The adsorption of endothelin 1 to phospholipids is governed by electrostatic interactions: A monolayer and fluorescence study

In order to elucidate the influence on the lipidic environment on the recognition process of its membrane associated receptor, the interactions of the vasoconstrictor peptide endothelin 1 with various phospholipids have been investigated using different lipidic model membranes: monolayers at constant surface pressure, vesicles and micelles. A monolayer study of ET1 adsorbed onto the water surface has shown that the C-terminus of the peptide points towards the aqueous phase. Penetration measurements into lipidic monolayers indicate that ET1 adsorbs to phospholipids with an orientation similar to that of the air–water interface and fluorescence measurements are in agreement with such an orientation of the peptide. This adsorption is selective for neutral phospholipids and indicates that the nature of the phospholipid headgroups is of major importance for the approach of the membrane associated receptor.     

Flexibility of the triblock copolymers modulating their penetration and expulsion mechanism in Langmuir monolayers of dihexadecyl phosphoric acid

The surface activity of the poly–[block (ethylene oxide)]–poly [block (propylene oxide)]–poly [block (ethylene oxide)] copolymers (EO)_x–(PO)_y–(EO)_x adsorbed together with dihexadecyl phosphoric acid (DHP), a synthetic phospholipid, is analyzed from their surface pressure and surface potential isotherms. The block copolymers of (EO)_x–(PO)_y–(EO)_x with variable molecular weight (1100–14 000) were dissolved in the subphase for DHP monolayers. The concentration of the copolymers within the aqueous subphase were selected to render an initial surface tension of 60 mN/m. The simultaneous adsorption of the copolymer and DHP is attested by the observation of a liquid expanded state at large areas, absent for pure DHP monolayers. Above some critical surface pressure all copolymers cited above are expelled from the interface. The surface potential isotherms, which give information on the component of the molecular dipole moment normal to the plane of the monolayer, are interpreted in terms of changes in the copolymer conformation as well as in terms of the copolymer desorption from the air–liquid interface. For an equal hydrophobic/hydrophilic ratio, the size of the chains or molecular weight is decisive in the mechanism of the copolymer expulsion from the air–liquid interface.     

Surface properties of hen egg yolk low-density lipoproteins spread at the air–water interface

Hen egg yolk is largely used as food ingredient notably because of its exceptional emulsifying properties. Low-density lipoproteins (LDL) are the main egg yolk constituent. LDL and particularly apoLDL are thought to control largely emulsifying properties of egg yolk-based products. Nevertheless, few studies have concerned the interfacial behaviour of these lipoproteins at the oil–water interface and nothing has been published about the air–water interface. Controversies still remain about LDL adsorption mechanism at the oil–water interface even if a widely spread theory suggests their breaking at the interface, allowing then their constituents to spread. The Langmuir film balance and atomic force microscopy (AFM) were used in this study in the aim to characterise LDL surface behaviour in dynamic conditions at the air–water interface. The understanding of LDL adsorption mechanism and surface organisation at the air–water interface should provide useful information about LDL behaviour at the oil–water interface. LDL and lipids extracted from LDL—neutral lipids, phospholipids and total lipids (mixture of the two previous species)—were spread at the air–water interface to clarify the role of each constituent in the lipoprotein film. Results clearly show that LDL are disrupted at the interface to release notably neutral lipids from the lipoprotein core, enabling then their spreading. Each lipid class has been identified on the LDL film isotherm and seems to behave independently and individually at the interface within the lipoprotein film.     

