Adsorbed Protein Molecules Research Articles

Model salad dressing emulsion stability as affected by the type of the lupin seed protein isolate

AbstractModel salad dressing emulsions of an oil volume fraction of 0.50 were prepared using two types of lupin seed protein isolate (LSPI) differing in the method applied for their isolation and their protein composition. The dressing stability against creaming and droplet coalescence were studied and correlated with data on oil droplet size, rheological characteristics and the amount of protein adsorption at the droplet surfaces. Model salad dressing emulsions containing the isolate, mainly composed of lupin globulins, exhibited higher stability and more pronounced rheological characteristics compared to those prepared with the isolate enriched in albumins or with the mixture of the two isolates. The lupin albumins appeared to displace the globulins from the droplet surfaces, following competitive adsorption from mixtures of the two types of the lupin isolates. The results are discussed in terms of droplet interaction and rearrangement as they are influenced by the presence of the adsorbed protein molecules and aggregates which appear to determine long‐term stability of the emulsion systems. Copyright © 2006 Society of Chemical Industry

Thermodynamic model for nonlinear electrostatic adsorption equilibrium of protein

AbstractA statistical thermodynamic model is developed for the nonlinear multicomponent protein adsorption equilibrium on ion exchanger. This model takes into account the electrostatic interactions between the adsorbent surface and protein molecules, as well as the lateral interactions between adsorbed protein molecules. The model is, therefore, in accord with the nonstoichiometric nature of the electrostatic adsorption of protein. There are two categories of model parameters: one corresponds to the adsorption affinity of the protein and the other is descriptive of the interactions between the adsorbed molecules. So, all the model parameters have definite physical meanings, and for the adsorption equilibria of single protein, there are only two model parameters. By comparison with batch adsorption equilibrium data of bovine serum albumin, the model is found to fit the experimental data well. The effects of buffer type, pH and ionic strength on the model parameters are reasonably interpreted by the electrostatic and thermodynamic theories. © 2006 American Institute of Chemical Engineers AIChE J, 2006

Effect of Thermal Treatment, Ionic Strength, and pH on the Short-Term and Long-Term Coalescence Stability of β-Lactoglobulin Emulsions

We present experimental results about the effects of thermal treatment, ionic strength, and pH on the protein adsorption and coalescence stability of freshly prepared (2 h after emulsification) and 6-day-stored emulsions, stabilized by the globular protein beta-lactoglobulin (BLG). In all emulsions studied, the volume fraction of the dispersed soybean oil is 30% and the mean drop diameter is d(32) approximately 40 microm. The protein concentration, C(BLG), is varied between 0.02 and 0.1 wt %, the electrolyte concentration, C(EL), between 1.5 mM and 1 M, and pH between 4.0 and 7.0. The emulsion heating is performed at 85 degrees C, which is above the denaturing temperature of BLG. The results show that, at C(BLG) > or = 0.04 wt %, C(EL) > or = 150 mM, and pH > or = 6.2, the heating leads to higher protein adsorption and to irreversible attachment of the adsorbed molecules, which results in enhanced steric repulsion between the protein adsorption multilayers and to higher emulsion stability. At low electrolyte concentration, C(EL) < or = 10 mM, the emulsion stability is determined by electrostatic interactions and is not affected significantly by the emulsion heating. The latter result is explained by electrostatic repulsion between the adsorbed protein molecules, which keeps them separated from each other and thus precludes the formation of disulfide covalent bonds in the protein adsorption layer. The coalescence stability of heated and nonheated emulsions is practically the same and does not depend on C(EL), when pH is around the isoelectric point (IEP) of the protein molecules. This is explained with the adsorption of uncharged BLG molecules, in compact conformation, which stores the reactive sulfhydryl groups hidden inside the molecule interior, thus preventing the formation of covalent intermolecular bonds upon heating. We studied also the effect of storage time on the stability of heated and nonheated emulsions. The stability of nonheated emulsions (C(BLG) = 0.1 wt %, C(EL) > or = 150 mM, and pH = 6.2) significantly decreases after 1 day of storage (aging effect). In contrast, no aging effect is observed after emulsion heating. FTIR spectra of heated and nonheated, fresh and aged emulsions suggest that the aging effect is caused by slow conformational changes of the protein molecules in the adsorption layer, accompanied with partial loss of the ordered secondary structure of the protein and with the formation of lateral noncovalent bonds (H-bonds and hydrophobic interactions) between the adsorbed molecules. After thermal treatment of the BLG emulsions, the molecules preserve their original secondary structure upon storage, which eliminates the aging effect.

