Expulsion of bovine serum albumin from the air/water interface by a sparingly soluble lecithin lipid

Dynamic surface tensiometry, ellipsometry, and infrared reflection-absorption spectroscopy (IRRAS) were used to study the dynamic adsorption and surface tensions of dilauroylphosphatidylcholine (DLPC) in the presence of bovine serum albumin (BSA). Results show that the equilibrium adsorbed layers consist mostly of DLPC, which can produce dynamic surface tensions (1 mN/m) as low as the more successful lung surfactant replacement formulations. When the aqueous surface expands and contracts sinusoidally, BSA can coadsorb and lead to slightly higher dynamic surface tensions than when DLPC is alone. Similar results were obtained with BSA and sodium myristate [McClellan and Franses, Colloids Surf. B 30 (2003) 1]. Expulsion of the BSA in the layer by DLPC can take from 5 to 15 min, depending on relative concentrations and history of solute addition. This is shown by tensiometry measurements on mixtures, and also by injecting aqueous DLPC underneath adsorbed BSA layers and probing the surface layer with ellipsometry and IRRAS. Albumin layers from buffer solutions aged up to 30 h can be expelled by DLPC. In pure water, there is an initial enhancement in protein adsorption after the DLPC is injected. This can be explained by the hypothesis that DLPC molecules bind with BSA molecules to form a hydrophobic lipoprotein complex, which is more hydrophobic than the protein itself. Since DLPC produces lower surface energy than BSA and--being slightly soluble--adsorbs to the surface by a molecular mechanism, it fulfills the thermodynamic and dynamic requirements for expelling the BSA from the surface. The results have implications for minimizing lung surfactant inhibition by serum proteins, as it occurs in the cases of adult or acute respiratory distress syndrome.     

Structural Conformation of Bovine Serum Albumin Layers at the Air-Water Interface Studied by Neutron Reflection

The adsorption of bovine serum albumin (BSA) at the air-water interface has been studied by specular neutron reflection. The variation of the adsorbed amount and the total thickness of the BSA layer with respect to bulk BSA concentration was determined at pH 5, close to its isoelectric point (IP). While the surface excess showed a steady increase with bulk concentration the thickness of the protein layer was found to be close to the short axial length of 40 ? of the globular solution structure of BSA at concentrations below 0.1 g dm-3, suggesting that BSA molecules adsorb with their long axes parallel to the surface of water. At 1 g dm-3 the adsorbed layer can be modeled as an upper layer of 40 ? with a volume fraction of 0.4 and a sublayer of 30 ? underneath the top main layer with a volume fraction of 0.12. The results suggest that, although there is some structural deformation accompanying adsorption, there is no denaturation. The extent of immersion of the BSA in water was determined by performing the measurements in D2O and in a mixture of H2O and D2O whose contrast matches that of BSA. The signal is then only from the part of the layer out of water. At pH 5 this layer was about 10 +/- 5 ? at a bulk concentration of 5 x 10(-4) g dm-3 and decreased to 5 +/- 3 ? at 1 g dm-3. The fraction of the BSA layer immersed in water therefore varies from about 70 to over 90%. The effect of pH on the adsorption was examined at two BSA concentrations. While pH had little effect on the adsorption at a low BSA concentration of 5 x 10(-3) g dm-3, both surface excess and layer thickness showed pronounced peaks at pH 5 at the higher concentration of 1 g dm-3. The increased adsorption at pH 5 is attributed to the reduced lateral electrostatic repulsion around the IP. This adsorption pattern became less pronounced when the total ionic strength was increased from 0.02 to 1 M, indicating that the electrolyte screens the electrostatic repulsions within the adsorbed layer. Copyright 1999 Academic Press.     