The effect of sucrose on the thermodynamic properties of ovalbumin and sodium caseinate in bulk solution and at air–water interface

This paper presents a study of the effect of sucrose on the molecular parameters and thermodynamic properties in a bulk aqueous medium and at the air–water interface for two proteins differing both in nature and structure, that is Na-caseinate and ovalbumin. To get more insight into the molecular nature of the effect of sucrose, mixing calorimetry, light scattering and tensiometry measurements have been made under different pHs (7.0 and 5.5) and temperatures (20–55°C) at an ionic strength of 0.005 mol dm⁻³. Combined temperature dependencies of light scattering and mixing calorimetry testify to hydrogen bonding (sucrose-protein and/or sucrose-water) as being the primary basis of the effect of sucrose on the molecular and thermodynamic properties of the proteins in the bulk and at interface of an aqueous medium. At pH 7.0, in the case of ovalbumin, the interaction with sucrose causes an increase in the protein hydrophilicity in the bulk aqueous medium followed by a decrease in the protein surface activity, whilst for Na-caseinate, there is an increase in the protein hydrophobicity due to Na-caseinate micelle dissociation and, consequently, to an increase in the protein surface activity. Lowering the pH to 5.5, accompanied by a strengthening of the competition between less charged proteins and sucrose for water molecules, induces a rise in the protein hydrophobic aggregation in the bulk. The special features of the latter process are probably mainly responsible for the changes in the surface activity of the proteins under influence of sucrose at pH 5.5.     

Effect of small molecule surfactants on molecular parameters and thermodynamic properties of legumin in a bulk and at the air–water interface depending on a protein structure in an aqueous medium

This paper presents the effect of fatty acid salts, namely, Na-caprate and Na-palmitate on the legumin (11S globulin of Vicia Faba broad beans) molecular and thermodynamic properties in the bulk aqueous medium and at the air–water interface under different molecular states of the protein. That are the native state of the protein globule (pH 7.2, ionic strength of 0.05 mol dm⁻³), as well as the acidic denatured (pH 3.0, ionic strength of 0.01 mol dm⁻³) and the heat denatured ones (after heating at 90°C for 30 min, pH 7.2, ionic strength of 0.05 mol dm⁻³). In turn, an importance of the state of the small molecule surfactants in a solution in reference to the critical concentrations of micelle formation (CMC), for their effect on the protein properties, was also under our studying. The peculiarities of the legumin structure in the aqueous medium appeared in the different nature of the interactions between the protein and the fatty acid salts, as was indicated by the mixing calorimetry data. So, the hydrophobic contacts provided a basis for interactions between both the native and heat denatured legumin with the fatty acid salts. At the same time, the electrostatic interactions between the oppositely charged functional groups of the fatty acid salts and the acidic denatured protein formed principally a basis of their interactions in an aqueous medium. In response to interactions of the fatty acid salts with legumin the essential changes in the protein conformational stability, depending on both the protein molecular state and concentration of the fatty acid salts, were found using differential scanning calorimetry (DSC). The rather high level of the protein association was observed by light scattering in the bulk aqueous medium in the presence of the fatty acid salts. As this takes place, the surface hydrophilicity of the protein increased under the formation of the associates. The combined data of mixing calorimetry, differential scanning calorimetry and light scattering suggested the complex formation between legumin and the fatty acid salts. The interactions of the fatty acid salts with the protein produced a change in the surface activity for the mixture of the protein with the fatty acid salts. That is a decrease in the protein surface tension at the air–water interface for the mixed solutions in comparison with ones for both the protein and small molecule surfactant alone in the case of Na-caprate, and those are the intermediate values of the surface tension in the case of Na-palmitate. These results were observed independently of the protein state (native or acidic/heat denatured) in an aqueous medium. As this took place, the most dramatic increase in the surface activity was found for the mixtures of the acidic denatured protein with Na-caprate as if the most hydrophobic species were formed in this case. The combined data of mixing calorimetry, DSC, light scattering and tensiometry showed that the effect of the fatty acid salts on the legumin thermodynamic properties in a bulk and at interfaces is governed by a number of the key factors such as: a structure of both the protein and fatty acid salt (a length of the hydrocarbon chain); a degree of the protein association in the bulk aqueous phase (as a result of the interactions with the small molecule surfactants); a change in the protein conformational stability (flexibility) under the influence of the small molecule surfactants; as well as by the nature (hydrophobic, electrostatic) of the protein–small molecule surfactant interactions, determining ultimately the hydrophilic–lipophilic balance of the protein surface.     