Protein Adsorption by Basal Plane Graphite Surfaces: Molecular Images and Nano-Structured Films

The adsorp0tion of human serum albumin (HSA) and human plasma fibronectin (Fn) from solutions in the respective concentration ranges 1–20 μg/ml and 0.1–10 μg/ml by highly orientated pyrolytic graphite (HOPG) surfaces was investigated using atomic force microscopy. Adsorption from solutions of relatively low concentrations (HSA, 1 μg/ml; Fn, 0.1 μg/ml) allowed individual adsorbed protein molecules to be imaged.HSA appeared to take one of two forms (N or F) depending upon the adsorption energy and site, whilst Fn appeared to be adsorbed in the coiled, bent form. AFM analysis of films obtained by adsorption from solutions of higher protein concentrations, which were dried slowly over a, period of 24 h, indicated the formation of network structures with the surface coverage increasing with solution concentration. The precise structure, which was very similar for both proteins, appeared to result from dewetting of the hydrophobic HOPG by the protein solution. A grainy structure resulted when films were dried r...

Structural coefficients in aggregates of protein-coated colloidal particles

The structural coefficients of bare and protein-coated polystyrene particles aggregating at different electrolyte concentrations were determined by light scattering techniques. The time dependence of the average diffusion coefficient could be fitted considering a simple form for the aggregation kernel. The fits allowed the structural coefficient to be determined. This parameter was then used to obtain the surface-to-surface separation between the monomers contained within the clusters. The results show that this distance is directly related to the size of the adsorbed protein molecules.

Equilibrium and Dynamic Characteristics of Protein Adsorption Layers at Gas-Liquid Interfaces: Theoretical and Experimental Data

Within the framework of a nonideal two-dimensional solution model, equations are derived for the state of a surface layer, adsorption isotherms, and the distribution function of adsorbed protein molecules with respect to their states characterized by different molar surface areas. The derived equations satisfactorily describe the known experimental dependences obtained for equilibrium adsorption layers of some proteins (serum albumin, β-casein, and β-lactoglobulin): the dependences of the surface pressure on concentration and adsorption, the surface layer thickness on adsorption, and the limiting high-frequency elastic modulus of an adsorption layer on the surface pressure. All dependences for a given protein are described by the same set of parameters of the theoretical model. It is shown that the kinetics of protein adsorption studied by dynamic tensiometry, ellipsometry, and the radiotracer technique is consistent with the diffusion model comprising the Ward-Tordai equation and the set of equations describing the equilibrium. The kinetics of protein desorption from the adsorption layer at a liquid-fluid interface is analyzed. The kinetics of β-lactoglobulin desorption is shown to be described by the barrier mechanism.

Study of protein adsorption on polymer coatings surface by combining quartz crystal microbalance with electrochemical impedance methods

