Interaction strength between proteins and polyelectrolyte brushes: a small angle X-ray scattering study

We present an investigation of β-lactoglobulin adsorption onto spherical polyelectrolyte brushes (SPBs) by small angle X-ray scattering (SAXS). The SPB consists of a polystyrene core onto which long chains of poly(styrene sulfonate) are grafted. The amount and the distribution of proteins adsorbed in the brush layer at low ionic strength can be derived from SAXS. The analysis of the SAXS data reveals additionally that some of the protein molecules form aggregates of about six monomers in the adsorbed state. Furthermore, the position and the amount of slightly bound protein can be detected by the combination of the SAXS results and the SPB loading after extensive ultrafiltration. The total amount of adsorbed protein is compared to data derived from isothermal titration calorimetry. The comparison of both sets of data demonstrates that the protein molecules in the inner layers of the spherical polyelectrolyte brush are firmly bound. Proteins located in the outer layers are only weakly bound and can be washed out by prolonged ultrafiltration.     

Controllable immobilization of proteins in quenched spherical polyelectrolyte brushes as observed by fluorescence spectroscopy and small angle X-ray scattering

The immobilization of lysozymes (pI = 11) onto anionic spherical polyelectrolyte brushes (SPB) which consist of a solid polystyrene core and a densely grafted poly(styrene sulfonate) (PSS) shell was systematically studied by fluorescence spectroscopy and small angle X-ray scattering. Results show that the capture of lysozyme by PSS brush is a dynamic process, which involves a quick agglomeration stage and a slow rearrangement one. And lysozyme inclines to immobilize in the inner layer of the brush, and saturation of lysozyme adsorption onto the SPB is gradually reached as the protein concentration increases, proceeding from the inside to the outside of the brush layers. As increasing the pH and ionic strength, the lysozyme previously adsorbed will be partially released and migrate from the inner to the outer layer of SPB. Last competitive adsorption tests between lysozyme and BSA or β-glucosidase were performed, indicating that besides electrostatic interaction counterion release force also plays an important role in protein adsorption. SPB was proved to be ideal candidate for controllable immobilization of protein, which can be extended into various applications, such as drug delivery and protein separation. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 1577–1588     

Interaction of dissolved proteins with spherical polyelectrolyte brushes

We consider the adsorption of bovine serum albumin (BSA) on spherical polyelectrolyte brushes (SPB). The SPB consist of a solid polystyrene core of 100nm diameter onto which linear polyelectrolyte chains (poly(acrylic acid), (PAA)) are grafted. The adsorption of BSA is studied at a pH of 6.1 at different concentrations of added salt and buffer (MES). We observe strong adsorption of BSA onto the SPB despite the effect that the particles as well as the dissolved BSA are charged negatively. The adsorption of BSA is strongest at low salt concentration and decreases drastically with increasing amounts of added salt. The adsorbed protein can be washed out again by raising the ionic strength. The various driving forces for the adsorption are discussed. It is demonstrated that the main driving force is located in the electrostatic interaction of the protein with the brush layer of the particles. All data show that the SPB present a new class of carrier particles whose interaction with proteins can be tuned in a well-defined manner.     

Binding of oppositely charged surfactants to spherical polyelectrolyte brushes: a study by cryogenic transmission electron microscopy

The formation of a complex between an anionic spherical polyelectrolyte brush (SPB) and the cationic surfactant cetyltrimethylammonium bromide (CTAB) is investigated. The SPB consists of long chains of the strong polyelectrolyte poly(styrene sulfonate) (PSS), which are bound chemically to a solid poly(styrene) core of 56 nm in radius. The SPB are dispersed in water, and the ionic strength is adjusted by addition of NaBr. The resulting complexes are investigated in dilute solution by dynamic light scattering, by electrophoretic light scattering, and by cryogenic transmission electron microscopy (cryo-TEM). The formation of the complex between the SPB and the surfactant can be monitored by a strong shrinking of the surface layer when adding CTAB to dilute suspensions (0.01 wt %) and by a decrease of the effective charge of the complexes. Complex formation starts at CTAB concentrations lower than the critical micelle concentration of this surfactant. If the ratio r of the charges on the SPB to the charge of the added surfactant is exceeding unity, the particles start to flocculate. Cryo-TEM images of the complexes at r = 0.6 measured in salt-free solution show that the surface layer composed of the PSS chains and the adsorbed CTAB molecules is partially collapsed: A part of the chains form a dense surface layer while another part of the chains or aggregates thereof are still sticking out. This can be deduced from the cryo-TEM micrographs as well as from the hydrodynamic radius, which is still of appreciable magnitude. The 1:1 complex (r = 1.0) exhibits a fully collapsed layer formed by the PSS chains and CTAB. If the complex is formed in the presence of 0.05 M NaBr, r = 0.6 leads to globular structures directly attached to the surface of the core particles. All structures seen in the cryo-TEM images can be explained by a collapse transition of the surface layer brought about by the hydrophobic attraction between the polyelectrolyte chains that became partially hydrophobic through adsorption of CTAB.     

