Protein adsorption at the oil/water interface: characterization of adsorption kinetics by dynamic interfacial tension measurements.

The dynamics of protein adsorption at an oil/water interface are examined over time scales ranging from seconds to several hours. The pendant drop technique is used to determine the dynamic interfacial tension of several proteins at the heptane/aqueous buffer interface. The kinetics of adsorption of these proteins are interpreted from tension/log time plots, which often display three distinct regimes. (I) Diffusion and protein interfacial affinity determine the duration of an initial induction period of minimal tension reduction. A comparison of surface pressure profiles at the oil/water and air/water interface reveals the role of interfacial conformational changes in the early stages of adsorption. (II) Continued rearrangement defines the second regime, where the resulting number of interfacial contacts per protein molecule causes a steep tension decline. (III) The final regime occurs upon monolayer coverage, and is attributed to continued relaxation of the adsorbed layer and possible build-up of multilayers. Denaturation of proteins by urea in the bulk phase is shown to affect early regimes.     

Kinetics of protein adsorption and desorption on surfaces with grafted polymers

The kinetics of protein adsorption are studied using a generalized diffusion approach which shows that the time-determining step in the adsorption is the crossing of the kinetic barrier presented by the polymers and already adsorbed proteins. The potential of mean-force between the adsorbing protein and the polymer-protein surface changes as a function of time due to the deformation of the polymer layers as the proteins adsorb. Furthermore, the range and strength of the repulsive interaction felt by the approaching proteins increases with grafted polymer molecular weight and surface coverage. The effect of molecular weight on the kinetics is very complex and different than its role on the equilibrium adsorption isotherms. The very large kinetic barriers make the timescale for the adsorption process very long and the computational effort increases with time, thus, an approximate kinetic approach is developed. The kinetic theory is based on the knowledge that the time-determining step is crossing the potential-of-mean-force barrier. Kinetic equations for two states (adsorbed and bulk) are written where the kinetic coefficients are the product of the Boltzmann factor for the free energy of adsorption (desorption) multiplied by a preexponential factor determined from a Kramers-like theory. The predictions from the kinetic approach are in excellent quantitative agreement with the full diffusion equation solutions demonstrating that the two most important physical processes are the crossing of the barrier and the changes in the barrier with time due to the deformation of the polymer layer as the proteins adsorb/desorb. The kinetic coefficients can be calculated a priori allowing for systematic calculations over very long timescales. It is found that, in many cases where the equilibrium adsorption shows a finite value, the kinetics of the process is so slow that the experimental system will show no adsorption. This effect is particularly important at high grafted polymer surface coverage. The construction of guidelines for molecular weight/surface coverage necessary for kinetic prevention of protein adsorption in a desired timescale is shown. The time-dependent desorption is also studied by modeling how adsorbed proteins leave the surface when in contact with a pure water solution. It is found that the kinetics of desorption are very slow and depend in a nonmonotonic way in the polymer chain length. When the polymer layer thickness is shorter than the size of the protein, increasing polymer chain length, at fixed surface coverage, makes the desorption process faster. For polymer layers with thickness larger than the protein size, increases in molecular weight results in a longer time for desorption. This is due to the grafted polymers trapping the adsorbed proteins and slowing down the desorption process. These results offer a possible explanation to some experimental data on adsorption. Limitations and extension of the developed approaches for practical applications are discussed.     

Thermal and Structural Stability of Adsorbed Proteins

Experimental evidence suggests that proteins adsorbed to hydrophobic surfaces at low coverages are stabilized relative to the bulk. For larger coverages, proteins unfold and form β-sheets. We performed computer simulations on model proteins and found that: 1), For weakly adsorbing surfaces, unfolded conformations lose more entropy upon adsorption than folded ones. 2), The melting temperature, both in the bulk and at surfaces, decreases with increasing protein concentration because of favorable interprotein interactions. 3), Proteins in the bulk show large unfolding free energy barriers; this barrier decreases at stronger adsorbing surfaces. We conjecture that typical experimental temperatures appear to be below the bulk melting temperature for a single protein, but above the melting temperature for concentrated protein solutions. Purely thermodynamic factors then explain protein stabilization on adsorption at low concentrations. However, both thermodynamic and kinetic factors are important at higher concentrations. Thus, proteins in the bulk do not denature with increasing concentration due to large kinetic barriers, even though the aggregated state is thermodynamically preferred. However, they readily unfold upon adsorption, with the surface acting as a heterogeneous catalyst. The thermal behavior of proteins adsorbed to hydrophobic surfaces thus appears to follow behavior independent of their chemical specificity.     

