J. Krägel

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.

Thermodynamics, adsorption kinetics and rheology of mixed protein-surfactant interfacial layers

Depending on the bulk composition, adsorption layers formed from mixed protein/surfactant solutions contain different amounts of protein. Clearly, increasing amounts of surfactant should decrease the amount of adsorbed proteins successively. However, due to the much larger adsorption energy, proteins are rather strongly bound to the interface and via competitive adsorption surfactants cannot easily displace proteins. A thermodynamic theory was developed recently which describes the composition of mixed protein/surfactant adsorption layers. This theory is based on models for the single compounds and allows a prognosis of the resulting mixed layers by using the characteristic parameters of the involved components. This thermodynamic theory serves also as the respective boundary condition for the dynamics of adsorption layers formed from mixed solutions and their dilational rheological behaviour. Based on experimental studies with milk proteins (beta-casein and beta-lactoglobulin) mixed with non-ionic (decyl and dodecyl dimethyl phosphine oxide) and ionic (sodium dodecyl sulphate and dodecyl trimethyl ammonium bromide) surfactants at the water/air and water/hexane interfaces, the potential of the theoretical tools is demonstrated. The displacement of pre-adsorbed proteins by subsequently added surfactant can be successfully studied by a special experimental technique based on a drop volume exchange. In this way the drop profile analysis can provide tensiometry and dilational rheology data (via drop oscillation experiments) for two adsorption routes--sequential adsorption of the single compounds in addition to the traditional simultaneous adsorption from a mixed solution. Complementary measurements of the surface shear rheology and the adsorption layer thickness via ellipsometry are added in order to support the proposed mechanisms drawn from tensiometry and dilational rheology, i.e. to show that the formation of mixed adsorption layer is based on a modification of the protein molecules via electrostatic (ionic) and/or hydrophobic interactions by the surfactant molecules and a competitive adsorption of the resulting complexes with the free, unbound surfactant. Under certain conditions, the properties of the sequentially formed layers differ from those formed simultaneously, which can be explained by the different locations of complex formation.

Dilational and shear rheology of adsorption layers at liquid interfaces

Abstract Dynamic properties of interfaces are of increasing interest in science and in practice as they give insight into interactions and processes at interfaces rather than equilibrium properties. The general ideas on mechanical interfacial properties as an important part of dynamic properties were established long ago by Gibbs and Boussinesq. Now on the basis of new techniques, better experiments can be performed which allow a more and more quantitative understanding. The mechanical behaviour of interfaces, modified by soluble adsorption layers or insoluble monolayers of surfactants or polymers, is the subject of many actual studies. Computer-driven instruments using new sensors and very sophisticated methodologies enable us to perform very complex and sensitive measurements which were impossible until recently. Numerous studies of interfacial shear and dilational rheology have been reported and use a large variety of techniques. Shear experiments are most useful for polymer and mixed polymer-surfactant adsorption layers and insoluble monolayers and give access to interaction forces in two-dimensional layers. Dilational interfacial properties however are most frequently studied for soluble adsorption layers of surfactants and mixtures of polymers and surfactants. This overview gives an introduction to the interfacial rheology and discusses some specific theoretical aspects necessary to interpret experiments. Experimental techniques to perform shear and dilational experiments at liquid interfaces are summarised and only the most recent developments are described in more detail. Examples are given to demonstrate how the experiments work and what output can be expected.

Interfacial shear rheology of protein-surfactant layers

The shear rheology of adsorbed or spread layers at air/liquid and liquid/liquid phase boundaries is relevant in a wide range of technical applications such as mass transfer, monolayers, foaming, emulsification, oil recovery, or high speed coating. Interfacial shear rheological properties can provide important information about interactions and molecular structure in the interfacial layer. A variety of measuring techniques have been proposed in the literature to measure interfacial shear rheological properties and have been applied to pure protein or mixed protein adsorption layers at air/water or oil/water interfaces. Such systems play for example an important role as stabilizers in foams and emulsions. The aim of this contribution is to give a literature overview of interfacial shear rheological studies of pure protein and protein/surfactant mixtures at liquid interfaces measured with different techniques. Techniques which utilize the damping of waves, spectroscopic or AFM techniques and all micro-rheological techniques will not discuss here.

Properties of mixed protein/surfactant adsorption layers

The adsorption isotherms, adsorption kinetics and surface rheological properties of β-lactoglobulin, β-casein, in the absence and presence of Tween 20 were measured. To study the adsorption process (isotherms and kinetics) at the water–air interface the pendant drop technique (axial drop shape analysis, ADSA), and ring tensiometry were used. The surface shear rheological parameters were measured with a torsion pendulum set-up. Also, data of the equilibrium film thickness and surface diffusion coefficients obtained from fluorescence recovery after photobleaching (FRAP) measurements are used to understand the competitive adsorption mechanism. The adsorption process and shear rheological behaviour of the studied systems show a rather complex behaviour which depends most of all on the systems composition. At high protein or surfactant content the behaviour is controlled by the main component while for the more mixed systems the adsorption process is complex and consists of partial adsorption, surfactant–protein interaction and protein rearrangement as a function of surface coverage. The results obtained illustrate that all these processes must be taken into account in future new theoretical models to be derived for such systems.