Structure and protein binding capacity of a planar PAA brush

We performed neutron reflectometry (NR) and total internal reflection fluorescence (TIRF) spectroscopy to characterize the structure and the protein binding capacity of a planar poly(acrylic acid) (PAA) brush at different temperatures. A PAA brush was prepared by spin-coating planar quartz or silicon wafers with a thin film of poly(styrene). Then, the diblock copolymer poly(styrene)-poly(acrylic acid) was deposited on these modified wafers using the Langmuir-Sch?fer or Langmuir-Blodgett technique. PAA grafting densities of about 0.1 chains per nm2 were obtained. The NR experiments indicate a remarkable swelling of the PAA brush in contact with a buffer solution, when it is heated to 40 degrees C for several hours. The swollen brush structure remains upon cooling back to 20 degrees C suggesting a disentanglement of the initially formed PAA brush by the temporary heating. At pD = 6.7, the protein bovine serum albumin (BSA) with a negative net charge is strongly adsorbed to the swollen PAA brush. From the scattering length density profiles obtained from the NR curves, an almost homogeneous filling of the whole PAA brush space with BSA molecules can be deduced corresponding to an average BSA volume fraction of about 7-10% and an adsorbed protein mass of about 1.4 mg m-2. By analyzing the TIRF experiments, it is found that BSA adsorption is enhanced when increasing the temperature which represents an evidence for an entropic driving force for protein adsorption. However, the mechanism of BSA adsorption at a PAA brush appears to be different at 20 and 40 degrees C.     

Selective uptake of different proteins by annealed and quenched cationic spherical polyelectrolyte brushes

The selective uptake of bovine serum albumin (BSA) and β-glucosidase (β-G) by annealed and quenched cationic spherical polyelectrolyte brushes (SPB) was systematically studied by combining turbidimetric titration, dynamic light scattering and small angle X-ray scattering (SAXS). These two kinds of SPB consist of a same polystyrene core and a dense shell of poly (2-aminoethyl methacrylate hydrochloride) (PAEMH) and poly [2-(methacryloyloxy) ethyl] trimethylammonium chloride (PMAETA), respectively. Results reveal that the adsorption/desorption of proteins on SPB can be easily controlled by changing external conditions (pH and ionic strength). For a particular annealed or quenched SPB, there is a significant difference of the interaction pH regions between the brush and the two proteins, and this difference can be tuned by ionic strength. At low ionic strength, quenched brushes were more suitable for selective adsorption of BSA and β-G, while annealed brushes performed better at high ionic strength. SAXS analysis demonstrated that volume exclusion effect played a remarkable role in protein uptake by both SPB, and larger proteins were more likely to be adsorbed on the outer layer of the brush. The unique core-shell structure and controllable chain types make SPB an excellent candidate in selective adsorption/separation of proteins of different sizes.     

The Adsorption-Desorption Cycle. Reversibility of the BSA-Silica System

The reversibility of the adsorption-desorption cycle was established by comparing the thermostability (determined by differential scanning calorimetry) and secondary structure (obtained by circular dichroism spectroscopy) of BSA before adsorption, adsorbed on, and exchanged from silica particles. Circular dichroism was also measured as a function of temperature at a given wavelength. Adsorbed BSA presents a higher thermostability and a lower alpha-helix content than the native protein while it regains its conformation when released from the surface back into the solution; the homomolecular exchange is reversible.The changes in ellipticity (at a given wavelength) as a function of the temperature show that the thermal denaturation of native, adsorbed, and exchanged BSA proceeds in two steps. For the dissolved protein, the first step up to 50 degrees C involves a slight change in the structure while in the 50-90 degrees C temperature range the actual unfolding takes place. For the adsorbed BSA, the first step proceeds up to 60 degrees C and includes some intermolecular association between the adsorbed protein molecules, which may be responsible for the increased thermostability. The unfolding occurs in the 60-90 degrees C range; it is less cooperative and involves a lower enthalpy change than the native protein. Adsorbed BSA presents the same secondary structure as that observed for dissolved BSA that has passed a heating-cooling cycle. Copyright 2001 Academic Press.     

