Adsorption of lysozyme to phospholipid and meibomian lipid monolayer films

It is believed that a lipid layer forms the outer layer of the pre-ocular tear film and this layer helps maintain tear film stability by lowering its surface tension. Proteins of the aqueous layer of the tear film (beneath the lipid layer) may also contribute to reducing surface tension by adsorbing to, or penetrating the lipid layer. The purpose of this study was to compare the penetration of lysozyme, a tear protein, into films of meibomian lipids and phospholipids held at different surface pressures to determine if lysozyme were part of the surface layer of the tear film. Films of meibomian lipids or phospholipids were spread onto the surface of a buffered aqueous subphase. Films were compressed to particular pressures and lysozyme was injected into the subphase. Changes in surface pressure were monitored to determine adsorption or penetration of lysozyme into the surface film. Lysozyme penetrated a meibomian lipid film at all pressures tested (max = 20 mN/m). It also penetrated phosphatidylglycerol, phosphatidylserine or phosphatidylethanolamine lipid films up to a pressure of 20 mN/m. It was not able to penetrate a phosphatidylcholine film at pressures ≥10 mN/m irrespective of the temperature being at 20 or 37 °C. However, it was able to penetrate it at very low pressures (<10 mN/m). Epifluorescence microscopy showed that the protein either adsorbs to or penetrates the lipid layer and the pattern of mixing depended upon the lipid at the surface. These results indicate that lysozyme is present at the surface of the tear film where it contributes to decreasing the surface tension by adsorbing and penetrating the meibomian lipids. Thus it helps to stabilize the tear film.     

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.     

Displacement of fibrinogen from the air/aqueous interface by dilauroylphosphatidylcholine lipid

Fibrinogen (FB) and other serum proteins leak into the aqueous alveolar lining layer due to lung injuries. The adsorption of these serum proteins at the air/aqueous interface can produce higher surface tensions than the pulmonary lipids, and acute respiratory distress syndrome (ARDS) can ensue. By having a molecular adsorption mechanism, as compared to a particulate adsorption mechanism of other longer chain lipids, dilauroylphosphatidylcholine (DLPC) lipid can expel FB from the air/aqueous interface at 25 degrees C, in water or in phosphate-buffered saline, as proven by tensiometry (also at 37 degrees C), ellipsometry, and infrared reflection-absorption spectroscopy. Moreover, before FB is displaced by DLPC at the interface, there is a substantial initial enhancement in the FB adsorption, consistent with some interaction or binding of DLPC with FB to produce a more hydrophobic protein surface. After the FB molecules have been displaced by DLPC, or when DLPC has already adsorbed at the interface, FB molecules are less favored to adsorb near the DLPC monolayer with the lecithin headgroups facing toward them. The results have implications for possible uses of DLPC lipid in potential lung surfactant formulations in treating patients with ARDS.     

Proteins at liquid interfaces: II. Adsorption isotherms

Adsorption isotherms for the three proteins β-casein, bovine serum albumin, and lysozyme at the air-water and oil-water interfaces have been determined independently using ellipsometry and surface radioactivity methods; the surface pressure and surface potential were also monitored. Saturated monolayer coverage occurs via irreversible adsorption of 2–3 mg M^?2 of protein; the resultant films generate surface pressures of about 20 mN m^?1 and are 50–60 Å thick. Molecules adsorbed in the first layer dominate the film pressures so that further adsorption causes no change in the pressure although the film thickness can increase to more than 100 Å. The molecules which give rise to this increase in film thickness are reversibly adsorbed with respect to aqueous substrate exchange. The experimental isotherm data and the Langmuir adsorption isotherm are in close agreement at low protein concentrations. However, comparison with the Gibbs adsorption equation is not valid, although reasonable agreement can be achieved if some account is taken of the fact that the protein molecules in the first layer are irreversibly adsorbed.     