Evaluation of the interactions between lipids and γ-globulin protein at the air–liquid interface

The interactions between lipids (cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine and sphingomyelin) and the γ-globulin protein have been analyzed using the monolayer technique at the air–liquid interface. The analysis has been carried out using both state equations and an adequate thermodynamic formulation for the surface pressure (π)–molecular area (a) isotherms. Different parameters as the virial coefficients, have been estimated. For the uncharged lipid monolayers, the interactions between the molecules are of an attractive nature, at medium and long distance, and of a steric repulsive nature at short distance. At low surface pressures the lipid molecules form small domains. The net force between γ-Globulin molecules in the monolayers has been found to be attractive. Finally, it can be concluded that when the lipid monolayers are uncharged, there is practically no interaction between the protein and lipid molecules at the mentioned interface.     

Surface thermodynamics study of monolayers formed with heteroacid phospholipids of biological interest

The interaction of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), two of the major components in biological membranes, were investigated using the monolayer technique at the air–water interface. The pressure–area isotherms indicate that both phospholipids are miscible through all range of compositions. POPE–POPC form stable mixtures, with a minimum for the Gibbs energy of mixing at X_POPC = 0.4. A virial equation of state was fitted to the experimental values. Positive values found for the second virial coefficient indicate repulsion between POPC and POPE. The interaction parameter was evaluated which indicated that a corresponding decrease in the repulsion occurs when POPC molar fraction is low. This effect suggests the existence of hydrogen bonds between POPE and the water beneath the interface.     

Incorporation conditions guiding the aggregation of a glycosylphosphatidyl inositol (GPI)-anchored protein in Langmuir monolayers

This work investigates the process of incorporation of a glycosylphosphatidyl inositol (GPI)-anchored alkaline phosphatase into Langmuir monolayers of dimyristoyl phosphatidic acid (DMPA). Three different methods of protein incorporation were assayed. When the protein solution was injected below the air–water interface after formation of the lipid monolayer a micro-heterogeneous distribution of alkaline phosphatase throughout the interface was observed. Adsorption kinetics studied by fluorescence microscopy, associated with surface pressure measurements, led to the proposition of a model in which the protein penetration is modulated by the surface packing of the monolayer and intermolecular interactions occurring between the phospholipid and the protein. At initial surface pressures higher than 20 mN m⁻¹, the protein is quickly adsorbed on the interface and the lateral diffusion drives the alkyl chains to turn towards the air phase while the polypeptide moiety faces the aqueous subphase.     

Effect of maltodextrins on the surface activity of small-molecule surfactants

We report on the effect of commercially important polysaccharides (maltodextrins with variable dextrose equivalent (Paselli SA-2, MD-6 and MD-10) on the surface activity at the air–water interface of small-molecule surfactants (sms), possessing different hydrophobic–lipophilic balance ((SSL (Na⁺), the main component is a sodium salt of stearol–lactoyl lactic acid, and PGE (080), polyglycerol ester of C18 fatty acid), and widely used in food products. A marked change of the surface activity of sms was found in the presence of maltodextrins by tensiometry. The combined data of laser multiangle light scattering and mixing calorimetry have suggested that this result is governed by specific complex formation between maltodextrins and sms in aqueous medium. Measurements have been made of the molar mass, the second virial coefficient and the enthalpy of intermolecular interactions in aqueous solutions. The implication of a degree of polymerization of maltodextrins in this phenomenon was shown. The interrelation between the molecular parameters of the formed complexes and their surface activity at the air–water interface has been revealed and discussed.     

