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.     

Adsorption of alkyl trimethylammonium bromides at the air/water interface

A number of features of the adsorption of alkyl trimethylammonium bromides with nc=10,12,14, and 16 at the air/water interface were studied. First, the adsorption isotherms were calculated from experimental surface tension vs concentration curves by means of the Gibbs equation. Second, a novel method was used to estimate the adsorption free energy change. From the analysis of these data it was concluded that the hydrophobic driving force for the adsorption first increases with increasing adsorbed amount and then levels off in a plateau, which holds true for all four homologues. This peculiar behavior was interpreted by the formation of a thin liquid-like alkane film at the air/water interface once a certain adsorbed amount is exceeded. The hydrophobic contribution to the standard free energy change of adsorption was compared with those values previously determined for alkyl sulfate homologues. This comparison suggests that the alkyl trimethylammonium type surfactants behave as if their alkyl chain was approximately one methylene group shorter than those of the corresponding alkyl sulfates.     

Fluorescence evidence that a phase transition causes the induction time in the reduction in dynamic tension during surfactant adsorption to a clean air/water interface and a kinetic-diffusive transport model for the phase-induced induction

An aqueous soluble surfactant adsorbing from solution onto an initially clean air/water interface often exhibits an induction period in the surface tension relaxation in which, as the adsorption begins, the tension remains near the clean interface value for an extended period of time before decreasing rapidly to the equilibrium value. In this study, using a model nonionic soluble surfactant, C14E6(CH3(CH2)13-(OCH2CH2)6-OH), we present direct fluorescence evidence that this induction is due to a first-order phase transition from a gaseous (G) to a liquid expanded (LE) phase that the assembling monolayer undergoes at constant surface pressure. An open channel flow cell is initially filled with water, and onto its air/water interface is spread an insoluble amphiphilic dye that fluoresces upon irradiation in the LE phase and whose fluorescence is quenched in the G phase. An aqueous solution of C14E(6) is then allowed to flow through the channel. We observe the immediate appearance of bright islands of the LE phase growing in a dark (G) background, confirming the presence of the G/LE phase transition. These islands eventually occupy the entire surface, after which the interface remains uniformly bright. We correlate this phase transition to the induction period by simultaneously measuring the tension of the interface of the open channel, and verifying that as the islands grow the tension remains at the clean value until the bright LE phase occupies the entire surface, whereupon the tension rapidly decreases. We further develop a phase transition surfactant transport model for the induction period in which surfactant diffuses toward and kinetically adsorbs onto the surface, and then rapidly equilibrates between the G and LE phases. For our model surfactant C14E6, we independently measure the surface concentration of the nucleating LE phase, the LE phase surfactant equation of state, the kinetic rate constants for adsorption into the LE phase, and the bulk diffusion coefficient. Using these measurements, we predict induction times for adsorption onto a clean surface without convection. We also measure these induction times in tension relaxation for adsorption onto a pendant bubble using axisymmetric shape analysis, and demonstrate agreement with the simulations with no adjustable constants.     

Monolayer behavior and Langmuir-Blodgett manipulation of CdS quantum dots

Cadmium sulfide (CdS) quantum dots (QDs) were prepared and surface modified by dodecanthiol or mercaptosuccinic acid (MSA) to render a surface with alkyl chains (C(12)-CdS) or carboxylic acid groups (MSA-CdS), respectively. Due to the hydrophobic property of C(12)-CdS, the nanoparticles disperse well in chloroform and stay stable at the air/water interface. However, 3-dimensional (3D) aggregative domains and particle-free pores were formed in the monolayer due to poor particle-water interaction. For the MSA-CdS nanoparticles, the surface was hydrophobized through physical adsorption of a cationic surfactant, cetyltrimethylammonium bromide (CTAB). The capped MSA on the CdS plays an important role in enhancing the adsorption of CTAB and improving the stability of the QDs at the air/water interface. Due to the reversible adsorption of CTAB on MSA-CdS, a hydrophilic area can be exposed in the water-contacting region of a nanoparticle when it stays at the air/water interface. Thus, the CTAB-MSA-CdS QD behaves as an amphiphilic compound at the air/water interface and has properties superior to those of C(12)-CdS QDs in fabrication of layer-by-layer 2D structure of particulate films. The distinct behaviors of the two QDs at the air/water interface and the related effect on the properties of LB films were studied using a number of methods, including pressure-area (pi-A) isotherm, relaxation and hysteresis experiments, in-situ observation of Brewster angle microscopy (BAM), the postdeposition analysis of atomic force microscopy (AFM), and UV-vis spectroscopy.     