Adsorption and direct probing of fibrinogen and sodium myristate at the air/water interface

Fibrinogen (FB), a serum protein, is considered a major inhibitor of lung surfactant function at the lining layer of the alveoli. In this study, the adsorption of aqueous bovine FB at the air/water interface was investigated with tensiometry and directly probed for the first time with ellipsometry and infrared reflection adsorption spectroscopy (IRRAS). The tension results show that FB has moderate surface activity. The surface densities of FB were calculated by using two different ellipsometry models to range from 3±0.2 to 17±2 mg/m², for 7.5 to 750 ppm of FB in water at 25°C. Although FB at concentrations from 75 to 750 ppm reached about the same steady surface tension value, the surface densities at 750 ppm FB were substantially larger. The same techniques were used for studying aqueous mixtures of 7.5 to 750 ppm FB with 2 mM of sodium myristate (SM) to investigate a possible interaction of the SM with the protein. The behavior of the FB/SM mixtures was found to be close to that of SM alone. The surface tension of the FB/SM mixtures reached values less than 10 mN/m under surface area oscillation at 20 or 80 rpm. These results and the ellipsometry and the IRRAS results indicate that at a concentration of 2 mM SM, FB, up to 750 ppm, does not inhibit the surfactant surface-tension-lowering function. In certain cases the results demonstrate that FB and SM may act cooperatively in lowering the surface tension.     

Bovine serum albumin adsorption on nano-rough platinum surfaces studied by QCM-D

The adsorption of bovine serum albumin (BSA) on platinum surfaces with a root-mean-square roughness ranging from 1.49nm to 4.62nm was investigated using quartz crystal microbalance with dissipation (QCM-D). Two different BSA concentrations, 50microg/ml and 1mg/ml, were used, and the adsorption studies were complemented by monitoring the antibody interaction with the adsorbed BSA layer. The adsorption process was significantly influenced by the surface nano-roughness, and it was observed that the surface mass density of the adsorbed BSA layer is enhanced in a non-trivial way with the surface roughness. From a close examination of the energy dissipation vs. frequency shift plot obtained by the QCM-D technique, it was additionally observed that the BSA adsorption on the roughest surface is subject to several distinct adsorption phases revealing the presence of structural changes facilitated by the nano-rough surface morphology during the adsorption process. These changes were in particular noticeable for the adsorption at the low (50microg/ml) BSA concentration. The results confirm that the nano-rough surface morphology has a significant influence on both the BSA mass uptake and the functionality of the resulting protein layer.     

Efficiency of blocking of non-specific interaction of different proteins by BSA adsorbed on hydrophobic and hydrophilic surfaces

The efficiency of a pre-absorbed bovine serum albumin (BSA) layer in blocking the non-specific adsorption of different proteins on hydrophobic and hydrophilic surfaces was evaluated qualitatively and quantitatively using infrared reflection spectroscopy supported by spectral simulations. A BSA layer with a surface coverage of 35% of a close-packed monolayer exhibited a blocking efficiency of 90–100% on a hydrophobic and 68–100% on a hydrophilic surface, with respect to the non-specific adsorption of concanavalin A (Con A), immunoglobulin G (IgG), and staphylococcal protein A (SpA). This BSA layer was produced using a solution concentration of 1 mg/mL and 30 min incubation time. BSA layers that were adsorbed at conditions commonly employed for blocking (a 12 h incubation time and a solution concentration of 10 mg/mL) exhibited a blocking activity that involved competitive adsorption–desorption. This activity resulted from the formation of BSA–phosphate surface complexes, which correlated with the conformation of adsorbed BSA molecules that was favourable for blocking. The importance of optimisation of the adsorbed BSA layer for different surfaces and proteins to achieve efficient blocking was addressed in this study.     