Understanding protein adsorption at electrode surface is important for biosensor development, in particular for piezoelectric quartz crystal (PQC) microbalance (QCM) sensor. Two proteins, bovine serum albumin (BSA) and fibrinogen were monitored in situ for their electrode-surface processes using both QCM and electrochemical impedance method. Conductive polymer films, which are based on de-doping polyaniline (PAN), ions-doped PAN (PAN + ClO 4 −) or sodium dodecyl sulfate (SDS) doped PAN (PAN + SDS) were prepared by electrochemical methods. The effect of the polymer film thickness and doping agents of PAN on protein adsorption were investigated. The maximum adsorptions of proteins onto PAN, PAN + ClO 4 − and PAN + SDS films were measured, with results showing 0.71, 0.65 and 0.99 μg/cm 2 for BSA and 2.73, 1.85 and 4.07 μg/cm 2 for fibrinogen, respectively. The results suggest an end-on type adsorption of BSA to produce a mono-layered structure onto all three PAN films. As for fibrinogen, only the ClO 4 −-doped PAN film was adsorbed in an end-on type mono-layered structure. For the adsorption of fibrinogen on the other two PAN films, the adsorbed protein molecules formed multi-layered structure. The adsorption of proteins onto the PAN films was found to follow the Langmuir isotherm. Results from adsorption kinetics studies showed two consecutive steps in the adsorption of BSA and fibrinogen on PAN films. BSA and fibrinogen molecules were firstly adsorbed onto the films before undergone rearrangement. The adsorption equilibrium constants were 1.64 × 10 6, 9.51 × 10 5 and 2.42 × 10 6 for BSA adsorption and 2.74 × 10 8, 2. 43 × 10 8 and 2.52 × 10 8 for fibrinogen adsorption onto ClO 4 −-doped PAN, de-doping PAN and SDS-doped PAN films, respectively.

Capillary isoelectric focusing--reproducibility and protein adsorption.

Capillary isoelectric focusing (CIEF) is an important tool for the quality assurance of biotechnologically maintained drugs and for proteome analysis. The critical performance parameters of this technique are the precisions of isoelectric point (pI) values and peak areas. Compared to capillary zone electrophoresis (CZE), where precise results can be obtained (e.g., 0.5% relative standard deviation (RSD) for peak areas, n = 60), only few data are available for CIEF experiments. So far, reproducible data of pI values (RSD = 0.5%) have been acquired, but peak areas show inferior results (about 3-15% RSD). Nonstable capillary coatings and protein adsorption have been discussed as possible reasons. Recent work of Righetti et al. [25, 27] has proven that the use of coated capillaries can reduce the adsorption of proteins by 50% but cannot prevent it. In our CIEF experiments irregular and poorly reproducible peak patterns have been observed. In a long-time experiment of 106 repeated runs, an overall RSD of 10% was obtained for peak areas, RSD of 2% only in series of about 10 consecutive replicates. Especially at higher concentrations the reproducibility deteriorates. This seems to be the result of a self-amplifying process, induced by adsorbed protein molecules, leading to further agglomerations. CZE control experiments in linear polyacrylamide (LPA)-coated capillaries proved a strong pH dependency of these effects within a small range. Compared to bare fused-silica surfaces, adsorption effects are reduced but not inhibited. An enhancement of reproducibility in CIEF experiments can be achieved only by controlling the interactions of proteins and capillary walls.

Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme.

Adsorption of chicken egg lysozyme on silica nanoparticles of various diameters has been studied. Special attention has been paid to the effect of nanoparticle size on the structure and function of the adsorbed protein molecules. Both adsorption patterns and protein structure and function are strongly dependent on the size of the nanoparticles. Formation of molecular complexes is observed for adsorption onto 4-nm silica. True adsorptive behavior is evident on 20- and 100-nm particles, with the former resulting in monolayer adsorption and the latter yielding multilayer adsorption. A decrease in the solution pH results in a decrease in lysozyme adsorption. A change of protein structure upon adsorption is observed, as characterized by a loss in alpha-helix content, and this is strongly dependent on the size of the nanoparticle and the solution pH. Generally, greater loss of alpha helicity was observed for the lysozyme adsorbed onto larger nanoparticles under otherwise similar conditions. The activity of lysozyme adsorbed onto silica nanoparticles is lower than that of the free protein, and the fraction of activity lost correlates well with the decrease in alpha-helix content. These results indicate that the size of the nanoparticle, perhaps because of the contributions of surface curvature, influences adsorbed protein structure and function.

Interpretation of Sum Frequency Generation Vibrational Spectra of Interfacial Proteins by the Thin Film Model

To interpret sum frequency generation (SFG) vibrational spectra collected from adsorbed protein molecules using structures of the interfacial proteins, the thin film model has been evaluated both theoretically and experimentally. The calculations indicate that nonlocal contributions to SFG spectra of interfacial proteins usually can be ignored. The experimental results indicate that for some polarization combinations, such as ssp and sps, local field variations across the adsorbed protein layer will not substantially affect SFG signals. Therefore, SFG spectra collected from adsorbed protein molecules are generally dominated by the electric-dipole contribution. From such spectra, quantitative structural or conformation information about interfacial proteins can be deduced using the thin film model. As an example, we analyzed the orientation distribution of the methyl groups of BSA molecules at different interfaces.