Protein-lipid interactions at the oil-water interface

The distribution of proteins and lipids in food emulsions and foams is determined by competitive and cooperative adsorption between the two types of emulsifiers at the fluid-fluid interfaces, and by the nature of protein-lipid interactions, both at the interface and in the bulk phase. The existence of protein-lipid interactions can have a pronounced impact on the surface rheological properties of these systems. Therefore, these results are of practical importance for food emulsion formulation, texture, and stability. In this study, the existence of protein-lipid interactions at the interface was determined by surface dynamic properties (interfacial tension and surface dilational modulus). Systematic experimental data on surface dynamic properties, as a function of time and at long-term adsorption, for protein (whey protein isolate (WPI)), lipids (monoglycerides), and protein-lipid mixed films at the oil-water interface were measured in an automated drop tensiometer. The dynamic behaviour of protein+lipid mixed films depends on the adsorption time, the lipid and the protein/lipid ratio in a rather complicated manner. The protein determined the interfacial characteristics of the mixed film as the protein at WPI>/=10(-2)% wt/wt saturated the film, no matter what the concentration of the lipid. However, there exists a competitive or cooperative adsorption of the emulsifier (WPI and monoglycerides), as the concentration of protein in the bulk phase is far lower than that for interfacial saturation.     

Mechanical properties of interfacial films formed by lysozyme self-assembly at the air-water interface

We present the first characterization of the mechanical properties of lysozyme films formed by self-assembly at the air-water interface using the Cambridge interfacial tensiometer (CIT), an apparatus capable of subjecting protein films to a much higher level of extensional strain than traditional dilatational techniques. CIT analysis, which is insensitive to surface pressure, provides a direct measure of the extensional stress-strain behavior of an interfacial film without the need to assume a mechanical model (e.g., viscoelastic), and without requiring difficult-to-test assumptions regarding low-strain material linearity. This testing method has revealed that the bulk solution pH from which assembly of an interfacial lysozyme film occurs influences the mechanical properties of the film more significantly than is suggested by the observed differences in elastic moduli or surface pressure. We have also identified a previously undescribed pH dependency in the effect of solution ionic strength on the mechanical strength of the lysozyme films formed at the air-water interface. Increasing solution ionic strength was found to increase lysozyme film strength when assembly occurred at pH 7, but it caused a decrease in film strength at pH 11, close to the pI of lysozyme. This result is discussed in terms of the significant contribution made to protein film strength by both electrostatic interactions and the hydrophobic effect. Washout experiments to remove protein from the bulk phase have shown that a small percentage of the interfacially adsorbed lysozyme molecules are reversibly adsorbed. Finally, the washout tests have probed the role played by additional adsorption to the fresh interface formed by the application of a large strain to the lysozyme film and have suggested the movement of reversibly bound lysozyme molecules from a subinterfacial layer to the interface.     

The adsorbed conformation of globular proteins at the air/water interface

External reflection FTIR spectroscopy and surface pressure measurements were used to compare conformational changes in the adsorbed structures of three globular proteins at the air/water interface. Of the three proteins studied, lysozyme, bovine serum albumin and beta-lactoglobulin, lysozyme was unique in its behaviour. Lysozyme adsorption was slow, taking approximately 2.5 h to reach a surface pressure plateau (from a 0.07 mM solution), and led to significant structural change. The FTIR spectra revealed that lysozyme formed a highly networked adsorbed layer of unfolded protein with high antiparallel beta-sheet content and that these changes occurred rapidly (within 10 min). This non-native secondary structure is analogous to that of a 3D heat-set protein gel, suggesting that the adsorbed protein formed a highly networked interfacial layer. Albumin and beta-lactoglobulin adsorbed rapidly (reaching a plateau within 10 min) and with little change to their native secondary structure.     