Interfacial and foaming properties of prolylenglycol alginates. Effect of degree of esterification and molecular weight

In the present work we have studied the characteristics of propylene glycol alginates (PGA) adsorption at the air–water interface and the viscoelastic properties of the films in relation to its foaming properties. To evaluate the effect of the degree of PGA esterification and viscosity, different commercial samples were studied—Kelcoloid O (KO), Kelcoloid LVF (KLVF) and Manucol ester (MAN). The temperature (20 °C) and pH (7.0) were maintained constant. For time-dependent surface pressure measurements and surface dilatational properties of adsorbed PGA at the air–water interface an automatic drop tensiometer was used. The foam was generated by whipping and then the foam capacity and stability was determined. The results reveal a significant interfacial activity for PGA due to the hydrophobic character of the propylene glycol groups. The kinetics of adsorption at the air–water interface can be monitored by the diffusion and penetration of PGA at the interface. The adsorbed PGA film showed a high viscoelasticity. The surface dilatational modulus depends on the PGA and its concentration in the aqueous phase. Foam capacity of PGA solutions increased in the order KO > MAN > KLVF, which followed the increase in surface pressure and the decrease in the viscosities of PGA solutions. The stability of PGA foams monitored by the drainage rate and collapse time follows the order MAN > KLVF > KO. The foam stability depends on the combined effect of molecular weight/degree of esterification of PGA, solution viscosity and viscoelasticity of the adsorbed PGA film.     

Influence of the saturation chain and head group charge of phospholipids in the interaction of hepatitis G virus synthetic peptides

Using the Langmuir technique, we have studied the properties at the air/water interface and the interaction of the hepatitis G virus synthetic peptide E1(53-66) and its palmitoyl derivative with membrane phospholipids. These phospholipids had different characteristics referring to the net charge and saturation of the acyl chain. The palmitoyl derivative was more stable at the air/water interface and in the kinetic at constant area measurements showed higher incorporation to the monolayer. The interaction was higher for saturated phospholipids and those with a negative net charge. When the peptides were in the subphase, they produced changes in the miscibility of mixed monolayers composed of DPPC/DPPG or DOPC/DOPG. It can be deduced from the results obtained that electrostatic interactions play a major role, but when the peptide is derivatized with the palmitoyl chain, hydrophobic interactions are added to the former ones. The interaction is also influenced by the saturation of the acyl chain.     

Interfacial characteristics of β-casein spread films at the air–water interface

Casein is well known to be a good protein emulsifier and β-casein is the major component of casein and commercial sodium caseinate. This work studies the behaviour of β-casein at the interface. The interfacial characteristics (structure and stability) of β-casein spread films have been examined at the air–water interface in a Langmuir-type film balance, as a function of temperature (5–40°C) and aqueous phase pH (pH 5 and 7). From surface pressure–area isotherms (π–A isotherms) as a function of temperature we can draw a phase diagram. β-Casein spread films present two structures and the collapse phase. That is, there is a critical surface pressure and a surface concentration at which the film properties change significantly. This transition depends on the temperature and the aqueous phase pH. The film structure was observed to be more condensed and β-casein interfacial density was higher at pH 5. β-Casein films were stable at surface pressures lower than equilibrium surface pressure. In fact, no hysteresis was observed in π–A isotherms after continuous compression-expansion cycles or over time. The relative area relaxation at constant surface pressure (10 or 20 mN m⁻¹) and the surface pressure relaxation at constant area near the monolayer collapse, can be fitted by two exponential equations. The characteristic relaxation times in β-casein films can be associated with conformation–organization changes, hydrophilic group hydration and/or surface rheology, as a function of pH.     

Monolayer formation of short helical turn forming peptide derivatives at the air–water and air–solid interfaces

Two tetrapeptide derivatives [peptide A (Boc–Ala–Ile–Ile–Gly–OMe) and peptide B (Boc–Ala–Ile–Leu–Ser–OMe)], that take helical turn conformation in solution, were shown to form monolayer at the air/water interface. Circular dichroism (CD) measurements indicate that peptide A has more helical turn propensity than peptide B in sodium dodecyl sulphate (SDS) micelles. Langmuir–Blodgget film study of peptides A and B suggest that both the peptides form stable monolayer at the air/water interface. Spectroscopic investigations reveal that peptide A forms helical turn assemblage on transferring the film into hydrophilic quartz and hydrophobic ZnSe surfaces. Whereas, peptide B adopts β-sheet structure on hydrophilic surface and a mixture of β-sheet and helical turn conformation on hydrophobic surface.     