Native and Hydrophobically Modified Human Immunoglobulin G at the Air/Water Interface

The adsorption of human immunoglobulin G (IgG) at the air/water interface was monitored both by the in situ radiotracer technique using [(14)C] labeled IgG and by surface tension measurements. The results reveal that adsorption of IgG from single protein systems displays bimodality due to molecular rearrangements at the interface. Above the threshold value of 1.5x10(-2) mg/ml solution concentration, adsorbed IgG molecules reoriented from the side-on to the end-on configuration. The existence of a lag time which did not appear in Gamma=f(t) curves, was observed in Pi=f(t) relationships at low protein concentrations and was due to the limits of the surface pressure technique to detect protein adsorption. The adsorption of native IgG was also carried out in the presence of a hydrophobized IgG obtained by grafting capryloyl residues to its lysine groups by reaction with N-hydroxysuccinimide ester of caprylic acid, which yielded 19 covalently bound alkyl chains to the IgG molecule (19C(8)-IgG). This modified IgG exhibited enhanced adsorption at the air/water interface, as manifested by its increased adsorption efficiency relative to the native protein. Sequential and competitive adsorption experiments from binary mixtures of native IgG and 19C(8)-IgG clearly demonstrate that the displacement of the native protein from the air/water interface strongly depended on the manner of how 19C(8)-IgG and native IgG competed with each other. When the two proteins competed simultaneously, 19C(8)-IgG predominantly occupied the available area but when native IgG was adsorbed first, for 2 h, the sequentially adsorbed 19C(8)-IgG was incapable of substantially displacing it from the interface. Copyright 2001 Academic Press.     

Interactions between nonpolar surfaces coated with the nonionic surfactant hexaoxyethylene dodecyl ether C12E6 and the origin of surface charges at the air/water interface

The interactions between nonpolar surfaces coated with the nonionic surfactant hexaoxyethylene dodecyl ether C12E6 were investigated using two techniques and three different types of surfaces. As nonpolar surfaces, the air/water interface, silanated negatively charged glass, and thiolated uncharged gold surfaces were chosen. The interactions between the air/water interfaces were measured with a thin film pressure balance in terms of disjoining pressure as a function of film thickness. The interactions between the solid/liquid interfaces were determined using a bimorph surface force apparatus. The influence of the nature of the surface on the interaction forces was investigated at surfactant concentrations below and above the cmc. The adsorption of the nonionic surfactant on the uncharged thiolated surface does not, as expected, lead to any buildup of a surface charge. On the other hand, adsorption of C12E6 on the charged silanated glass and the charged air/water interface results in a lowering of the surface charge density. The reduction of the surface charge density on the silanated glass surfaces is rationalized by changes in the dielectric permittivity around the charged silanol groups. The reason for the surface charge observed at the air/water interface as well as its decrease with increasing surfactant concentration is discussed and a new mechanism for generation of OH- ions at this particular interface is proposed.     

SURFACE PROPERTIES OF SOME LOW MOLECULAR WEIGHT PROTEINS

Surface properties of four proteins having molecular weights less than 5,000 are reported at air/water and alumina/water interface at pH 7.0. Reversibility in the adsorption of these proteins at the alumina/water interface is tested. The adsorption on alumina/water interface has been found to be controlled by electrostatic interaction. Positive adsorption was obtained when protein and alumina surface had opposite charges and negative adsorption was obtained when both protein and surface had same charges. Of the four proteins reversibility in adsorption was observed with the one having the lowest molecular weight of 3100. The adsorption behavior apparently had no correlation with their surface hydrophobic!ty. Time dependent changes in air/water interfacial tension was observed for all the four proteins indicating time dependent loosening of compact protein structure and surface unfolding.     