Competitive adsorption from mixed nonionic surfactant/protein solutions

A thermodynamic model is derived which is suitable to describe adsorption from a mixed protein/surfactant solution. The comparison with experimental data for HSA mixed with the nonionic surfactant decyl dimethyl phosphine oxide shows good agreement. Some model calculations are discussed in terms of the competitive character of the process of adsorption from mixed protein/surfactant solutions. The behavior of globular (HSA) and flexible ( β-casein) proteins appears to be quite different due to the possibility of changing the molar area of adsorbed protein molecules.

Protein adsorption: Kinetics and history dependence

The adsorption of protein and other macromolecular species is often strongly history dependent owing to the slow relaxation of nonequilibrium structures. The rate at which molecules adsorb is exquisitely sensitive to, and thus a sensitive measure of, interfacial structure; adsorption kinetics may therefore serve both to identify and to quantify history dependence. Optical waveguide lightmode spectroscopy (OWLS) in multistep mode, where an adsorbing surface is alternately exposed to a protein solution and one free of protein, enables the comparison of adsorption rates onto interfacial layers of identical composition but different formation histories. Contributions to the overall kinetics from the apparent adsorption rate constant, the desorption rate constants of molecules in different adsorbed states, and the interfacial one-body cavity function (a measure of the probability of a “cavity” on the surface free of protein) may be isolated from OWLS kinetic data. We find the rate of adsorption and the corresponding cavity function to increase significantly—for systems of fibronectin, cytochrome c, and lysozyme adsorbing onto Si(Ti)O 2—from a first to a subsequent adsorption step and attribute this to a clustering transition among adsorbed protein molecules. From the kinetic data, we determine important structural and temporal information on this event.

Coverage-Dependent Orientation of Lysozyme Adsorbed on Silica

We use total internal reflection fluorescence (TIRF), streaming current, and optical reflectometry measurements, under a variety of solution conditions, to examine the mechanism of the chicken egg lysozyme reorientation on silica surfaces, originally proposed by Robeson and Tilton (Robeson, J. L.; Tilton, R. D. Langmuir 1996, 12, 6104). The TIRF data suggest that a two-stage reorientation occurs in the lysozyme layer during adsorption. The first stage involves a reorientation to optimize lysozyme interaction with the charged surface and to reduce lateral repulsions between adsorbed protein molecules. This stage occurs when the adsorbed layer reaches a critical surface coverage and indicates the importance of lateral interactions between adsorbed proteins. Moreover, the reorientation rate depends on both the bulk protein concentration and the fluid wall shear rate, indicating that incoming protein molecules from the bulk solution also participate in the reorientation. The preferred orientation puts the act...

Measurement of mechanical properties and adsorption of protein molecules

Measurement of mechanical properties and adsorption of protein molecules

Novel resonant-frequency sensor to detect the kinetics of protein adsorption

Proteins prefer interfaces, and in aqueous solutions they rapidly adsorb to available solid–liquid interfaces. The adsorption process often involves a change in protein conformation at the surface that can result in functional inactivation of the protein. These changes in protein conformation, which are thought to lead to the formation of an entangled gel-like layer of denatured protein, are responsible for a number of deliterious processes, including biofouling on contact lenses and medical implants. The adsorption process is generally irreversible; dilution of protein in the solution phase does not result in protein desorption from the solid. Presumably, this is due to the effects of the protein denaturation and entanglement process on the rate constant for desorption. Nonspecific protein adsorption to solid–liquid interfaces is, therefore, a kinetically controlled process. Hence, measuring and understanding the kinetics of protein adsorption to solid surfaces, including the kinetics of protein conformational changes, is of considerable interest. We have developed a sensor that responds to protein adsorption kinetics and also, we believe, is sensitive to protein conformational changes during adsorption. The device is operated by monitoring the change in resonant frequency of an elastomeric film (25 μm thick), as an aqueous protein solution is exposed to the surface. Since the mass of a monolayer of the protein or other adsorbent is an extremely small fraction of the mass of the film, the observed change in resonant frequency is due almost entirely to changes in the surface tension of the film. Upon exposing the elastomeric film to a protein solution, we observe a continuing change in resonant frequency for more than 24 h, which is well beyond the time it would take for the population of proteins on the surface to equilibrate under diffusion-limited kinetics. This prolonged response is likely due to the surface energy changes of the sensor as the adsorbed protein molecules change their conformation. We present a description of the basic operation of this sensor as well as some examples of its response to bulk protein and surfactant concentrations.