The influence of pH on phosphatidylcholine monolayer at the air/aqueous solution interface

The measurements of the interfacial tension at the air/aqueous subphase interface as the function of pH were performed. The interfacial tension of the air–aqueous subphase interface was divided into contributions of individuals. A simple model of the influence of pH on the phosphatidylcholine monolayer at the air/hydrophobic chains of phosphatidylcholine is presented. The contributions of additive phosphatidylcholine forms (both interfacial tension values and molecular area values) depend on pH. The interfacial tension values and the molecular areas values for LH⁺, LOH⁻ forms of phosphatidylcholine were calculated. The assumed model was verified experimentally.     

Emulsification of caraway essential oil in water by lecithin and β-lactoglobulin: emulsion stability and properties of the formed oil–aqueous interface

The stability and droplet size of protein and lipid stabilised emulsions of caraway essential oil as well as the amount of protein on the emulsion droplets have been investigated. The amount of added protein (β-lactoglobulin) and lipid (phosphatidylcholine from soybean (sb-PC)) were varied and the results compared with those obtained with emulsions of a purified olive oil. In general, emulsions with triglyceride oil proved to be more stable compared with those made with caraway essential oil as the dispersed phase. However, the stability of the emulsions can be improved considerably by adding sb-PC. An increase in the protein concentration also promoted emulsion stability. We will also present how ellipsometry can be used to study the adsorption of the lipid from the oil and the protein from the aqueous phase at the oil–water interface. Independently of the used concentration, close to monolayer coverage of sb-PC was observed at the caraway oil–aqueous interface. On the other hand, at the olive oil–aqueous interface, the presence of only a small amount of sb-PC lead to an exponential increase of the layer thickness with time beyond monolayer coverage. The amounts of β-lactoglobulin adsorbed at the caraway oil–aqueous interface and at the olive oil–aqueous interface were similar, corresponding roughly to a protein monolayer coverage.     

Temperature effect of hydrophobically modified polyethylene oxide at the air–water interface

Surface pressure (π)–area (A) isotherms of hydrophobically modified polyethylene oxide (HEUR) at the air–water interface was examined. Conformational transitions between pancake, mushroom, and brush states of the hydrophilic backbone influence the intermolecular interaction between the hydrophobic chains. We choose relatively long (18 carbons) hydrophobic ends, which have large hydrophobic interactions, and investigate the main chain effect by change in the length of the hydrophilic PEO chain. At high surface concentration region, the temperature coefficient of surface pressure, dπ/dT, was larger by increasing the portion of the hydrophobicity. This indicates an increase in surface energy and a decrease in surface enthalpy at high surface concentrations. As alkyl chains on both sides of HEURs are anchored at the air–water interface, restriction caused by the alkyl chain would be smaller for the long PEO chain, but the larger for the short PEO chain length.     

Adsorption at the air-water interface and emulsification properties of grain legume protein derivatives from pea and broad bean

Functional properties of native and modified (through induced autolysis) pea (Pisum sativum L.) and broad bean (Vicia faba L.) protein derivatives are studied. In specific, protein solubility and behavior at the air–water interface through surface pressure measurements are investigated. Furthermore the ability of the protein products to act as emulsifying agents and to stabilize emulsions is studied through oil droplet size distribution measurements and by the protein adsorbed at the oil–water interface. The data reveal that the ability of the proteins to act as surfactants and build up a rigid film around the oil droplets, mainly depends on their suitable molecular configuration and structure. Hydrolysis did not promote the functionality of the legume proteins. Broad bean exhibited better functionality than pea, before and after hydrolysis. Some comparisons were also made with lupin (Lupinus albus L.) protein isolate.     