Dynamics of protein and mixed protein/surfactant adsorption layers at the water/fluid interface

The adsorption behaviour of proteins and systems mixed with surfactants of different nature is described. In the absence of surfactants the proteins mainly adsorb in a diffusion controlled manner. Due to lack of quantitative models the experimental results are discussed partly qualitatively. There are different types of interaction between proteins and surfactant molecules. These interactions lead to protein/surfactant complexes the surface activity and conformation of which are different from those of the pure protein. Complexes formed with ionic surfactants via electrostatic interaction have usually a higher surface activity, which becomes evident from the more than additive surface pressure increase. The presence of only small amounts of ionic surfactants can significantly modify the structure of adsorbed proteins. With increasing amounts of ionic surfactants, however, an opposite effect is reached as due to hydrophobic interaction and the complexes become less surface active and can be displaced from the interface due to competitive adsorption. In the presence of non-ionic surfactants the adsorption layer is mainly formed by competitive adsorption between the compounds and the only interaction is of hydrophobic nature. Such complexes are typically less surface active than the pure protein. From a certain surfactant concentration of the interface is covered almost exclusively by the non-ionic surfactant. Mixed layers of proteins and lipids formed by penetration at the water/air or by competitive adsorption at the water/chloroform interface are formed such that at a certain pressure the components start to separate. Using Brewster angle microscopy in penetration experiments of proteins into lipid monolayers this interfacial separation can be visualised. A brief comparison of the protein adsorption at the water/air and water/n-tetradecane shows that the adsorbed amount at the water/oil interface is much stronger and the change in interfacial tension much larger than at the water/air interface. Also some experimental data on the dilational elasticity of proteins at both interfaces measured by a transient relaxation technique are discussed on the basis of the derived thermodynamic model. As a fast developing field of application the use of surface tensiometry and rheometry of mixed protein/surfactant mixed layers is demonstrated as a new tool in the diagnostics of various diseases and for monitoring the progress of therapies.     

Electrochemical quartz crystal nanobalance study of the adsorption/displacement phenomena of proteins and lipids on Pt

The electrochemical quartz crystal nanobalance (EQCN) was used to measure the adsorption behavior of a series of lipids (stearate, oleate, linoleate, and gamma-linolenate) on a Pt surface from a phosphate buffer pH 7.0 solution at 295 K and to investigate their adsorption/displacement behavior with the proteins, beta-lactoglobulin and alpha-lactalbumin, which are known to cause fouling during milk processing. The EQCN technique and the complementary technique of cyclic voltammetry measured simultaneously provided information on the efficiency of solubilization of the proteins by these lipids. Excellent agreement was obtained for the surface concentration of adsorbed lipid from the surface charge density from cyclic voltammetry measurements and the change in mass from the EQCN frequency measurements. The Gibbs energy of adsorption showed the lipids to have a strong affinity for the platinum surface. Addition of protein to a preadsorbed lipid layer showed alpha-lactalbumin to be able to coadsorb with the lipids, while beta-lactoglobulin was able to desorb some of the unsaturated lipids but appeared to coadsorb with the saturated lipid, stearate. Addition of lipid to a preadsorbed protein layer showed the unsaturated lipids to be able to displace some of the protein. A comparison of the desorption ability of the lipids showed stearate to be very inefficient at removing protein, while the other three lipids were able to remove each of the proteins, with the order of efficiency for protein desorption being oleate > linoleate > gamma-linolenate.     

Spreading of proteins and its effect on adsorption and desorption kinetics

The kinetics of adsorption of lysozyme and alpha-lactalbumin from aqueous solution on silica and hydrophobized silica has been studied. The initial rate of adsorption of lysozyme at the hydrophilic surface is comparable with the limiting flux. For lysozyme at the hydrophobic surface and alpha-lactalbumin on both surfaces, the rate of adsorption is lower than the limiting flux, but the adsorption proceeds cooperatively, as manifested by an increase in the adsorption rate after the first protein molecules are adsorbed. At the hydrophilic surface, adsorption saturation (reflected in a steady-state value of the adsorbed amount) of both proteins strongly depends on the rate of adsorption, but for the hydrophobic surface no such dependency is observed. It points to structural relaxation ("spreading") of the adsorbed protein molecules, which occurs at the hydrophobic surface faster than at the hydrophilic one. For lysozyme, desorption has been studied as well. It is found that the desorbable fraction decreases after longer residence time of the protein at the interface.     