Surface characterization of human serum albumin and sodium perfluorooctanoate mixed solutions by pendant drop tensiometry and circular dichroism

The interfacial behavior of mixed human serum albumin (HSA)/sodium perfluorooctanoate (C8FONa) solutions is examined by using two experimental techniques, pendant drop tensiometry and circular dichroism spectroscopy. Through the analysis of the surface tension of the mixed solutions, surface competitive adsorption at the air-water interface between C8FONa and HSA is detected. The dynamic adsorption curves exhibit the distinct regimes in their time-dependent surface tension. The nature of these regimes is further analyzed in terms of the variation of the molecules surface areas. As a consequence, a compact and dense structure was formed where protein molecules were interconnected and overlapped. Thus, a reduction of the area occupied per molecule from 100 to 0.2 nm(2) is interpreted as a gel-like structure at the surface. The presence of the surfactant seems to favor the formation of this interfacial structure. Finally, measurements of circular dichroism suggests a compaction of the protein due to the association with the surfactant given by an increase of alpha-helix structure in the complexes as compared to that of pure protein.     

The investigation of protein adsorption behaviors on different functionalized polymers films

The adsorption of BSA and fibrinogen onto plasma-polymerized di-(ethylene glycol) vinyl ether, allylamine, and maleic anhydride films were investigated in detail by surface plasmon resonance spectroscopy (SPR). The chemical properties of the plasma polymers were initially determined by the plasma deposition conditions during the generation procedure. The analysis of the chemical structure of the films and the refractive index of plasma polymers in aqueous solution was carried out using Fourier transform infrared spectroscopy and waveguide mode spectroscopy, respectively. Using water contact angle measurement, the surface wettability of plasma polymers was also characterized. These properties have a critical influence on the behavior of protein adsorption on the surface of the plasma polymers. Protein adsorption was found to depend not only on the types of functionalized groups, but also on the plasma polymer thickness since the protein molecules penetrate into the plasma polymer network bulk. According to the size of protein molecules in aqueous solution and the amount of adsorbed proteins observed by SPR, the conformational changes of proteins could be deduced.     

Dissociation coefficients of protein adsorption to nanoparticles as quantitative metrics for description of the protein corona: A comparison of experimental techniques and methodological relevance

Protein adsorption to nanoparticles is described as a chemical reaction in which proteins attach to binding sites on the nanoparticle surface. This process is defined by a dissociation coefficient, which tells how many proteins are adsorbed per nanoparticle in dependence of the protein concentration. Different techniques to experimentally determine dissociation coefficients of protein adsorption to nanoparticles are reviewed. Results of more than 130 experiments in which dissociation coefficients have been determined are compared. Data show that different methods, nanoparticle systems, and proteins can lead to significantly different dissociation coefficients. However, we observed a clear tendency of smaller dissociation coefficients upon less negative towards more positive zeta potentials of the nanoparticles. The zeta potential thus is a key parameter influencing protein adsorption to the surface of nanoparticles. Our analysis highlights the importance of the characterization of the parameters governing protein–nanoparticle interaction for quantitative evaluation and objective literature comparison.     

Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability

The influence of protein stability on the adsorption and desorption behavior to surfaces with fundamentally different properties (negatively charged, positively charged, hydrophilic, and hydrophobic) was examined by surface plasmon resonance measurements. Three engineered variants of human carbonic anhydrase II were used that have unchanged surface properties but large differences in stability. The orientation and conformational state of the adsorbed protein could be elucidated by taking all of the following properties of the protein variants into account: stability, unfolding, adsorption, and desorption behavior. Regardless of the nature of the surface, there were correlation between (i) the protein stability and kinetics of adsorption, with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability. All of the above correlations could be explained by the rate of denaturation and the conformational state of the adsorbed protein. In conclusion, protein engineering for increased stability can be used as a strategy to decrease irreversible adsorption on surfaces at a liquid-solid interface.     

Mobility of adsorbed proteins: a Brownian dynamics study

We simulate the adsorption of lysozyme on a solid surface, using Brownian dynamics simulations. A protein molecule is represented as a uniformly charged sphere and interacts with other molecules through screened Coulombic and double-layer forces. The simulation starts from an empty surface and attempts are made to introduce additional proteins at a fixed time interval that is inversely proportional to the bulk protein concentration. We examine the effect of ionic strength and bulk protein concentration on the adsorption kinetics over a range of surface coverages. The structure of the adsorbed layer is examined through snapshots of the configurations and quantitatively with the radial distribution function. We extract the surface diffusion coefficient from the mean square displacement. At high ionic strengths the Coulombic interaction is effectively shielded, leading to increased surface coverage. This effect is quantified with an effective particle radius. Clustering of the adsorbed molecules is promoted by high ionic strength and low bulk concentrations. We find that lateral protein mobility decreases with increasing surface coverage. The observed trends are consistent with previous theoretical and experimental studies.     