Langmuir and Gibbs magnetite NP layers at the air/water interface

The interfacial properties of Fe(3)O(4)@MEO(2)MA(90)-co-OEGMA(10) NPs, recently developed and described as promising nanotools for biomedical applications, have been investigated at the air/water interface. These Fe(3)O(4) NPs, capped with catechol-terminated random copolymer brushes of 2-(2-methoxyethoxy) ethyl methacrylate (MEO(2)MA) and oligo(ethylene glycol) methacrylate (OEGMA), with molar fractions of 90% and 10%, respectively, proved to be surface active. Surface tension measurements of aqueous dispersions of the NPs showed that the adsorption of the NPs at the air/water interface is time- and concentration-dependent. These NPs do not behave as classical amphiphiles. Once adsorbed at the air/water interface, they do not exchange with NPs in bulk, but they are trapped at the interface. This means that all NPs from the bulk adsorb to the interface until reaching maximum coverage of the interface, which corresponds to values between 6 × 10(-4) and 8 × 10(-4) mg/cm(2) and a critical equilibrium surface tension of ～47 mN/m. Moreover, Langmuir layers of Fe(3)O(4)@MEO(2)MA(90)-co-OEGMA(10) NPs have been investigated by measuring surface pressure-area compression-expansion isotherms and in situ X-ray fluorescence spectra. The compression-expansion isotherms showed a plateau region above a critical surface pressure of ～25 mN/m and a pronounced hysteresis. By using a special one-barrier Langmuir trough equipped with two surface pressure microbalances, we have shown that the NPs are squeezed out from the interface into the aqueous subphase, and they readsorb on the other side of the barrier. The results have been supported by TEM as well as AFM experiments of transferred Langmuir-Schaefer films on solid supports. This study shows the ability of Fe(3)O(4)@MEO(2)MA(90)-co-OEGMA(10) NPs to transfer from hydrophilic media (an aqueous solution) to the hydrophobic/hydrophilic interface (air/water interface) and back to the hydrophilic media. This behavior is very promising, opening studies of their ability to cross biological membranes.     

Adsorption of bovine serum albumin at the air/water interface and its effect on the formation of DPPC surface film

The adsorption of bovine serum albumin (BSA) at the air/water interface and its effect on the transport of dipalmitoylphosphatidylcholine (DPPC) to form a surface film were studied with tensiometry, infrared reflection absorption spectroscopy (IRRAS), and ellipsometry. For 1, 10, 100, and 1000 ppm BSA solutions, the steady-state tension ranges from 55 to 50 mN m⁻¹. At pulsating area (at 20 cycles min⁻¹), both the minimum and maximum tensions decrease with increasing bulk concentration. Even though the steady-state tension is similar for 100 and 1000 ppm BSA, IRRAS and ellipsometry results indicate that the adsorbed density is higher for 1000 ppm BSA. For 1000 ppm/1000 ppm BSA/DPPC mixture, the tension behavior was found to be similar to that of 1000 ppm BSA when alone. Results from IRRAS and ellipsometry also demonstrate that BSA is the dominant adsorbed component at the air/water interface. Thus, at 1000 ppm, by adsorbing fast and possibly irreversibly, BSA interferes with the transport and adsorption of DPPC and inhibits its ability to lower the surface tension. However, when DPPC is introduced via a spread monolayer mechanism, DPPC expels partly or completely the adsorbed BSA monolayer and then controls the tension behavior with little or no inhibition by BSA. Thus, the competitive adsorption of DPPC and BSA depends strongly on the path or mechanism of introducing DPPC to the surface and involves path-dependent nonequilibrium adsorption phenomena.     

The reorientation of poly(2-dimethylamino ethyl methacrylate) after environment stimuli improves hydrophilicity and resistance of protein adsorption

There is a dense poly(2-dimethylamino ethyl methacrylate) (PDMAEMA) layer on the surface of poly(acrylonitrile and 2-dimethylamino ethyl methacrylate) (PAN-DMAEMA) membrane. The contact angle measurement indicated that the hydrophilicity of membrane increases at higher pH value and ionic strength. The reorientation of PDMAEMA after environment stimuli results in further enrichment of ester groups on the membrane surface according to XPS analysis. The amount of adsorbed bovine serum albumin (BSA) on PAN-DMAEMA membrane is dramatically decreased at higher pH value and ionic strength. A viewpoint based on the minimizing electrostatic interactions between PDMAEMA groups after environment stimuli leading to the conformation switch of PDMAEMA chains from stretched to shrunk states, which results in higher surface enrichment of ester groups enhancing hydrophilic property of the PAN-DMAEMA membrane, was put forward to explain the resistance of protein adsorption on the membrane.     