Polyelectrolyte complex layers: a promising concept for anti-fouling coatings verified by in-situ ATR-FTIR spectroscopy

In-situ attenuated total reflection (ATR)-FTIR spectroscopy enabled studies on the interaction between the differently charged model proteins human serum albumin, lysozyme, immunoglobulin G and multilayer assemblies, which were deposited by alternating adsorption of poly(ethyleneimine) and poly(acrylic acid) onto Si crystals. Low adsorbed protein amounts were observed if the top polyelectrolyte layer and the protein were equally charged, whereas enhanced protein adsorption occurred for electrostatic attraction between protein and top polyelectrolyte layer.     

Manipulation of the adsorption of ionic surfactants onto hydrophilic silica using polyelectrolytes

This paper demonstrates the use of polyelectrolytes to modify and manipulate the adsorption of ionic surfactants onto the hydrophilic surface of silica. We have demonstrated that the cationic polyelectrolyte poly(dimethyl diallylammonium chloride), poly-dmdaac, modifies the adsorption of cationic and anionic surfactants to the hydrophilic surface of silica. A thin robust polymer layer is adsorbed from a dilute polymer/surfactant solution. The resulting surface layer is cationic and changes the relative affinity of the cationic surfactant hexadecyl trimethylammonium bromide, C16TAB, and the anionic surfactant sodium dodecyl sulfate, SDS, to adsorb. The adsorption of C16TAB is dramatically reduced. In contrast, strong adsorption of SDS was observed, in situations where SDS would normally have a low affinity for the surface of silica. We have further shown that subsequent adsorption of the anionic polyelectrolyte sodium poly(styrene sulfonate), Na-PSS, onto the poly-dmdaac coated surface results in a change back to an anionic surface and a further change in the relative affinities of the cationic and anionic surfactants for the surface. The relative amounts of C16TAB and SDS adsorption depend on the coverage of the polyelectrolyte, and these preliminary measurements show that this can be manipulated.     

Salt-regulated attraction and repulsion of spherical polyelectrolyte brushes towards polyelectrolyte multilayers

Adsorption of colloidal particles presents an interesting alternative to the modification of surfaces using covalent coupling or physisorption of molecules. However, to tailor the properties of these materials full control over the effective particle-substrate interactions is required. We present a systematic investigation of the adsorption of spherical polyelectrolyte brushes (SPB) onto polyelectrolyte multilayers (PEM). A brush layer grafted from colloidal particles allows the incorporation of various functional moieties as well as the precise adjustment of their adsorption behaviour. In the presence of oppositely charged surfaces the amount of adsorbed SPB monotonically increases with the ionic strength, whereas equally charged substrates efficiently prevent colloidal attachment below a threshold salt concentration. We found that the transition from the osmotic to the salted brush regime at approximately 100 mM coincided with a complete loss of substrate selectivity. In this regime of high ionic strength, attractive secondary interactions become dominant over electrosteric repulsion. Due to the soft polyelectrolyte corona a surface coverage exceeding the theoretical jamming limit could be realized. Both the adsorption kinetics and the resulting thin film morphologies are discussed. Our study opens avenues for the production of two-dimensional arrays and three-dimensional multilayered structures of SPB particles.     