Adsorption of globular proteins on locally planar surfaces: models for the effect of excluded surface area and aggregation of adsorbed protein on adsorption equilibria.

Equilibrium statistical-thermodynamic models are presented for the surface adsorption of proteins modeled as regular convex hard particles. The adsorbed phase is treated as a two-dimensional fluid, and the chemical potential of adsorbed protein is obtained from scaled particle theory. Adsorption isotherms are calculated for nonassociating and self-associating adsorbing proteins. Area exclusion broadens adsorption isotherms relative to the Langmuir isotherm (negative cooperativity), whereas self-association steepens them (positive cooperativity). The calculated isotherm for adsorption of hard spheres using scaled particle theory for hard discs agrees well with that calculated from the hard disc virial expansion. As the cross section of the adsorbing protein in the plane of the surface becomes less discoidal, the apparent negative cooperativity manifested in the isotherm becomes more pronounced. The model is extended to the case of simultaneous adsorption of a tracer protein at low saturation and a competitor protein with a different size and/or shape at arbitrary fractional saturation. Area exclusion by competitor for tracer (and vice versa) is shown to substantially enhance the displacement of tracer by competitor and to qualitatively invalidate the standard interpretation of ligand competition experiments, according to which the fractional displacement of tracer by competitor is equal to the fractional saturation by competitor.     

Approach for an equation of state for adsorbed protein surfaces.

A surface equation of state for polymer interfaces is obtained by equating the chemical potentials of the solvent in the bulk and surface phases. This equation of state contains the fraction of the surface covered with polymer and the surface activity coefficient as parameters. These parameters may be obtained by measuring the thermodynamic modulus of elasticity. Moreover, if the amount of protein in the interface is known the degree of folding in the surface may be evaluated. The present theory was applied to adsorbed beta-lactoglobulin in surfaces. The results are in agreement with qualitative equations and results from other sources.     

Overcoming rapid inactivation of lung surfactant: Analogies between competitive adsorption and colloid stability

Lung surfactant (LS) is a mixture of lipids and proteins that line the alveolar air-liquid interface, lowering the interfacial tension to levels that make breathing possible. In acute respiratory distress syndrome (ARDS), inactivation of LS is believed to play an important role in the development and severity of the disease. This review examines the competitive adsorption of LS and surface-active contaminants, such as serum proteins, present in the alveolar fluids of ARDS patients, and how this competitive adsorption can cause normal amounts of otherwise normal LS to be ineffective in lowering the interfacial tension. LS and serum proteins compete for the air-water interface when both are present in solution either in the alveolar fluids or in a Langmuir trough. Equilibrium favors LS as it has the lower equilibrium surface pressure, but the smaller proteins are kinetically favored over multi-micron LS bilayer aggregates by faster diffusion. If albumin reaches the interface, it creates an energy barrier to subsequent LS adsorption that slows or prevents the adsorption of the necessary amounts of LS required to lower surface tension. This process can be understood in terms of classic colloid stability theory in which an energy barrier to diffusion stabilizes colloidal suspensions against aggregation. This analogy provides qualitative and quantitative predictions regarding the origin of surfactant inactivation. An important corollary is that any additive that promotes colloid coagulation, such as increased electrolyte concentration, multivalent ions, hydrophilic non-adsorbing polymers such as PEG, dextran, etc. added to LS, or polyelectrolytes such as chitosan, also promotes LS adsorption in the presence of serum proteins and helps reverse surfactant inactivation. The theory provides quantitative tools to determine the optimal concentration of these additives and suggests that multiple additives may have a synergistic effect. A variety of physical and chemical techniques including isotherms, fluorescence microscopy, electron microscopy and X-ray diffraction show that LS adsorption is enhanced by this mechanism without substantially altering the structure or properties of the LS monolayer.     

The Adsorption of Whey Proteins on the Surface of Emulsified Fat

Whey proteins adsorbed on fat globule surfaces during emulsification with coconut oil at pH 3 ~ 9 were examined. The amount of proteins adsorbed on the fat surface was dependent on the pH during emulsification. At any pH examined here, however, tightly-adsorbed proteins which were not extracted from the fat surface with urea or guanidine-HCl were 2 ~ 3 mg/m². Marked selectivities in the adsorption of individual whey proteins were observed at any pH. No correlation between the adsorbabilities and the surface hydrophobicities of individual whey proteins was observed. Whey proteins adsorbed on the emulsified fat were much more easily digested with proteases compared to the native whey proteins, indicating that conformational changes of whey proteins occurred at the fat surface. The results suggested that conformational properties, such as flexibility of the structure, of whey proteins are important in the adsorption and possibly affect their emulsifying ability.     