Adsorption of bovine serum albumin at solid/aqueous interfaces

Adsorption of soluble serum proteins on hydrophilic and hydrophobic solid surfaces is important for biomaterials and chromatographic separations of proteins. The adsorption of bovine serum albumin (BSA) from aqueous solutions was studied with in situ ATR-IR spectroscopy, and with ex situ ATR-IR, ellipsometry, and water wettablity measurements. The results were used to quantitatively determine the adsorbed film thickness and surface density of BSA on hydrophilic silicon oxide/silicon surfaces, and on these surfaces covered with a hydrophobic lipid monolayer of dipalmitoylphosphatidylcholine (DPPC). The water contact angles were 5° for silicon oxide, 47° ± 1° for the DDPC monolayer, and 53° ± 1° for the BSA monolayers. At 25 °C, and with 0.01–1 wt% BSA in water, the surface densities range from Γ = 2.6–5.0 mg/m², and the film thicknesses range from d = 2.0–3.8 nm, on the assumption that the film is as dense as bulk protein. These results, and certain changes in the IR amide I and II bands of the protein, indicate that the protein adsorbs as a side-on monolayer, with some flattening due to unfolding or denaturation. The estimated -helical content for protein in buffer solutions is 15% higher than for solutions in water. The adsorption density reaches a steady-state value within 10 min for the lowest concentration, but does not appear to reach a steady-state value after 3 h f‘or the higher concentrations. Adsorption of BSA on a silicon oxide surface covered with a monolayer of DPPC leads to an adsorbed protein film of about half the thickness and surface density than on silicon oxide, but the same contact angle, indicating more protein unfolding on the hydrophobic than on the hydrophilic surface.     

The effect of surface coverage on conformation changes of bovine serum albumin molecules at the air-solution interface detected by sum frequency generation vibrational spectroscopy

The air-BSA solution interface has been investigated by various techniques for years. From these studies we know that BSA molecules segregate at the BSA solution-air interface, and the surface coverage increases with the increase of the bulk solution concentration. However, questions still remain as to whether the protein changes conformation, orientation, or a combination of the two upon adsorption. In this paper, by using sum frequency generation (SFG) vibrational spectroscopy we found that the conformation of interfacial BSA molecules changes dramatically at the solution-air interface, compared to that of the native BSA in solution. The hydrophobic methyl groups of BSA molecules at this interface tend to align along the surface normal. The degree of such conformational changes of surface BSA molecules depend on the surface coverage, indicating that the protein-protein interaction plays a very important role in determining the conformation of interfacial protein molecules. At very low surface concentration, the adsorbed BSA molecules unfold substantially. Our results can provide a molecular interpretation of results obtained from other studies such as protein layer thickness and surface tension measurements of protein solution.     

Modes of conformational changes of proteins adsorbed on a planar hydrophobic polymer surface reflecting their adsorption behaviors

Infrared spectra of hen egg white lysozyme and bovine serum albumin (BSA) adsorbed on a solid poly tris(trimethylsiloxy)silylstyrene (pTSS) surface in D2O solution were measured using attenuated total reflection (ATR) Fourier transform infrared spectroscopy. From the area and shape of the amide I' band of each spectrum, the adsorption amount and the secondary structure were determined simultaneously, as a function of adsorption time. We could show that the average conformation for all the adsorbed lysozyme molecules was solely determined by the adsorption time, and independent of the bulk concentration, while the adsorption amount increased with the bulk concentration as well as the adsorption time. These results suggest that lysozyme molecules form discrete assemblies on the surface, and that the surface assemblies grow over several hours to have a definite architecture independent of the adsorption amount. As for BSA, the extent of the conformational change was solely determined by the adsorption amount, regardless of the bulk concentration and the adsorption time. These differences in the adsorption properties of lysozyme and BSA may reflect differences in their conformational stabilities.     

Adsorption-desorption mechanism of phosphate by immobilized nano-sized magnetite layer: interface and bulk interactions

Phosphate adsorption mechanism by a homogenous porous layer of nano-sized magnetite particles immobilized onto granular activated carbon (nFe-GAC) was studied for both interface and bulk structures. X-ray Photoelectron Spectroscopy (XPS) analysis revealed phosphate bonding to the nFe-GAC predominantly through bidentate surface complexes. It was established that phosphate was adsorbed to the magnetite surface mainly via ligand exchange mechanism. Initially, phosphate was adsorbed by the active sites on the magnetite surface, after which it diffused into the interior of the nano-magnetite layer, as indicated by intraparticle diffusion model. This diffusion process continues regardless of interface interactions, revealing some of the outer magnetite binding sites for further phosphate uptake. Desorption, using NaOH solution, was found to be predominantly a surface reaction, at which hydroxyl ions replace the adsorbed phosphate ions only at the surface outer biding sites. Five successive fix-bed adsorption/regeneration cycles were successfully applied, without significant reduction in the nFe-GAC adsorption capacity and at high regeneration efficiency.     