Adsorption of CETP on monolayers formed from HDL extracted lipids

To obtain information on the interactions between CETP and HDL3 lipoproteins, we have studied (by surface tension measurements) the adsorption of the CETP at the air–water interface and at the interface between the water and monolayers formed by spreading of lipids extracted from HDL3. We have compared the interfacial behavior of CETP and ApoA-1 (the constitutive protein of HDL3); and the influence of monolayers composition and pressure on the kinetics of the CETP adsorption. The results obtained show that CETP was more expanded than the ApoA-1 which adsorbed more strongly at the air–water interface. CETP adsorbs more and quickly at the lipid interface that at the air–interface, specially for 20% fraction of cholesterol in the monolayer. Our results show that the adsorption of the CETP at the HDL3 surface lipids are strongly dependent of the composition of the monolayer and that the exclusion pressure of CETP varied from 31 to 33.7 mN m⁻¹ with the addition of cholesterol. Finally, the kinetics of the adsorption at water–lipid interface exhibited two steps (quick increase followed by slow decrease of the excess surface pressure) which should indicate a penetration into monolayer followed by a partial desorption of phospholipids with or without cholesterol corresponding to a proteolipid association.     

Reciprocal influence between the protein and lipid components of a lipid-protein membrane model

The crystallization of bacterial surface layer proteins (S-layer proteins) at phosphoethanolamine monolayers on aqueous (buffer) surfaces has been investigated with dual label fluorescence microscopy, FTIR spectroscopy, and electron microscopy. The phase state of the lipid exerts a marked influence on protein crystallization: when the surface monolayer is in the phase-separated state between the isotropic and anisotropic fluid phases, the S-layer protein is preferentially adsorbed at the isotropic phase. Protein crystals nucleate at the boundary lines between the coexisting lipid phases and crystallization proceeds underneath the anisotropic fluid. Crystal growth is much slower under the fluid lipid and the entire interface is overgrown only after prolonged protein incubation. In turn, as indicated by characteristic frequency shifts of the methylene stretch vibrations on the lipids, protein crystallization affects the order of the alkane chains and drives the fluid lipid into a state of higher order. Most probably, the protein does not interpenetrate the lipid surface monolayer and the coupling between protein and lipid occurs via the lipid head groups.     

髓鞘碱性蛋白对细胞膜脂质分子DPPC、DPPE单层膜影响机理研究

本文通过Langmuir单层膜的表面压力-平均分子面积(π-A)曲线的测定与分析,分别对髓鞘碱性蛋白(MBP)与细胞膜中不同头部基团脂质分子二棕榈酰基磷脂胆碱(DPPC)和二棕榈酰基磷脂酰乙醇胺(DPPE)在空气/液体界面上的相互作用过程进行了系统研究.实验结果表明:(1)当界面上脂质含量一定时,亚相中随着MBP浓度的增大,DPPC、DPPE单层膜的等温线向平均分子面积较大的方向移动;(2)在单层膜表面压力为10 mN/m时,一个MBP分子分别结合140±3个DPPC分子和100±3个DPPE分子,随着表面压力增大,当MBP分子分别与两种磷脂分子相互作用时,MBP插入到磷脂单层界面的个数逐渐减少;(3)随着蛋白质浓度的增加,脂分子形成的单层膜变得较为疏松,且MBP分子易于插入到分子头部较小的DPPE单层膜中;(4)蛋白质的存在使DPPC单层膜的表面压力逐渐减小,且蛋白质浓度越大表面压力降低越多,DPPC被MBP带入到亚相中越多;(5)对于DPPE单层膜,蛋白质通过与DPPE相互作用插入到界面膜中,引起表面压力增大,且蛋白质浓度越高,压力变化量越大.     