Kinetic studies of protein-surface interactions: A two-stage model of surface-induced protein transitions in adsorbed biofilms

The irreversible adsorption of proteins on artificial surfaces plays an important role in a wide variety of practical problems. The simple analytical models based on definite concepts regarding the mechanisms of interfacial evolution can be used efficiently for characterization of protein-surface interactions by analyzing the intrinsic kinetics of the process. In this article, analytical expressions are derived for the adsorption kinetics that take into account the presence of more than one adsorbed state for proteins in biofilms. It is shown that the experimentally observed dependence of the adsorbed mass on the concentration of protein in solution can be reproduced with this model, and the approach provides a rapid method for obtaining quantitative parameters for the adsorption process. It is shown by analytical approximation of the kinetic curves for fibrinogen adsorption onto an unmodified gold surface studied by a surface plasmon resonance biosensor that this model is in good quantitative agreement with experiments. It is found that the rate of adsorption, controlled mainly by the mass flow from the solution, determines the contribution both to self-assembling and spreading, resulting in variations of adsorbed fibrinogen interfacial structures.     

Adsorption of frog foam nest proteins at the air-water interface

The surfactant properties of aqueous protein mixtures (ranaspumins) from the foam nests of the tropical frog Physalaemus pustulosus have been investigated by surface tension, two-photon excitation fluorescence microscopy, specular neutron reflection, and related biophysical techniques. Ranaspumins lower the surface tension of water more rapidly and more effectively than standard globular proteins under similar conditions. Two-photon excitation fluorescence microscopy of nest foams treated with fluorescent marker (anilinonaphthalene sulfonic acid) shows partitioning of hydrophobic proteins into the air-water interface and allows imaging of the foam structure. The surface excess of the adsorbed protein layers, determined from measurements of neutron reflection from the surface of water utilizing H(2)O/D(2)O mixtures, shows a persistent increase of surface excess and layer thickness with bulk concentration. At the highest concentration studied (0.5 mg ml(-1)), the adsorbed layer is characterized by three distinct regions: a protruding top layer of approximately 20 angstroms, a middle layer of approximately 30 angstroms, and a more diffuse submerged layer projecting some 25 angstroms into bulk solution. This suggests a model involving self-assembly of protein aggregates at the air-water interface in which initial foam formation is facilitated by specific surfactant proteins in the mixture, further stabilized by subsequent aggregation and cross-linking into a multilayer surface complex.     

Adsorption of HSA, IgG and laminin-1 on model hydroxyapatite surfaces--effects of surface characteristics

Ellipsometry and mechanically assisted sodium dodecyl sulphate elution was utilized to study the adsorption of human serum albumin (HSA), human immunoglobulin G (IgG), and laminin-1, as well as competitive adsorption from a mixture of these proteins on spin-coated and sintered hydroxyapatite (HA) surfaces, respectively. The HA surfaces were characterized with respect to wettability and roughness by means of water contact angles and atomic force microscopy, respectively. Both surface types were hydrophilic, and the average roughness (Sa) and surface enlargement (Sdr) were lower for the sintered compared to the spin-coated HA surfaces. The adsorbed amounts on the sintered HA increased as follows: HSA < laminin-1 < IgG < the protein mixture. For the competitive adsorption experiments, the adsorbed fractions increased accordingly: HSA < laminin-1 < IgG on both types of HA substratum. However, a higher relative amount of HSA and laminin-1 and a lower relative amount of IgG was found on the spin-coated surfaces compared to the sintered surfaces. The effects observed could be ascribed to differences in surface roughness and chemical composition between the two types of HA substratum, and could have an influence on selection of future implant surface coatings.     

C3 adsorbed to a polymer surface can form an initiating alternative pathway convertase

Contact between blood and a biomaterial surface induces an immediate complement-mediated inflammatory response. Under these conditions, the alternative pathway of complement is often initiated and amplified on the biomaterial surface. Adsorption of a protein such as C3 to a polymer surface induces conformational changes in the protein. Based on the expression on adsorbed C3 of conformational neoepitopes specific for bound C3 fragments, we have hypothesized that adsorbed C3 is able to bind factor B and form a functional C3,Bb convertase. Using a quartz crystal microbalance to monitor binding of proteins to a polymer surface, we have demonstrated that a functional C3-containing alternative pathway convertase can be formed, in particular, in the presence of properdin. These data indicate that adsorption of C3 induces conformational changes that turn C3 into a C3b-like molecule that is able to participate in the functioning of the alternative convertase, and they suggest a new mechanism for complement activation on a biomaterial surface.     