Characterization of bovine serum albumin adsorption on chromium and AISI 304 stainless steel, consequences for the Pseudomonas fragi K1 adhesion

Adsorption of BSA on the surface of chromium and 304 stainless steel, has been characterized by Contact Angle Measurements, X-ray Photoelectron Spectroscopy (XPS) and Infrared Reflection Absorption Spectroscopy (IRRAS). Bacterial adhesion has been tested and compared on these two materials before and after pre-conditioning the surface with BSA. Chromium and stainless steel surfaces, when covered by a natural oxide layer, exhibit different energetic characteristics as shown by their γ_s^- and γ_s^LW respective values. These data vary upon immersion in BSA solutions, tending towards common values for duration of immersions. After immersion in BSA solutions, the evolution of the N 1s XPS signal, specific for the BSA, suggests that the surface is nearly saturated in a few minutes. Longer times of immersion only lead to a re-ordering of the adsorbed layer. Immersion tests in dilute BSA solutions (0.01 g/l) enabled us to make clear a higher reactivity of chromium towards the protein compared to stainless steel. These differences are cancelled at higher BSA concentrations (1 g/l). IRRAS spectra of BSA adsorbed on the two substrates demonstrated the appearance of amide I and amide II bands with small shifts and intensity variations supporting orientation changes of the protein when the concentration or immersion time varies. A model for the building up of the BSA layer is proposed, which accounts for these data. Chromium and stainless steel surfaces, also have different behaviours towards adhesion of Pseudomonas fragi K1, whereas surfaces that are pre-conditioned by BSA behave in a similar way. The overall number of adherent bacteria is decreased on stainless steel, whereas it is hardly affected on chromium. On both surfaces, the fraction of viable cells is increased.     

The role of bentonite addition in UF flux enhancement mechanisms for oil/water emulsion

The main problem in treating oil/water emulsion from car wash waste-water by ultrafiltration (UF) is fouling caused by oil adsorption on the membrane surface and internal pore walls. This study demonstrates that the addition of bentonite clay can reduce the adsorption layer on cellulose acetate UF membrane, resulting in a reduction of total membrane resistance (R_t). Experiments were conducted to identify and describe three possible mechanisms: (i) bulk oil emulsion concentration reduction; (ii) particle aggregation and (iii) detachment of the adsorbed gel layer by shear force. Adsorption of oil emulsion by bentonite can lead to a significant reduction of bulk oil emulsion concentration, one of the major causes of flux enhancement. Results show that contact of oil emulsion with bentonite forms larger particles resulting in flux increment. An optimum particle size of 37 μm, corresponds with a bentonite concentration of 300 mg/l and provided the highest flux. Beyond this limiting concentration, flux improvement gradually declined, possibly due to the formation of packed cake of particles on the membrane surface. The presence of bentonite in the oil emulsion promotes high shear stress which acts against the gel layer. This high shear stress, caused by bentonite particles and cross-flow velocity, reverses the adsorbed gel layer to the bulk of the liquid phase.     

Noninteracting versus interacting poly(N-isopropylacrylamide)-surfactant mixtures at the air-water interface