Adsorption of poly(vinyl formamide-co-vinyl amine) onto silica particle surfaces and stability of the formed hybrid materials

In order to produce silica/polyelectrolyte hybrid materials the adsorption of the polyelectrolyte poly(vinyl formamide-co-vinyl amine), P(VFA-co-VAm) was investigated. The adsorption of the P(VFA-co-VAm) from an aqueous solution onto silica surface is strongly influenced by the pH value and ionic strength of the aqueous solution, as well as the concentration of polyelectrolyte. The adsorption of the positively charged P(VFA-co-VAm) molecules on the negatively charged silica particles offers a way to control the surface charge properties of the formed hybrid material. Changes in surface charges during the polyelectrolyte adsorption were studied by potentiometric titration and electrokinetic measurements. X-ray photoelectron spectroscopy (XPS) was employed to obtain information about the amount of the adsorbed polyelectrolyte and its chemical structure. The stability of the adsorbed P(VFA-co-VAm) was investigated by extraction experiments and streaming potential measurements. It was shown, that polyelectrolyte layer is instable in an acidic environment. At a low pH value a high number of amino groups are protonated that increases the solubility of the polyelectrolyte chains. The solvatation process is able to overcompensate the attractive electrostatic forces fixing the polyelectrolyte molecules on the substrate material surface. Hence, the polyelectrolyte layer partially undergoes dissolving process.     

Structure and dynamics of alpha-lactalbumin adsorbed at a charged brush interface

We have studied the adsorption of alpha-lactalbumin at a planar poly(acrylic acid) (PAA) brush using neutron reflectometry (NR) and total internal reflection fluorescence (TIRF) spectroscopy. The PAA brush has been prepared by spin-coating silicon or quartz plates with a hydrophobic poly(styrene) film and by transferring the copolymer poly(styrene)-poly(acrylic acid) onto the modified surface. In the case of NR, the poly(styrene) film and the poly(styrene) chain ends of the copolymer were perdeuterated in order to generate a high contrast to the non-deuterated PAA brush. alpha-Lactalbumin was chosen as the model protein because it is a relatively small globular protein with a negative net charge at neutral pH-values, as chosen in the experiments. Thus, it is interacting with the PAA brush on the 'wrong' side of its isoelectric point. In addition, the effects of temperature on the volume fraction profile and the reorientational mobility of the protein within the PAA brush were determined. From the analysis of the NR data, it has been found that despite of its negative net charge, alpha-lactalbumin is penetrating into the PAA brush. Its volume fraction profile parallels that of the PAA brush, indicating an exclusive interaction between the protein and the PAA. No protein accumulation is found at the inner poly(styrene) or the outer solution interface of the PAA brush. When increasing the temperature from 20 to 40 degrees C, less protein is adsorbed, suggesting the presence of enthalpic interaction contributions. From the analysis of the TIRF data, a high degree of reorientational mobility of alpha-lactalbumin within a PAA brush can be inferred. The reorientational correlation time of alpha-lactalbumin labeled with the Alexa Fluor 488 dye was found to increase from 5.5 to 32 ns upon adsorption, which can well be explained by the higher viscosity inside the PAA brush. Overall, the results of this study quantify for the first time the molecular details of the unique interaction of a protein on the 'wrong' side of its isoelectric point with a planar charged brush interface. It is concluded that the high mobility of alpha-lactalbumin within a PAA brush can partially be understood by the presence of repulsive electrostatic interactions. There is no 'freezing' of the protein dynamics, which is a precondition for biological activity.     

A modified box model including charge regulation for protein adsorption in a spherical polyelectrolyte brush

Recent experiments showed significant adsorption of bovine serum albumin (BSA) in spherical polyelectrolyte brushes (SPB) consisting of polyacrylic acid, even for pH values above the isoelectric point of the protein, when both protein and polyion are negatively charged. To describe these experimental findings theoretically, we have constructed a spherical box model for an annealed brush consisting of a weak polyelectrolyte that includes the adsorption of BSA. At equilibrium the chemical potential of BSA in solution equals that at each location in the brush, while the net force on the polyions (including osmotic, stretching, and excluded volume terms) is zero at each location. Protein adsorption is predicted above the isoelectric point and--in agreement with experimental data--is a strong function of ionic strength and pH. Adsorption of protein in the brush is possible because the pH in the brush is below the isoelectric point and protein reverses its charge from negative to positive when it adsorbs.     