Interfacial characteristics of food emulsifiers (proteins and lipids) at the air-water interface

Factors affecting the interfacial characteristics (structure, stability, interfacial rheology, molecular diffusion, and rate of film formation) of food emulsifiers (polar lipids and proteins) at the air-aqueous phase interface are reviewed. The effect of interfacial and aqueous phase (water, and aqueous solutions of ethanol, glycerol, sugars, electrolytes, and pH) compositions have been analyzed as variables. Many measurement methods—such as tensiometry (Wilhelmy plate and pendant drop methods), and Langmuir- and Wilhelmy-type film balances—have been used in the experiments. The effect of the interfacial, aqueous phase composition, and operational conditions (surface density, surface pressure, and temperature) of food emulsifiers (lipids and proteins) at the air-aqueous phase interface are discussed.     

Reversible switching of protein uptake and release at polyelectrolyte multilayers detected by ATR-FTIR spectroscopy

The reversible switching of uptake and release of the proteins lysozyme (LYZ, IEP = 11.1) and human serum albumin (HSA, IEP = 4.8) at the surface attached polyelectrolyte multilayer (PEM) consisting of poly(ethylene-imine) (PEI) and poly(acrylic acid) (PAC) is shown. Protein adsorption could be switched by pH setting due to electrostatic interaction. Adsorption of positively charged LYZ at PEM-6 took place at pH = 7.3, where the outermost PAC layer was negatively charged. Complete desorption was obtained at pH = 4, where the outermost PAC layer was neutral. Additionally the charge state of the last adsorbed PAC layer in dependence of the pH of the medium could be determined in the ATR-FTIR difference spectra by the ν(COO⁻) and ν(C=O) band due to carboxylate and carboxylic acid groups. Adsorption of negatively charged HSA at PEM-7 was achieved at pH = 7.3, where the outermost PEI layer was positively charged. Part desorption was obtained at pH = 10, where the outermost PEI layer was neutral. PEM of PEI/PAC may be used for the development of bioactive and bionert materials and protein sensors.     

Proteins at liquid interfaces: I. Kinetics of adsorption and surface denaturation

The rates of change of film pressure (π) and surface concentration (Γ) of protein during the adsorption of β-casein, bovine serum albumin (BSA), and lysozyme at the air-water interface have been monitored by the Wilhelmy plate and surface radioactivity methods, respectively. The increases in π and Γ for the relatively flexible β-casein molecule occur simultaneously with both parameters attaining their steady-state values at about the same time. In contrast, π and Γ follow different time courses for the globular lysozyme molecule; Γ can reach a steady state value while π is still increasing significantly. The kinetics indicate that initially adsorption is diffusion-controlled but at higher surface coverages there is an energy barrier to adsorption. Under these conditions, the ability of the protein molecules to create space in the existing film and penetrate and rearrange in the surface is rate-determining. Two kinetic regions exist: the relaxation time τ₁ (typically ～2 hr when Γ ～2 mg m^?2) describes the adsorption when both π and Γ are increasing whereas τ₂ (in the range 1–8 hr for all three proteins) relates to the situation when π is increasing at constant Γ because the protein molecules are changing conformation in the surface.     

Strong improvement of interfacial properties can result from slight structural modifications of proteins: the case of native and dry-heated lysozyme

Identification of the key physicochemical parameters of proteins that determine their interfacial properties is still incomplete and represents a real stake challenge, especially for food proteins. Many studies have thus consisted in comparing the interfacial behavior of different proteins, but it is difficult to draw clear conclusions when the molecules are completely different on several levels. Here the adsorption process of a model protein, the hen egg-white lysozyme, and the same protein that underwent a thermal treatment in the dry state, was characterized. The consequences of this treatment have been previously studied: net charge and hydrophobicity increase and lesser protein stability, but no secondary and tertiary structure modification (Desfougères, Y.; Jardin, J.; Lechevalier, V.; Pezennec, S.; Nau, F. Biomacromolecules 2011, 12, 156-166). The present study shows that these slight modifications dramatically increase the interfacial properties of the protein, since the adsorption to the air-water interface is much faster and more efficient (higher surface pressure). Moreover, a thick and strongly viscoelastic multilayer film is created, while native lysozyme adsorbs in a fragile monolayer film. Another striking result is that completely different behaviors were observed between two molecular species, i.e., native and native-like lysozyme, even though these species could not be distinguished by usual spectroscopic methods. This suggests that the air-water interface could be considered as a useful tool to reveal very subtle differences between protein molecules.     