Two polymer-surfactant mixtures have been studied at the air-water interface using neutron reflectivity and surface tension techniques. For the noninteracting system poly(N-isopropylacrylamide) (PNIPAM)/octaethyleneglycol mono n-decyl ether (C10E8), the adsorption behavior is competitive and driven purely by surface pressure (pi). When pi(polymer) > pi(surfactant), the surface layer consists of almost pure polymer, and for pi(polymer) < pi(surfactant), the polymer is displaced from the surface by the increasing pressure of the surfactant. Beyond the CMC, the polymer is completely displaced from the surface. For the interacting system PNIPAM/sodium dodecyl sulfate (SDS) where the two species interact strongly in the bulk beyond the critical aggregation concentration (CAC), the surface behavior is more original. Earlier neutron reflectivity studies investigated PNIPAM adsorption behavior where the SDS was contrast-matched to the solvent. In the present study, complementary measurements of SDS adsorption where PNIPAM is contrast-matched to the solvent give a complete view of the surface composition of the mixed system. At a constant polymer concentration, with increasing SDS, three main regimes are obtained. For C(SDS) < CAC, adsorption is governed by simple competition and PNIPAM is predominant at the interface. At intermediate SDS concentration (CAC < C(SDS) < x2, where x2 indicates the predominance of free SDS micelles), interfacial behavior is governed by bulk polymer-surfactant interaction. Adsorbed polymer is displaced from the interface to form PNIPAM-SDS complex in the bulk. SDS adsorption remains weak since most of the SDS molecules are used to form bulk polymer-surfactant aggregates. Further increase in SDS concentration results in continued displacement of PNIPAM and an abrupt increase in SDS adsorption. This is a result of saturation of bulk polymer chain with adsorbed micelles. Interestingly, beyond x2, PNIPAM is not completely displaced from the surface. A mixed PNIPAM-SDS adsorbed layer with enhanced packing of the SDS monolayer is formed.     

Thermal Analysis of Adsorbed Poly(methyl methacrylate) on Silica

Modulated differential scanning calorimetry has been used to quantify the glass transitions of small adsorbed amounts of poly(methyl methacrylate) (PMMA) on silica. While a relatively narrow, single glass transition was found for bulk PMMA, broader two-component transitions were found for the adsorbed polymer. A two-state model based on loosely bound polymer (glass transition similar to bulk) and more tightly bound polymer (glass transition centered around 156 degrees C) was used to interpret the thermograms. On the basis of this model, the amount of tightly bound polymer was found to be approximately 1.3 mg/m2, corresponding to a 1.1 nm thick layer. The change in heat capacity for the tightly bound polymer at the glass transition temperature was estimated to be about 16% of that of the bulk polymer.     

Probing protein adsorption onto mercaptoundecanoic acid stabilized gold nanoparticles and surfaces by quartz crystal microbalance and zeta-potential measurements

The adsorption characteristics of three proteins [bovine serum albumin (BSA), myoglobin (Mb), and cytochrome c (CytC)] onto self-assembled monolayers of mercaptoundecanoic acid (MUA) on both gold nanoparticles (AuNP) and gold surfaces (Au) are described. The combination of quartz crystal microbalance measurements with dissipation (QCM-D) and pH titrations of the zeta-potential provide information on layer structure, surface coverage, and potential. All three proteins formed adsorption layers consisting of an irreversibly adsorbed fraction and a reversibly adsorbed fraction. BSA showed the highest affinity for the MUA/Au, forming an irreversibly adsorbed rigid monolayer with a side-down orientation and packing close to that expected in the jamming limit. In addition, BSA showed a large change in the adsorbed mass due to reversibly bound protein. The data indicate that the irreversibly adsorbed fraction of CytC is a monolayer structure, whereas the irreversibly adsorbed Mb is present in form of a bilayer. The observation of stable BSA complexes on MUA/AuNPs at the isoelectric point by zeta-potential measurements demonstrates that BSA can sterically stabilize MUA/AuNP. On the other hand, MUA/AuNP coated with either Mb or CytC formed a reversible flocculated state at the isoelectric point. The colloidal stability differences may be correlated with weaker binding in the reversibly bound overlayer in the case of Mb and CytC as compared to BSA.     