Dynamic properties of soy globulin adsorbed films at the air-water interface

In this paper we present surface dilatational properties of soy globulins (beta-conglycinin, glycinin, and reduced glycinin with 10 mM of dithiothreitol (DTT)) adsorbed onto the air-water interface, as a function of adsorption time. The experiments were performed at constant temperature (20 degrees C), pH (8.0), and ionic strength (0.05 M). The surface rheological parameters were measured as a function of protein concentration (ranging from 1 to 1x10(-3)% wt/wt). We found that the surface dilatational modulus, E, increases, and the phase angle, phi, decreases with time, theta, which may be associated with protein adsorption. These phenomena have been related to protein adsorption, unfolding, and/or protein-protein interactions (at long-term adsorption) as a function of protein concentration in solution. From a rheological point of view, the surface viscoelastic characteristics of soy globulin films adsorbed at the air-water interface are practically elastic. The main conclusion is that the dilatational properties of the adsorbed films depend on the molecular structure of the protein.     

Cytochrome c unfolding on an anionic surface

It is now well accepted that the adsorption of proteins to solid supports sometimes involves surface-mediated unfolding. A detailed understanding of the adsorption and surface-mediated unfolding process is lacking. We selected a well studied protein, horse heart cytochrome c, and a weakly ionic support to examine some of the characteristics of protein adsorption under near-physiological conditions. We used high-performance liquid chromatography (HPLC) to investigate the effect of temperature on surface-mediated unfolding. Samples of cytochrome c were introduced to an anionic support, and a NaCl gradient was used to desorb the protein at different times and temperatures. The profiles and retention times were monitored to examine the adhesive properties of cytochrome c to the anionic support. We found that protein retention increased with time at temperatures as low as 0 degrees C, and a significant loss of cytochrome c occurred between 55 degrees C and 70 degrees C. The loss of recovery of cytochrome c indicates irreversible surface-mediated unfolding. The changes in retention time may indicate more subtle transitions, including reversible surface-mediated unfolding of cytochrome c. These results suggest that perturbations in the structure as well as unfolding of cytochrome c can be detected at a lower temperature on an anionic surface than in solution thereby acting like a catalyst for protein unfolding.     

Adsorption and structural change of beta-lactoglobulin at the diacylglycerol-water interface

Diacylglycerol (DAG)/water and triacylglycerol (TAG)/water emulsions were prepared using beta-lactoglobulin (beta-LG) as an emulsifier. The oil phase (20% in emulsion) was mixed with beta-LG solution (1% beta-LG in water, pH 7) to prepare the emulsions. A fine oil-in-water emulsion was produced from both DAG and TAG oils. The interfacial protein concentration of the TAG emulsion was higher than that of the DAG emulsion. The zeta potential of the DAG oil droplet was higher than that of the TAG oil droplet. The front-surface fluorescence spectroscopy results revealed that tryptophan residues in beta-LG moved to the more hydrophobic environment during the adsorption of protein on the oil droplet surfaces. Changes in secondary structure of beta-LG during the adsorption were determined by FT-IR spectroscopy. Decreases in the beta-sheet content concomitant with increases in the alpha-helix content were observed during the adsorption to the oil droplets, and the degree of structural change was greater for beta-LG in the TAG emulsion than in the DAG emulsion, indicating the increased unfolding of adsorbed beta-LG on the TAG oil droplet surface. Results of interfacial tension measurement supported this speculation, that is, the increased unfolding of the protein at the TAG-water interface. Trypsin- and proteinase K-catalyzed proteolysis was used to probe the topography of the adsorbed beta-LG on the oil droplet surface. SDS-PAGE analyses of liberated peptides after the proteolysis indicated the higher susceptibility of beta-LG adsorbed on the DAG oil droplet surface than on the TAG oil droplet surface. On the basis of all the results, we discussed the conformation of the adsorbed beta-LG on the two oil droplet surfaces.     

Polyelectrolyte-mediated protein adsorption: fluorescent protein binding to individual polyelectrolyte nanospheres

We have used confocal fluorescence microscopy with single molecule sensitivity to characterize uptake and release of fluorescent protein (mEosFP) molecules by individual spherical polyelectrolyte brush (SPB) nanoparticles that were immobilized on a glass surface. The SPB particles consisted of a solid core particle of 100 nm diameter onto which long polyelectrolyte chains were affixed. They could be loaded with up to 30 000 mEosFP molecules in a solvent of low ionic strength. The concentration dependence of protein loading can be described with a simple bimolecular binding model, characterized by an equilibrium dissociation coefficient of 0.5 microM. Essentially complete release of the bound proteins was observed after increasing the ionic strength by adding 250 mM NaCl to the solvent. Fluorescence emission spectra and time-resolved fluorescence intensity decays were measured on individual, mEosFP-loaded SPB nanoparticles, and also on the dissolved mEosFP before and after adsorption. These results indicate that the mEosFP molecules remained structurally intact in this procedure. Hence, the present investigation demonstrates unambiguously that polyelectrolyte-mediated protein adsorption onto SPB particles presents a viable process for protein immobilization.     