Adsorption and surface tension of fibrinogen at the air/water interface

Static and dynamic surface tensiometry, ellipsometry, and infrared reflection absorption spectroscopy (IRRAS) were used to probe adsorption of bovine (I-S) fibrinogen (FB) at the air/water interface. Tensiometry data were obtained at 25, 37, or 60 °C and at concentrations from 7.5 to 750 ppm, in water, saline, or phosphate/saline buffer (PBS). The steady state tension decreased with concentration up to 75 ppm and then remained nearly constant at about 50±2 mN m−1. The dynamic surface tension at pulsating area conditions, at 20 or 80 rpm (cycles per min), showed interesting and complex patterns, but tension at minimum area of 38 mN m−1 or higher. Thus, if FB covers the surface, it can inhibit an essential function of effective lung surfactants, which must produce equilibrium tensions between 20 and 30 mN m−1 and minimum tensions below 10 mN m−1. Ellipsometry at 633 nm revealed that the adsorbed layers had refractive indices from 1.35 to 1.39, indicating a high water content, as expected from the non-compact molecular structure of the protein, which has dimensions 5×5×46 nm. The surface layer thicknesses ranged from 14 to 46 nm, and the surface densities ranged from 3 to 16 mg m−2, indicating that if the surface layer is a monolayer, then its average orientation increases with increasing FB concentration. The thicknesses and surface densities were slightly smaller in a PBS solution than in water, with the difference increasing with increasing concentration of FB. This can be attributed to the higher stability, or weaker intermolecular interactions, of the protein molecules in the PBS solution than in water. Unlike ellipsometry and IRRAS, which probe the whole surface layer, tensiometry provides no reliable measures of adsorption densities, probably because only a small portion of the adsorbed protein molecule affects the surface tension.

Formation of Protein–Birnessite Complex: XRD, FTIR, and AFM Analysis

Limited information is available on formation chemistry of enzyme–Mn oxide complexes. Adsorption isotherm of protein molecules (tyrosinase) on birnessite (δ-MnO2) at pH 6.0 and room temperature (23°C) was of H type, indicating a very high affinity of the enzyme protein molecules to the birnessite mineral surfaces. After thorough washing of the protein–mineral complex with deionized-distilled water, up to 89% of adsorbed protein molecules remained bound to the mineral surfaces. When a high amount of the protein was immobilized, the X-ray diffractogram shows a significant decrease in the intensity of characteristic d-spacings of birnessite. No shift to higher values of the d-spacings of protein–birnessite complex was observed, indicating that the enzyme molecules were not intercalated in the mineral structure but immobilized at the external surfaces and the edges of the mineral oxide. By comparison to the free enzyme, infrared absorption spectra of the protein–birnessite complexes show a shift by up to 11 cm−1 to lower frequencies in the absorption bands characteristic of amide I and II modes of the polypeptides chains. The mineral surfaces exerted some strain on the protein structure, resulting in an alteration of the protein molecular conformation after binding to the mineral colloid surfaces. In the free state, the globular protein molecules had a spheroid shape with an average cross-sectional diameter of 70±6 nm. The unfolding and flattening of the protein molecules after immobilization is clearly shown in atomic force micrographs. Compared to the tyrosinase–birnessite complex, similar FTIR spectra and atomic force micrographs were observed for the pure protein, bovine serum albumin (BSA), after immobilization on birnessite. The information obtained in this study is of fundamental significance for understanding birnessite as an adsorbent of biopolymers and the catalytic role of the enzyme–birnessite complex.