Circular dichroism studies on conformational changes in protein molecules upon adsorption on ultrafine polystyrene particles

The conformational changes in well-characterized model proteins [bovine ribonuclease A (RNase A), horseradish peroxidase, sperm-whole myoglobin, human hemoglobin, and bovine serum albumin (BSA)] upon adsorption on ultrafine polystyrene (PS) particles have been studied using circular dichroism (CD) spectroscopy. These proteins were chosen with special attention to molecular flexibility. The ultrafine PS particles were negatively charged and have average diameters of 20 or 30 nm. Utilization of these ultrafine PS particles makes it possible to apply the CD technique to determine the secondary structure of proteins adsorbed on the PS surface. Effects of protein properties and adsorption conditions on the extent of the changes in the secondary structure of protein molecules upon adsorption on ultrafine PS particles were studied. The CD spectrum changes upon adsorption were significant in the "soft" protein molecules (myoglobin, hemoglobin, and BSA), while they were insingnificant in the "rigid" proteins (RNase A and peroxidase). The soft proteins sustained a marked decrease in alpha-helix content upon adsorption. Moreover, the native alpha-helix content, which is given as the percentage of the alpha-helix content in the free proteins, of adsorbed BSA was found to decrease with decreasing pH and increase with increasing adsorbed amount. These observations confirm some well-known hypotheses for the confirmational chages in protein molecules upon adsorption. (c) 1992 John Wiley & Sons, Inc.     

Interaction forces and morphology of a protein-resistant poly(ethylene glycol) layer

The molecular interactions on a protein-resistant surface coated with low-molecular-weight poly(ethylene glycol) (PEG) copolymer brushes are investigated using the extended surface forces apparatus. The observed interaction force is predominantly repulsive and nearly elastic. The chains are extended with respect to the Flory radius, which is in agreement with qualitative predictions of scaling theory. Comparison with theory allows the determination of relevant quantities such as brush length and adsorbed mass. Based on these results, we propose a molecular model for the adsorbed copolymer morphology. Surface-force isotherms measured at high resolution allow distinctive structural forces to be detected, suggesting the existence of a weak equilibrium network between poly(ethylene glycol) and water--a finding in accordance with the remarkable solution properties of PEG. The occurrence of a fine structure is interpreted as a water-induced restriction of the polymer's conformational space. This restriction is highly relevant for the phenomenon of PEG protein resistance. Protein adsorption requires conformational transitions, both in the protein as well as in the PEG layer, which are energetically and kinetically unfavorable.     

Surface denaturation and amyloid fibril formation of insulin at model lipid-water interfaces

We consider the effects that different lipid surfaces have upon the denaturation and subsequent formation of amyloid fibrils of bovine insulin. The adsorption and unfolding kinetics of insulin being adsorbed onto the different lipid surfaces under denaturing conditions are studied using FTIR ATR spectroscopy and are compared to the bulk solution behavior of the protein. Atomic force microscopy studies are also performed to compare the fibrils growing on the different surfaces. This study shows that both the adsorption and unfolding kinetics of insulin can be described by a sum of exponential processes and that different surfaces behave differently, with respect both to one another and to the bulk protein solution. The proteins adsorbed onto the surfaces are observed to have faster unfolding kinetics than those in the bulk, and the fibril-like structures formed at the surfaces are shown to be different in a number of ways from those found in bulk solution. The beta-sheet content and growth kinetics of the adsorbed proteins also differ from those of the bulk system. An attempt is made to describe the observed behavior in terms of simple physical arguments involving adsorption, unfolding, and aggregation of the proteins.     

Limited conformational change of beta-lactoglobulin when adsorbed at the air-water interface

Detailed insight can be obtained from proteins at and near the air-water interface using external reflection IR and circular dichroism techniques. Besides information on local protein concentrations and surface layer thickness, it is shown that beta-lactoglobulin displays a limited unfolding at the interface. The conformational change is comparable to that observed upon heat-induced aggregation of the protein and can be understood in view of the high surface concentration of the protein (approximately 40% volume fraction). The layer thickness and the conformational properties of the protein do not depend on the bulk concentration. After adsorption of beta-lactoglobulin to a preformed lipid monomolecular layer a similar conformational change is induced, suggesting that the folding properties of the protein itself determine the extent of conformational changes at the interfaces.