Surface potential of spherical polyelectrolyte brushes in the presence of trivalent counterions

We consider the ζ-potential and the effective charge of spherical polyelectrolyte brushes (SPBs) in aqueous solution in the presence of trivalent europium ions. The SPB consists of a polystyrene core of ca. 250 nm diameter onto which long chains of the strong polyelectrolyte poly(styrene sulfonate) are grafted (contour length: 82 nm). At low concentration of EuCl₃ the chains are stretched to nearly full length. If the concentration of the trivalent ions is raised, the surface layer of the polyelectrolyte chains collapses. The ζ-potential of the SPB is calculated from the electrophoretic mobilities measured at different concentrations of EuCl₃. At the collapse, ζ decreases by the partial neutralization of the charges by the trivalent ions. The experimental ζ-potential thus obtained agrees with the theoretical surface potential Ψ_theo calculated for the effective shear plane by a variational free energy model of the SPB.     

Polyelectrolyte blend multilayer films: surface morphology, wettability, and protein adsorption characteristics

We report the influence of polyelectrolyte (PE) multilayer films prepared from poly(styrene sulfonate)-poly(acrylic acid) (PSS-PAA) blends, deposited in alternation with poly(allylamine hydrochloride) (PAH), on film wettability and the adsorption behavior of the protein immunoglobulin G (IgG). Variations in the chemical composition of the PAH/(PSS-PAA) multilayer films, controlled by the PSS/PAA blend ratio in the dipping solutions, were used to systematically control film thickness, surface morphology, surface wettability, and IgG adsorption. Spectroscopic ellipsometry measurements indicate that increasing the PSS content in the blend solutions results in a systematic decrease in film thickness. Increasing the PSS content in the blend solutions also leads to a reduction in film surface roughness (as measured by atomic force microscopy), with a corresponding increase in surface hydrophobicity. Advancing contact angles (theta) range from 7 degrees for PAH/PAA films through to 53 degrees for PAH/PSS films. X-ray photoelectron spectroscopy measurements indicate that the increase in film hydrophobicity is due to an increase in PSS concentration at the film surface. In addition, the influence of added electrolyte in the PE solutions was investigated. Adsorption from PE solutions containing added salt favors PSS adsorption and results in more hydrophobic films. The amount of IgG adsorbed on the multilayer films systematically increased on films assembled from blends with increasing PSS content, suggesting strong interactions between PSS in the multilayer films and IgG. Hence, multilayer films prepared from blended PE solutions can be used to tune film thickness and composition, as well as wetting and protein adsorption characteristics.     

On the Use of the Hypothesis of Statistically Homogeneous Suspensions in the Calculation of Their Conductivity

Electrostatic effects on protein adsorption were investigated using differential scanning calorimetry (DSC) and adsorption isotherms. The thermal denaturation of lysozyme, ribonuclease A (RNase), and alpha-lactalbumin in solution and adsorbed onto silica nanoparticles was examined at three concentrations of cations: 10 and 100 mM of sodium and 100 mM of sodium to which 10 mM of calcium was added. The parameters investigated were the denaturation enthalpy (DeltaH), the temperature at which the denaturation transition was half-completed (T(m)), and the temperature range of the denaturation transition. For lysozyme and RNase, adsorption isotherms depend strongly on the ionic strength. At low ionic strength both proteins have a high affinity for the silica particles and adsorption is accompanied by a 15-25% reduction in DeltaH and a 3-6 degrees C decrease in T(m), indicating that the adsorbed state of the proteins is destabilized. Also, an increase in the width of the denaturation transition is observed, signifying a larger conformational heterogeneity of the surface bound proteins. At higher ionic strengths, both with and without the addition of calcium, no significant adsorption-induced alteration in DeltaH was observed for all three proteins. The addition of calcium, however, decreases the width of the denaturation transition for lysozyme and RNase in the adsorbed state. Copyright 2001 Academic Press.