Monitoring entering and spreading of emulsion droplets at an expanding air/water interface: a novel technique

The entering and spreading of emulsion droplets at quiescent and expanding air/water interfaces was studied using a new apparatus consisting of a modified Langmuir trough in which the air/water interface can be continuously expanded by means of rollers in the place of traditional barriers. When sodium caseinate and whey protein isolate-stabilized emulsion droplets were injected under the surface of sodium caseinate and whey protein isolate solutions, respectively, it appeared that the droplets entered the air/water interface only if the air/water surface pressure did not exceed a threshold value of approximately 15 mN/m. This condition was satisfied either under quiescent conditions for low protein concentrations or by continuous expansion of the interface at higher protein concentrations. According to equilibrium thermodynamics, entering of the droplets and the formation of lenses should occur for all the systems investigated, but this was not observed. At surface pressures higher than approximately 15 mN/m, immersed emulsion droplets were metastable. This is probably due to a kinetic barrier caused by the formation of a thin water film bounded by protein adsorption layers between the emulsion droplet and the air/water interface.     

Micellization of non-surface-active diblock copolymers in water. Special characteristics of poly(styrene)-block-poly(styrenesulfonate)

Strongly ionized amphiphilic diblock copolymers of poly(styrene)-b-poly(styrenesulfonate) with various hydrophilic and hydrophobic chain lengths were synthesized by living radical polymerization, and their properties and self-assembling behavior were systematically investigated by surface tension measurement, foam formation, hydrophobic dye solubilization, X-ray reflectivity, dynamic light scattering, small-angle neutron scattering, small-angle X-ray scattering, and atomic force microscope techniques. These copolymer solutions in pure water did not show a decrease of surface tension with increasing polymer concentration. The solutions also did not show foam formation, and no adsorption at the air/water interface was confirmed by reflectivity experiments. However, in 0.5 M NaCl aq solutions polymer adsorption and foam formation were observed. The critical micelle concentration (cmc) was observed by the dye solubilization experiment in both the solutions with and without added salt, and by dynamic light scattering we confirmed the existence of polymer micelles in solution, even though there was no adsorption of polymer molecules at the water surface in the solution without salt. By the small-angle scattering technique, we confirmed that the micelles have a well-defined core-shell structure and their sizes were 100-150 A depending on the hydrophobic and hydrophilic chain length ratio. The micelle size and shape were unaffected by addition of up to 0.5 M salt. The absence of polymer adsorption at the water surface with micelle formation in a bulk solution, which is now known as a universal characteristic for strongly ionized amphiphilic block copolymers, was attributed to the image charge effect at the air/water interface due to the many charges on the hydrophilic segment.     

有机反离子对C12-s-C12·2Br水溶液表面活性的影响

溶液中添加的苯磺酸钠(SNzS)和萘磺酸钠(SNphS)与C12-s-C12·2Br产生强烈结合, 增大了Gemini表面活性剂分子的疏水性, 明显促进其在气/液界面的吸附和在溶液中的聚集. 这使得体系降低水表面张力的效率和能力大大提高, 并且在表面活性剂浓度很低时就生成了小聚集体. 因而, 此时表面张力法测得的cmc仅具有表观上的意义, 只反映了表面活性剂在气/液界面达到饱和吸附时的临界浓度. SNphS的疏水性强于SNzS, 更有效地促进了C12-s-C12·2Br的吸附和聚集.     

The adsorption process of some acetic acid derivatives at the water/air interface

The adsorption of trifluoro-, trichloro-, tribromo-, and trimethylacetic acid at the water/air interface is discussed on the basis of surface tension measurements. The process of adsorption is described by Henry's and Langmuir's isotherm equations. The obtained results allow calculation of the standard free energy of adsorption of investigated molecules and the contribution to this energy of hydrophobic groups of these molecules.     

X型与线型嵌段聚醚在空气/水和正庚烷/水界面上聚集行为的比较研究

通过阴离子聚合方法合成了环氧乙烷(EO)含量和分子量均相同的线型聚氧丙烯(PEO)-聚氧乙烯(PPO) (LPE)和X型聚氧丙烯-聚氧乙烯(TPE)嵌段聚醚,考察了它们在空气/水及正庚烷/水界面上聚集行为的差异. 界面活性的研究结果表明,TPE降低水、正庚烷界面张力的效率和效能均低于LPE的. 聚醚分子在正庚烷/水界面达到吸附平衡的时间比在空气/水表面短. 由于正庚烷分子插入到聚醚吸附层中,聚醚分子可以在正庚烷/水界面上采取更为直立的状态,因此聚醚分子在正庚烷/水界面扩散较快. 聚醚在正庚烷/水界面的扩张弹性高于空气/水表面的.     