Interfacial Properties of Mixed β-Lactoglobulin-SDS Layers at the Water/Air and Water/Oil Interface

The adsorption behavior of the beta-lactoglobuline has been studied in the presence of the anionic surfactant sodium dodecylsulfate (SDS) and compared for two different interfaces, water/air and water/hexane. The fitting of experimental data (adsorption isotherms) by a mixed adsorption model and the determination of structural parameters such as the molecular area occupied by the protein-surfactant complex and the surfactant molecules at the interface allowed to have a better understanding of the composition and as a consequence the behavior of the mixed interfacial layer. The parameters obtained for the mixtures are similar to those obtained separately for the single components, but the comparison of the both interfaces has shown significant differences. Much higher concentration of complex is found at the water/hexane interface, which is the result of a better affinity of the protein for this interface. A higher penetration of the protein into the oil phase and the presence of interactions between protein-surfactant complexes and free surfactant molecules stabilize the interface preventing its replacement by the SDS molecules. Rheological experiments show a decrease of the visco-elastic modulus at both interfaces with increasing SDS concentration. But at the water/oil interface, contrary to the water/air interface at which the replacement of the protein has been clearly observed, this decrease is attributed to changes of complex properties. At high SDS concentrations, an increase of the hydrophilic character due to hydrophobic interactions with the surfactant molecules leads to an increase in the mobility of the complex, which favors its desorption upon increased competition by the surfactant.

Competition between lipases and monoglycerides at interfaces

Tensiometry (the pendant drop technique), interfacial shear rheology, and ellipsometry have been used to study the effect of polar lipids that are generated during fat digestion on the behavior of lipases at the oil-water interface. Both Sn-1,3 regiospecific and nonregiospecific lipases have been used, and a noncatalytically active protein, beta-lacloglobulin, has been used as reference in the interfacial shear rheology experiments. The results from the pendant drop measurements and the interfacial rheology studies were in agreement with each other and demonstrated that the Sn-2 monoglyceride, which is one of the lipolysis products generated when a Sn-1,3 regiospecific lipase catalyzes triglyceride hydrolysis, is very interfacially active and efficiently expels the enzyme from the interface. Ellipsometry conducted at the liquid-liquid interface showed that the lipase forms a sublayer in the aqueous phase, just beneath the monoglyceride-covered interface. Sn-1/3 monoglycerides do not behave this way because they are rapidly degraded to fatty acid and glycerol and the fatty acid (or the fatty acid salt) does not have enough interfacial activity to expel the lipase from the interface. Since the lipases present in the gastrointestinal tract are highly Sn-1,3 regiospecific, we believe that the results obtained can be transferred to the in vivo situation. The formation of stable and amphiphilic Sn-2 monoglycerides can be seen as a self-regulatory process for fat digestion.

Dynamic surface tension and surface shear rheology studies of mixed β-lactoglobulin/Tween 20 systems

Abstract The mechanical behaviour of β-lactoglobulin (BLG) and BLG/Tween 20 adsorption layers at the air/water interface has been studied by dynamic surface tension and surface shear rheological measurements. The adsorption properties were measured using different surface tension methods (ring tensiometry, drop volume tensiometry and axisymmetric drop shape analysis). The surface shear rheological measurements were performed with a torsion pendulum set-up which simultaneously provides information about the surface shear viscosity and elasticity. The adsorption layer structure is controlled by the interactions between protein and surfactant molecules. The corresponding dynamic surface tension measurements confirm the peculiarities foun adsorption layer. With increasing surfactant concentration at constant protein concentration the mixed surface film gradually approaches a state identical to a pure surfactant adsorption layer, from which the protein is completely repelled.

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

Abstract 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.

Measurement of interfacial shear rheological properties: An apparatus

Abstract A new surface shear rheometer was designed on the basis of the principle of a torsion pendulum. The torsion wire transfers the deformation, produced by a stepper motor, via a sharp edge onto the surface. The movement of the edge is registered by a position-sensitive photodiode with an accuracy to ±0.01°. After an impulsive torque is applied by a rapid movement of the torsion head the pendulum performs damped oscillations with a damping factor α and a radian frequency β. This type of apparatus provides information on the surface shear coefficient of viscosity and the surface shear modulus of rigidity in one single experiment. Different instrumental parameters are discussed which influence the range of application and the accuracy of the measurements. Different applications of the set-up to protein solutions (gelatine and human albumin) alone and mixed with a surfactant are discussed. The experimental data demonstrate the high reproducibility and accuracy of the apparatus.