Structure and orientation of puroindolines into wheat galactolipid monolayers

Puroindolines (PINs), basic and cysteine-rich proteins of wheat endosperm, are composed of two proteins, puroindoline-a (PIN-a) and puroindoline-b (PIN-b). Using a monolayer assay at the air/liquid interface, both PIN-a and PIN-b were studied in pure components and mixed with wheat galactolipids, 1,2-di-O-acyl-3-O-(beta-d-galactopyranosyl)- sn-glycerol (MGDG) and 2-di-O-acyl-3-O-(beta-d-galactopyranosyl-1,6-beta-d-galactopyranosyl)-sn-glycerol (DGDG). Following the adsorption of PINs at the air/liquid interface thanks to surface pressure increases, we concluded that PIN-a displays a more amphipathic character than PIN-b. Compression isotherms combined with ellipsometric measurements showed that the area per molecule is smaller and the protein film is more condensed for PIN-a than for PIN-b. According to the polarization modulation-infrared reflection-absorption spectroscopy data, both proteins display a highly alpha-helical structure and the alpha-helices are oriented rather parallel to the interface. By measuring the overpressure due to PIN adsorption into MGDG and DGDG monolayers, we observed that PIN-a interacts more strongly into lipid films than PIN-b. The observation by atomic force microscopy of mixed protein/lipid films showed that the nature of the lipid plays a significant role in the organization of PINs, particularly for PIN-a. The presence of galactolipids at the interface stabilizes the alpha-helical structure of PINs, but significant changes were observed concerning the orientation of the alpha-helices. They adopt a perfect parallel orientation to the interface in the MGDG monolayer, whereas the bundle of alpha-helices orients normal to the interface in the DGDG film.     

Structure and dynamics of crystalline protein layers bound to supported lipid bilayers

We study proteins at the surface of bilayer membranes using streptavidin and avidin bound to biotinylated lipids in a supported lipid bilayer (SLB) at the solid-liquid interface. Using X-ray reflectivity and simultaneous fluorescence microscopy, we characterize the structure and fluidity of protein layers with varied relative surface coverages of crystalline and noncrystalline protein. With continuous bleaching, we measure a 10-15% decrease in the fluidity of the SLB after the full protein layer is formed. We propose that this reduction in lipid mobility is due to a small fraction (0.04) of immobilized lipids bound to the protein layer that create obstacles to membrane diffusion. Our X-ray reflectivity data show a 40 A thick layer of protein, and we resolve an 8 A layer separating the protein layer from the bilayer. We suggest that the separation provided by this water layer allows the underlying lipid bilayer to retain its fluidity and stability.     

Mechanical properties of protein adsorption layers at the air/water and oil/water interface: A comparison in light of the thermodynamical stability of proteins

Over the last decades numerous studies on the interfacial rheological response of protein adsorption layers have been published. The comparison of these studies and the retrieval of a common parameter to compare protein interfacial activity are hampered by the fact that different boundary conditions (e.g. physico-chemical, instrumental, interfacial) were used. In the present work we review previous studies and attempt a unifying approach for the comparison between bulk protein properties and their adsorption films. Among many common food grade proteins we chose bovine serum albumin, β-lactoglobulin and lysozyme for their difference in thermodynamic stability and studied their adsorption at the air/water and limonene/water interface. In order to achieve this we have i) systematically analyzed protein adsorption kinetics in terms of surface pressure rise using a drop profile analysis tensiometer and ii) we addressed the interfacial layer properties under shear stress using an interfacial shear rheometer under the same experimental conditions. We could show that thermodynamically less stable proteins adsorb generally faster and yield films with higher shear rheological properties at air/water interface. The same proteins showed an analog behavior when adsorbing at the limonene/water interface but at slower rates.