Structure of mixed beta-lactoglobulin/pectin adsorbed layers at air/water interfaces; a spectroscopy study

Based on earlier reported surface rheological behaviour two factors appeared to be important for the functional behaviour of mixed protein/polysaccharide adsorbed layers at air/water interfaces: (1) protein/polysaccharide mixing ratio and (2) formation history of the layers. In this study complexes of beta-lactoglobulin (positively charged at pH 4.5) and low methoxyl pectin (negatively charged) were formed at two mixing ratios, resulting in negatively charged and nearly neutral complexes. Neutron reflection showed that adsorption of negative complexes leads to more diffuse layers at the air/water interface than adsorption of neutral complexes. Besides (simultaneous) adsorption of protein/polysaccharide complexes, a mixed layer can also be formed by adsorption of (protein/)polysaccharide (complexes) to a pre-formed protein layer (sequential adsorption). Despite similar bulk concentrations, adsorbed layer density profiles of simultaneously and sequentially formed layers were persistently different, as illustrated by neutron reflection analysis. Time resolved fluorescence anisotropy showed that the mobility of protein molecules at an air/water interface is hampered by the presence of pectin. This hampered mobility of protein through a complex layer could account for differences observed in density profiles of simultaneously and sequentially formed layers. These insights substantiated the previously proposed organisations of the different adsorbed layers based on surface rheological data.     

Molecular details of ovalbumin-pectin complexes at the air/water interface: a spectroscopic study

To stabilize air-water interfaces, as in foams, the adsorption of surface-active components is a prerequisite. An approach to controlling the surface activity of proteins is noncovalent complex formation with a polyelectrolyte in the bulk phase. The molecular properties of egg white ovalbumin in a complex with pectin in the bulk solution and at air/water interfaces were studied using drop tensiometry (ADT) and time-resolved fluorescence anisotropy techniques. The complex formation of ovalbumin with pectin in the bulk resulted in the formation of a compact structure with a different spatial arrangement depending on the protein/pectin ratio. Complex formation did not provide an altered protein structure, whereas the conformational stability was slightly increased in the complex. In excess pectin, an overall condensed complex structure is formed, whereas at limited pectin concentrations the structure of the complex is more "segmental". The characteristics of these structures did not depend on pH in the 7.0 to 4.5 regime. Interaction with pectin in the bulk solution resulted in a significantly slower adsorption of the protein to the air/water interface. The limited mobility of the protein at the interface was found for both ovalbumin and ovalbumin-pectin complexes. From both the rotational dynamics and total fluorescence properties of the protein in the absence and presence of pectin, it was suggested that the complex does not dissociate at the interface. Ovalbumin in the complex retains its initial "aqueous" microenvironment at the interface, whereas in the absence of pectin the microenvironment of the protein changed to a more nonpolar one. This work illustrates a more general property of polyelectrolytes, namely, the ability to retain a protein in its microenvironment. Insight into this property provides a new tool for better control of the surface activity of complex biopolymer systems.     

界面扩张流变法研究C_(12)mimBr对Gemini12-2-12在空气/水界面聚集行为的影响

采用界面扩张流变技术研究了季铵盐偶联表面活性剂C12-(CH2)2-C12·2Br(Gemini12-2-12)及其与离子液体表面活性剂溴化1-十二烷基-3-甲基咪唑(C12mim Br)复配体系的动态界面张力、扩张流变性质和界面弛豫过程等,探讨了C12mim Br对C12mim Br/Gemini12-2-12混合体系界面性质的影响及C12mim Br对Gemini12-2-12界面聚集行为影响的机制.结果表明,随着离子液体表面活性剂的不断引入,体系界面吸附达到平衡所需的时间逐渐缩短,扩张模量和相角明显降低,界面吸附膜由粘弹性膜转变为近似纯弹性膜;同时,界面及其附近的弛豫过程也发生显著变化,慢弛豫过程消失,快弛豫过程占主导地位,且离子液体浓度越高,快弛豫的贡献越大.这些界面性质的变化主要归因于离子液体表面活性剂C12mim Br参与界面形成及两表面活性剂在界面竞争吸附的结果.少量离子液体表面活性剂C12mim Br的加入可以填补疏松的Gemini12-2-12界面上的空位,形成混合界面吸附膜.随着C12mim Br含量的增加,嵌入界面的C12mim Br分子数不断增多,导致界面上相互缠绕的Gemini12-2-12烷基链"解缠",在体相和界面分子扩散交换的过程中"解缠"的Gemini12-2-12分子从界面上解吸回到体相,与此同时,C12mim Br分子相对较小的空间位阻及较强的疏水作用促使其优先扩散至界面进而取代Gemini12-2-12分子,最终界面几乎完全被C12mim Br分子所占据.     