Biocatalytic Langmuir–Blodgett assemblies based on penicillin G acylase

The recently developed ‘protective plate’ method offers the possibility to include protein layers into a Langmuir–Blodgett (LB) assembly without contact of protein molecules with the air–water interface thus avoiding their denaturation. In the present work, this technique was applied for the deposition of biocatalysts with active layers of penicillin G acylase (PGA), an enzyme widely used for medicine production. Easy selection of LB and adsorbed layers resulted in the creation of appropriate environments for the preservation of PGA functions. Two structures were tested regarding such performances as the enzymatic activity value and the level of PGA detachment in aqueous solutions. It was shown that they satisfy the requirements for biocatalytic applications. The enzymatic activity of PGA monolayer incorporated into the film reached 25–30% of the activity value of the equivalent amount of protein in the solution, which is a good result for an immobilized enzyme. Further modification of the deposition procedure resulted in increasing the effective activity per unit of the substrate surface due to adsorption of a thicker protein layer in one cycle. Probably, a three-dimensional frame-like structure was formed, which allowed the substrate molecules to penetrate into the film. The enzymatic activity of such films per unit of the substrate surface was 20–25 times higher than that of the assemblies with one adsorbed monolayer. Finally, the method is proposed of biocatalytic LB assembly deposition onto flexible supports of practically unlimited length without the exposure of protein layer to air medium.     

Interaction of the meso-tetrakis (4-N-methylpyridyl) porphyrin with gel and liquid state phospholipid vesicles

The interaction of the cationic meso-tetrakis 4-N-methylpyridyl porphyrin (TMPyP) with large unilamellar vesicles (LUVs) was investigated in the present study. LUVs were formed by mixtures of the zwitterionic 1,2-dipalmitoyl-sn-glycero-phosphatidylcholine (DPPC) and anionic 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) phospholipids, at different DPPG molar percentages. All investigations were carried out above (50 °C) and below (25 °C) the main phase transition temperature of the LUVs (~41 °C). The binding constant values, K(b), estimated from the time-resolved fluorescence study, showed a significant increase of the porphyrin affinity at higher mol% DPPG. This affinity is markedly increased when the LUVs are in the liquid crystalline state. For both situations, the increase of the K(b) value was also followed by a higher porphyrin fraction bound to the LUVs. The displacement of the vesicle-bound porphyrins toward the aqueous medium, upon titration with the salt potassium chloride (KCl), was also studied. Altogether, our steady-state and frequency-domain fluorescence quenching data results indicate that the TMPyP is preferentially located at the LUVs Stern layer. This is supported by the zeta potential studies, where a partial neutralization of the LUVs surface charge, upon porphyrin titration, was observed. Dynamic light scattering (DLS) results showed that, for some phospholipid systems, this partial neutralization leads to the LUVs flocculation.     

Phase behavior and surface tensions of amphiphilic fluorinated random copolymer aqueous solutions

Phase behavior and surface tension of aqueous solutions of fluorinated random copolymers [perfluoroalkylacrylate]–[poly(ethyleneoxide)methacrylate], [C_mRf-acrylate]-[EO_n-methacrylate] with fluroalkyl carbon number m = 8, 6, 4, 2 and number of ethyleneoxide unit, n = 9 and 4.5 were investigated as a function of composition and different combinations of m and n. Isotropic solutions are formed at lower temperatures over wide concentration range of copolymer but at higher temperature phase separation occurs. The cloud point of copolymer decreases with decreasing n as well as m, and also with decreasing the number of poly(ethyleneoxide)methacrylate chain per perfluorinatedalkylacrylate chain, suggesting that the copolymers become more hydrophobic on decreasing m and n. Equilibrium and dynamic surface tension measurements show that copolymers become increasingly surface active as m as well as n decrease but the adsorption at the air–water interface is very slow due to bulkiness of the molecules. No clear evidence of the formation of micellar aggregates could be obtained from surface tension–composition curves.