Fibronectin and bovine serum albumin adsorption and conformational dynamics on inherently conducting polymers: a QCM-D study

Quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to characterize the adsorption of the model proteins, bovine serum albumin (BSA) and fibronectin (FN), to polypyrrole doped with dextran sulfate (PPy-DS) as a function of DS loading and surface roughness. BSA adsorption was greater on surfaces of increased roughness and was above what could be explained by the increase in surface area alone. Furthermore, the additional mass adsorbed on the rough films was concomitant with an increase in the rigidity of the protein layer. Analysis of the dynamic viscoelastic properties of the protein adlayer reveal BSA adsorption on the rough films occurs in two phases: (1) arrival and initial adsorption of protein to the polymer surface and (2) postadsorption molecular rearrangement to a more dehydrated and compact conformation that facilitates further recruitment of protein to the polymer interface, likely forming a multilayer. In contrast, FN adsorption was independent of surface roughness. However, films prepared from solutions containing the highest concentration of DS (20 mg/mL) demonstrated both an increase in adsorbed mass and adlayer viscoelasticity. This is attributed to the higher DS loading in the conducting polymer film resulting in presentation of a more hydrated molecular structure indicative of a more unfolded and bioactive conformation. Modulating the redox state of the PPy-DS polymers was shown to modify both the adsorbed mass and viscoelastic nature of FN adlayers. An oxidizing potential increased both the total adsorbed mass and the adlayer viscoelasticity. Our findings demonstrate that modification of polymer physicochemical and redox condition alters the nature of protein-polymer interaction, a process that may be exploited to tailor the bioactivity of protein through which interactions with cells and tissues may be controlled.     

Modulating surface rheology by electrostatic protein/polysaccharide interactions

There is a large interest in mixed protein/polysaccharide layers at air-water and oil-water interfaces because of their ability to stabilize foams and emulsions. Mixed protein/polysaccharide adsorbed layers at air-water interfaces can be prepared either by adsorption of soluble protein/polysaccharide complexes or by sequential adsorption of complexes or polysaccharides to a previously formed protein layer. Even though the final protein and polysaccharide bulk concentrations are the same, the behavior of the adsorbed layers can be very different, depending on the method of preparation. The surface shear modulus of a sequentially formed beta-lactoglobulin/pectin layer can be up to a factor of 6 higher than that of a layer made by simultaneous adsorption. Furthermore, the surface dilatational modulus and surface shear modulus strongly (up to factors of 2 and 7, respectively) depend on the bulk -lactoglobulin/pectin mixing ratio. On the basis of the surface rheological behavior, a mechanistic understanding of how the structure of the adsorbed layers depends on the protein/polysaccharide interaction in bulk solution, mixing ratio, ionic strength, and order of adsorption to the interface (simultaneous or sequential) is derived. Insight into the effect of protein/polysaccharide interactions on the properties of adsorbed layers provides a solid basis to modulate surface rheological behavior.     

Importance of physical vs. chemical interactions in surface shear rheology

The stability of adsorbed protein layers against deformation has in literature been attributed to the formation of a continuous gel-like network. This hypothesis is mostly based on measurements of the increase of the surface shear elasticity with time. For several proteins this increase has been attributed to the formation of intermolecular disulfide bridges between adsorbed proteins. However, according to an alternative model the shear elasticity results from the low mobility of the densely packed proteins. To contribute to this discussion, the actual role of disulfide bridges in interfacial layers is studied. Ovalbumin was thiolated with S-acetylmercaptosuccinic anhydride (S-AMSA), followed by removal of the acetylblock on the sulphur atom, resulting in respectively blocked (SX) and deblocked (SH) ovalbumin variants. This allows comparison of proteins with identical amino acid sequence and similar globular packing and charge distribution, but different chemical reactivity. The presence and reactivity of the introduced, deblocked sulfhydryl groups were confirmed using the sulfhydryl-disulfide exchange index (SEI). Despite the reactivity of the introduced sulfhydryl groups measured in solution, no increase in the surface shear elasticity could be detected with increasing reactivity. This indicates that physical rather than chemical interactions determine the surface shear behaviour. Further experiments were performed in bulk solution to study the conditions needed to induce covalent aggregate formation. From these studies it was found that mere concentration of proteins (to 200 mg/mL, equivalent to a surface concentration of around 2 mg/m(2)) is not sufficient to induce significant aggregation to form a continuous network. In view of these results, it was concluded that the adsorbed layer should not be considered a gelled network of aggregated material (in analogy with three-dimensional gels formed from heating protein solutions). Rather, it would appear that the adsorbed proteins form a highly packed system of proteins with net-repulsive interactions.