are not formed.     

In situ observation of gamma-Fe2O3 nanoparticle adsorption under different monolayers at the air/water interface

We studied the adsorption of gamma-Fe 2O 3 nanoparticles from an aqueous solution under different charged Langmuir monolayers (stearic acid, stearyl alcohol, and stearyl amine). The aqueous subphase was composed of a colloidal suspension of gamma-Fe 2O 3 nanoparticles. The average hydrodynamic diameter of the nanoparticles measured by dynamic light scattering measurements was 16 nm. The observed zeta potential of +40 mV (at pH 4) results in a long-term stability of the colloidal dispersion. The behavior of the different monolayer/nanoparticle composites were studied with surface pressure/area (pi/ A) isotherms. The adsorption of the nanoparticles under the different monolayers induced an expansion of the monolayers. These phenomena depended on the charge of the monolayers. After the Langmuir/Blodgett transfer on glass substrates, the nanoparticle/monolayer composite films were studied by means of UV-vis spectroscopy. The spectra pointed to increasing adsorption of the nanoparticles with increasing electronegativity of the monolayers. On the basis of these results, we studied the in situ adsorption of nanoparticles under the different monolayers by X-ray reflectivity measurements. Electron density profiles of the liquid/gas interfaces were obtained from the X-ray reflectivity data. The results gave clear evidence for the presence of electrostatic interaction between the differently charged monolayers and the positively charged nanoparticles. While the adsorption process was favored by the negatively charged stearic acid monolayer, the positively charged layer of stearyl amine prevented the formation of ultrathin nanoparticle layers.     

A technique to estimate the apparent surface pressure of emulsion particles using apolipoproteins as probes

The apparent pressures in the surface monolayer of emulsion particles can be estimated by comparing the absorption of an apolipoprotein to planar lipid monolayers and to emulsions. Lipids are spread at an air-water interface in a Pockels/Langmuir surface balance and the adsorption of [¹⁴C]-labeled apolipoproteins placed in the subphase is studied as a function of surface pressure using the surface radioactivity method. An apoprotein surface concentration/initial lipid surface pressure curve (Γ/gp_i) is constructed. The maximum apolipoprotein surface concentration Γ_e of emulsions is derived from standard emulsion/apolipoprotein binding isotherms. The apparent emulsion surface pressure is then estimated by comparing Γ_e to the Γ/π_i curve. Apolipoprotein A-I has been used as an example of a probe to estimate the effective surface pressure in ~1000 Å diameter egg yolk phosphatidylcholine/cholesterol/triolein emulsion particles. When the cholesterol content of emulsions is low, the surface pressure of the emulsion is about 17 dyne cm⁻¹. At high cholesterol concentrations (0.49 cholesterol/phospholipid mole ratio) the surface pressure is increased to 25 dyne cm⁻¹. The addition of the maximum amounts of apoA-I to these particles raises the effective surface pressure of the emulsion to about 30 dyne cm⁻¹ and stabilizes the particles.     

Amphiphilic peptides as models for protein-membrane interactions: interfacial behaviour of sequential Lys- and Leu-based peptides and their penetration into lipid monolayers

Synthetic peptides constructed with doublets of hydrophobic residues tandemly repeated with doublets of positively charged residues, (Leu-Lys-Lys-Leu)_n, were used as models for the study of protein-membrane interactions. Their behaviour has been compared with that of their strictly alternating iso peptides, (Leu-Lys)_n. Both peptides present a random coil structure in pure water. In saline solutions, (Leu-Lys-Lys-Leu)_n peptides adopt an α-helical structure whereas (Leu-Lys)_n transit into a β-sheet structure. These peptides form multilayer assemblies on a pure water subphase but they are organized in monomolecular films on a saline aqueous subphase. The stability of these films increases with the peptide length. Structured peptides (α helices and β sheets) penetrate readily into lipid monolayers, whereas the penetration of unordered peptides is very slow. We have not observed any significant difference between the behaviour of a helices and β-sheet structures.     