Sequential adsorption of bacteriophage T4 lysozyme stability variants at solid–water interfaces

The sequential adsorption of the wild type T4 lysozyme and one of its structural stability variants was studied, using ellipsometry and 125I radioisotope labeling techniques. The mutant lysozyme was produced by substitution of the isoleucine residue at position 3 in the wild type with a tryptophan residue, resulting in a protein with lower structural stability. The mutant protein was more resistant to surfactant-mediated elution, and apparently adsorbed at the interfaces with a greater interfacial area/molecule than the wild typeT4 lysozyme. However, the results of each type of experiment suggested that sequential adsorption and exchange of proteins occurred only in the case of the less stable mutant followed by the wild type. This suggests that, in these exchange reactions, properties of the adsorbing protein (e.g. its ability to adsorb when a relatively small amount of unoccupied area is present) were more important than the apparent binding strength of the adsorbed protein molecules.

Dynamical Behavior of Human Serum Albumin Adsorbed on or Embedded in Polyelectrolyte Multilayers

We investigate the lateral diffusion of Human Serum Albumin−Fluorescein Isothiocyanate (HSA) adsorbed “on” or embedded “in” poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) multilayers. Special attention is brought to the evolution of the diffusion coefficient with the surface HSA concentration. We find that while on PSS terminating films the diffusion coefficient of adsorbed HSA is independent of the protein surface concentration in the explored range, it decreases strongly with the surface concentration when HSA is adsorbed on PAH ending films. On both films, the mobile fraction of adsorbed protein molecules decreases when the surface concentration increases. At low surface coverage, up to 90% of the adsorbed protein molecules are mobile both on PSS and PAH terminating films. The decrease of the mobile fraction with the HSA surface concentration is more pronounced on PSS than on PAH reaching, respectively, 50% and 70% of mobile HSA molecules at high surface coverage. This behavio...

Characterization of Ultraflat Titanium Oxide Surfaces

In this work we investigated the physical and chemical nature of ultraflat titanium dioxide (TiO2) samples which we had previously used as substrates for the investigation of adsorbed protein molecules (Cacciafesta, P.; Humphris, A. D. L.; Jandt, K. D.; Miles, M. J. Langmuir 2000, 16, 8167). Titanium films were prepared by thermal evaporation on a heated mica surface and either separated from the mica to investigate the resulting surface or left in contact with the mica to analyze the titanium−mica interface. Atomic force microscopy (AFM) of the surfaces exposed after removal of the mica showed very flat surfaces [root-mean-square roughness of 0.29 nm ± 0.03 nm] and the presence of disordered as well as ordered structures. Different degrees of order were observed, such as a square lattice with spacing of a0 = 0.49 nm, a regular pattern with spacings of a0 = 0.47 nm and b0 = 0.35 nm with an angle of ∼80° between a0 and b0, or the presence of surface defects. These ordered structures were not stable upon long-term AFM imaging and could be damaged by the scanning tip. To further investigate the nature of the ordered regions, X-ray diffraction (XRD) was performed for the titanium−mica samples. XRD showed the crystalline structures of both the mica and the titanium film but did not detect any order in the titanium−mica interface. Possible causes for the formation of the ordered regions are discussed, in the cases of formation both after removal of mica or during sample preparation. The chemical nature of the titanium−mica interface was investigated with secondary ion mass spectrometry and X-ray photoelectron spectroscopy (XPS) via depth profiling of the titanium−mica samples from the titanium side. The metal film was found to be mainly composed of titanium and to a lesser extent of oxygen and carbon. After the metal film was sputtered and the titanium−mica interface was reached, the titanium concentration decreased and the concentration of the typical mica elements, such as silicon, aluminum, and potassium, increased. XPS was also used to investigate the chemical composition of the surfaces obtained after mica removal, which were found to be mainly composed of TiO2 with a small percentage of Ti2O3. The possible applications of this simple method for preparing ultraflat TiO2 surfaces are discussed.