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.     

Dynamics of mixed protein–surfactant layers adsorbed at the water/air and water/oil interface

Drop and bubble shape tensiometry experiments are performed at the water/air and water/hexane interfaces in order to get more information about the differences in the adsorption layer structure of mixed protein/surfactant systems. For mixtures of β-lactoglobulin and sodium dodecyl sulphate the adsorption at the water/air interface is essentially a competitive process between protein/surfactant complexes and free surfactant molecules, while the water/oil interface is essentially covered by the complexes.     

Kinetic study of adsorption of some biocompounds at the oil/water interface

The adsorption kinetics of some local anesthetics, like dibucaine and tetracaine, and of stearic acid from bulk solutions at the oil/water interface was studied by using the pendent drop and ring methods. The anesthetics were dissolved in aqueous solutions (pH 2), and the fatty acid was dissolved in benzene, each biocompound at several different concentrations in bulk solutions. Kinetic equations for Langmuir mechanism of adsorption at oil/water interface were tested. The kinetic analysis shows that Langmuir kinetic approach describes the dynamic interfacial pressures within the limits of the experimental errors over a wide range of time and for different surfactant concentrations in bulk solutions. It is also concluded that this approach allows the calculation of the ratio of the adsorption and desorption rate constants of these biocompounds at the oil/water interface. Obtained results are in substantial agreement with earlier reported data for the surfactant adsorption as, well as with their molecular structure.     

Effect of sucrose on the properties of caffeine adsorption layers at the air/solution interface

Sweet and bitter tastes are known to be mediated by G-protein-coupled receptors. The relationship between the chemical structure of gustable molecules and their molecular organization in saliva (aqueous solution) near the surface of the tongue provides a useful tool for elucidating the mechanism of chemoreception. The interactions between stimulus and membrane receptors occur in an anisotropic system. They might be influenced by the molecular packing of gustable molecules within an aqueous solvent (saliva) close to the receptor protein. To investigate the molecular organization of a sweet molecule (sucrose), a bitter molecule (caffeine), and their mixture in an aqueous phase near a "wall", a hydrophobic phase, we modeled this using an air/liquid interface as an anisotropic system. The experimental (tensiometry and ellipsometry) data unambiguously show that caffeine molecules form an adsorption layer, whereas sucrose induces a desorption layer at the air/water interface. The adsorption of caffeine molecules at the air/water interface gradually increases with the volume concentration and is delayed when sucrose is added to the solution. Spectroscopic ellipsometry data show that caffeine in the adsorption layer has optical properties practically identical to those of the molecule in solution. The results are interpreted in terms of molecular association of caffeine with itself at the interface with and without sucrose in the subphase, using the theory of ideal gases.     

Partition and water/oil adsorption of some surfactants

Adsorption isotherms have been determined at the water/oil interface for five biphasic systems involving surfactants (non-ionic and ionic) present in both phases at partition equilibrium. The systems studied were polyoxyethylene(23)lauryl ether (Brij35) in water/hexane and four ionic surfactants, hexadecyltrimethylammonium bromide (CTAB), and a series of three tetraalkylammonium dodecylsulfate (TEADS, TPADS, and TBADS) in water/CH 2Cl 2. Interfacial tension measurements performed at the water/air and water/oil interfaces provided all the necessary information for the determination of the adsorption parameters by taking partition into account. These measurements also allowed the comparison of the adsorption properties at both interfaces which showed an increase of the adsorption equilibrium constant and a decrease of the maximum surface concentration at the water/oil interface compared to water/air. The values of the critical aggregation concentration showed, in all cases, that only the surfactant dissolved in the aqueous phase contribute to the decrease of the water/oil interfacial tension. In the case of the four ionic surfactants, the critical aggregation concentration obtained in biphasic conditions were lowered because of the formation of mixed surfactant-CH 2Cl 2 aggregates.