Glycoprotein adsorption into bilirubin/cholesterol mixed monolayers at the air-water interface

The adsorption of α₁-acid glycoprotein into bilirubin/cholesterol mixed monolayers with various component molar ratios is investigated using surface pressure-area (π-A) isotherms and (dπ/dA)-A curves. The results showed that the surface area per molecule increased after the adsorption/insertion of glycoprotein molecules into the monolayers. The compressibility of mixed monolayers increased as a result of hydrogen bonding between bilirubin and glycoprotein molecules, while the interactions between bilirubin and cholesterol are weakened. The adsorption of glycoprotein into a monolayer induced changes in molecular surface area depending on the surface pressure and molar fraction of bilirubin. The transmission electron microscopy of mixed monolayers confirmed the insertion of glycoprotein particles of spherical shape with an average diameter of about 80 nm into the monolayer. The text was submitted by the authors in English.     

Monolayers of Chiral Calix[4]Resorcinarenes: Surface Pressure and Surface Potential Studies

Abstract

Chiral amphiphilic C-undecylcalix[4]resorcinarenes substituted with phenylethyl group or L(-)nore-phedrine were found to form well-organized mono-layers at the aqueous solution-air interface. The substituents, L(-)norephedrine and phenylethyl group, determined the area occupied by the molecule on the water subphase. Introduction of these substituents lead also to perpendicular dipole moments of the molecules in the monolayers ca. 6 times larger than those of the parent amphiphilic calixresorcinarene, CAL11. Interactions of the compounds with K⁺ were detected by the increase of the surface potential values measured at maximum packing of the monolayer. Addition of amino acids to the subphase lead to conformational changes in the monolayers evidenced by increased surface mean molecular area of the unmodified C-undecyl-calix[4]resorcinarene. These changes were explained by the formation of hydrogen bonds with the amino acids at the expense of hydrogen bonding between the calixarene molecules in the monolayer. In contrast to unsubstituted calixresorcinarenes, interactions of the L(-)norephedrine-and phenylethyl-substituted molecules with amino acids could be easily recognized by the decrease of surface potential and dipole moment in monolayers formed by these calixarenes on subphases containing amino acids. A significant drop in the surface potential and an increased area per molecule demonstrated more specific interactions with selected amino acids: L(-)norephedrine-substituted calixarene interacted with D-valine and the phenylethyl-substituted, with D-tryptophan.     

Penetration and interactions of the antimicrobial peptide, microcin J25, into uncharged phospholipid monolayers

Microcin J25 forms stable monolayers at the air-water interface showing a collapse at a surface pressure of 5 mN/m, 220 mV of surface potential, and 6 fV per squared centimeter of surface potential per unit of molecular surface density. The adsorption of microcin J25 from the subphase at clean interfaces leads to a rise of 10 mN/m in surface pressure and a surface potential of 220 mV. From these data microcin appears to be a poor surfactant per se. Nevertheless, the interaction with the lipid monolayer further increase the stability of the peptide at the interface depending on the mode in which the monolayer is formed. Spreading with egg PC leads to nonideal mixing up to 7 mN/m, with hyperpolarization and expansion of components at the interface, with a small excess free energy of mixing caused by favorable contributions to entropy due to molecular area expansion compensating for the unfavorable enthalpy changes arising from repulsive dipolar interactions. Above 7 mN/m microcin is squeezed out, leaving a film of pure phospholipid. Nevertheless, the presence of lipid at 10 and 20 mN/m stabilize further microcin at the interface and adsorption from the subphase proceeds up to 30 mN/m, equivalent to surface pressure in bilayers.     

Incorporation of blood clotting factor X into phospholipid model membranes: fluorescence microscopy imaging and subphase effects surrounding the lipid phase transition region

Factor X is a blood clotting protein that associates at membrane surfaces to become activated during the coagulation cascade. A molecular level understanding of the protein-membrane phospholipid interactions has not been reached, although it is thought that the protein binds to phospholipids in the presence of calcium through a bridge with the Gla (gamma-carboxyglutamic acid) domain on the protein. In this work, phospholipid Langmuir monolayers have been utilized as model membranes to study factor X association with phospholipid membrane components. Surface pressure measurements indicate that subphase addition of sodium, magnesium, and calcium ions enhances protein penetration of the lipid monolayer, with the largest association found with calcium ions in the subphase. Fluorescence microscopy images collected after protein penetration of lipid monolayers indicate monolayer condensation in the presence of sodium and magnesium ions. Aggregation of lipid domains is induced when calcium is in the subphase, indicating binding-induced flocculation of surface lipid aggregates. Calcium binding to factor X likely causes a conformational change which allows protein-membrane interaction via hydrophobic association with lipid molecules.