Tommy Nylander

Protein interactions at solid surfaces

Abstract In this review article we discuss a large number of the studies of interactions between protein-coated surfaces that has been presented in the literature. We also demonstrate how to relate surface force data to results from other techniques in order to provide a more full picture of protein behaviour at interfaces. One aim of the article is to discuss the experimental procedure and the difficulties with surface force measurements in protein systems. It is particularly important to point out how the sensitivity of this technique differs from that of other techniques, e.g. in determining structural changes in adsorbed proteins and in detecting proteins adsorbed on top of an inner firmly bound layer. It is also important to realize which surface force data cannot easily be compared with findings from other techniques (one example is the kinetics of adsorption and desorption). We have tried to group proteins into different classes depending on their size and structure, and to try to find results that are common within these classes. It was found that some observations for unordered proteins with amphiphilic character, and for the small compact proteins, appear consistently within the respective class. Hence, for these types of protein common features/principles of the interfacial behaviour are identified. The very large and flexible glycoproteins behave in a similar way to synthetic polymers, but we found it hard to draw any firm conclusions based on the surface force studies presented so far. Perhaps, the most complicated surface behaviour is observed for soft globular proteins that undergo large-scale conformational changes upon adsorption and when the layers are held under a high compressive force.

Addition of hydrophilic and lipophilic compounds of biological relevance to the monoolein/water system. I. Phase behavior.

The solubilization of hydrophilic and lipophilic molecules, with biological relevance, in the monoolein/water (MO/W) system has been investigated for phase behavior. Small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) and optical microscopy (OM) have been used to characterize the microstructure of the liquid crystalline phases. Partial phase diagrams of the MO/W system in the presence of sodium decanoate, 1-adamantanamine hydrochloride, decanoic and dodecanoic acids, acetyl salicilic acid and retinol have been determined. The stability of the various phases has been followed for at least eight months. The polarity and the molecular structure of the additive determine whether it is located at the polar interface or in the apolar region of the lipid layer. Therefore, the additive affects the interfacial curvature of the lipid layer differently, which in turn will trigger transition to disparate phases. A cubic-to-reverse hexagonal phase transition has been observed with time for most of the ternary systems, with the exception of 1-adamantanamine hydrochloride and retinol. The release of free glycerol and oleic acid due to MO hydrolysis has been clearly demonstrated by 13C NMR. This would account for the changes in phase behavior observed with time. The released oleic acid, located in the MO acyl chain region, favors the inverse interfacial curvature. The average lipid dimensions in the cubic and in the reverse hexagonal phases have been calculated from SAXS data.

Adsorption behaviour of amphiphilic polymers at hydrophobic surfaces: Effects on protein adsorption

The adsorption of four different amphiphilic polymers to a model surface has been studied, and the effects of the adsorbed amphiphiles on the subsequent adsorption of fibrinogen (Fg) and human serum albumin (HSA) at the surfaces were investigated. The amphiphilic polymers were one commercially available ABA block copolymer, Pluronic PE9400 (PE94), composed of poly(ethylene oxide) (A-blocks) and poly(propylene oxide) (B-block), and three graft copolymers, two with backbones of poly(styrene-co-acrylamide) (STY) and one with a backbone of poly(methyl methacrylate-co-ethylhexyl methacrylate) (ACRY). The backbones carried poly(ethylene oxide) (PEO) grafts. The model surface was a hydrophobic methylated silica surface (HMS). The amphiphilic polymers were adsorbed at the HMS surface from an ethanol/water solution. The adsorption process was monitored by ellipsometry. After rinsing with phosphate buffered saline (PBS), protein was added and the continued adsorption measured by ellipsometry. Surfaces modified by adsorption of the amphiphilic polymers were also characterized by contact angle measurements and X-ray photoelectron spectroscopy (XPS). According to these measurements the amphiphilic polymers adsorbed in significant amounts at the HMS surface. A limited study by atomic force microscopy (AFM), as well as the XPS measurements, suggests that both single molecules and micellar aggregates adsorb at the surface. ACRY and PE94 gave the highest levels of adsorption. As compared to the Pluronic block copolymer the graft copolymers were more strongly attached to the HMS surface, as shown by less desorption on rinsing with solvent. The ellipsometric results show that the adsorption of HSA and Fg at HMS surfaces containing preadsorbed amphiphilic polymer was significantly reduced as compared to the bare HMS surface. ACRY and PE94 showed the largest effects. Both polymers gave more than a 20-fold reduction of the Fg adsorption and a 10-fold reduction of the HSA adsorption. The STY polymers reduced the protein adsorption by a factor of 2-3.

Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes

Fengycin is a biologically active lipopeptide produced by several Bacillus subtilis strains. The lipopeptide is known to develop antifungal activity against filamentous fungi and to have hemolytic activity 40-fold lower than that of surfactin, another lipopeptide produced by B. subtilis. The aim of this work is to use complementary biophysical techniques to reveal the mechanism of membrane perturbation by fengycin. These include: 1), the Langmuir trough technique in combination with Brewster angle microscopy to study the lipopeptide penetration into monolayers; 2), ellipsometry to investigate the adsorption of fengycin onto supported lipid bilayers; 3), differential scanning calorimetry to determine the thermotropic properties of lipid bilayers in the presence of fengycin; and 4), cryogenic transmission electron microscopy, which provides information on the structural organization of the lipid/lipopeptide system. From these experiments, the mechanism of fengycin action appears to be based on a two-state transition controlled by the lipopeptide concentration. One state is the monomeric, not deeply anchored and nonperturbing lipopeptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. The mechanism, thus, appears to be driven mainly by the physicochemical properties of the lipopeptide, i.e., its amphiphilic character and affinity for lipid bilayers.

Electrochemical biosensors for glucose, lactate, urea, and creatinine based on enzymes entrapped in a cubic liquid crystalline phase

Abstract A novel method to construct enzyme-based biosensors, where the enzyme is entrapped in a lipid matrix (cubic liquid crystalline phase) is presented. The cubic phases were made of monoolein (1-monooleyl-glycerol) and 35% (w/w) of water-based enzyme solutions. The biocatalytic layers of the sensors consisted of a thin layer of the cubic phase with entrapped enzyme, which was coated with a dialysis membrane. The idea was used to construct and investigate the performance of amperometric β- d -glucose and l -lactate, and pH-sensitive urea and creatinine bioelectrodes based on glucose oxidase, lactate oxidase, urease, and creatinine deiminase. The amperometric enzyme electrodes generate an anodic biocatalytic current due to the oxidation of H 2 O 2 at the Pt electrode. Using the urease- and creatinine deiminase-based pH electrodes, proton consumption in the enzymatic reactions is measured. The data for the amperometric sensors are presented on the influence of substrate concentration, potential, pH, and temperature upon the electrodes, Urease- and creatinine deiminase-pH electrodes are tested in relation to the substrate and enzyme concentration, and the buffer capacity. Results of the electrodes long-term stability are discussed in relation to the structural features of the cubic phase and the enzyme.

Adsorption of α-lactalbumin and β-lactoglobulin on metal surfaces versus temperature

Abstract The adsorption of α-lactalbumin and β-lactoglobulin on a hydrophilic chromium surface was followed in situ, using ellipsometry. The experiments were performed at temperatures up to and exceeding (in the case of α-lactalbumin) the thermal denaturation temperatures of the proteins. The denaturation temperatures and the reversibility of the denaturation of the proteins were estimated by differential scanning calorimetry (DSC). The residual transition enthalpy for the thermal denaturation of β-lactoglobulin, as a function of preheat time at temperatures dose to the denaturation temperature, was also recorded. In agreement with earlier reports, the denaturation of β-lactoglobulin was found to be irreversible, whereas the denaturation of α-lactalbumin was highly reversible. When approaching its denaturation temperature, the adsorption behavior of β-lactoglobulin indicates that an aggregation occurs at the surface starting after a time lag of several minutes. No such behavior was found for α-lactalbumin, where both the initial and the plateau values of the adsorbed amount increased gradually as a function of temperature. Preheated solutions of β-lactoglobulin had nearly the same adsorption behavior at 25°C as the native protein, while the thermally denatured form of α-lactalbumin seemed to be more surface active than the native.

Sequential and competitive adsorption of β-lactoglobulin and κ-casein on metal surfaces☆

The adsorbed amount of K-casein and β-lactoglobulin on metal surfaces was measured in situ by ellipsometry as a function of time. The measurements were made in phosphate-buffered solutions. Glass slides with vacuum-deposited chromium, treated in different ways to obtain hydrophilic as well as hydrophobic surfaces, were used. The effect of a monolayer of one protein on the subsequent adsorption of the other protein was studied, and competitive adsorption of the two proteins was followed using ellipsometry and 14C-labeled β-lactoglobulin. The results of the sequential adsorption indicate that κ-casein adsorbs after the plateau value of adsorption of β-lactoglobulin has been reached, whereas no adsorption of β-lactoglobulin takes place after the plateau value of κ-casein has been reached. When the two proteins were added simultaneously the surface energy of the substrate influences both the total adsorbed amount and the composition of the adsorbed layer.

A study of entrapped enzyme stability and substrate diffusion in a monoglyceride-based cubic liquid crystalline phase

Our recent results have shown that enzymes with molecular weights of up to 590 kDa can be entrapped in cubic liquid crystalline phases in lipid/aqueous systems. In the present study, both pure monoolein and monoolein/phosphatidylcholine mixtures were used for the preparation of the cubic phases. Electrochemical measurements of the enzyme activity show that the entrapment in the cubic phase is liable to stabilise the enzyme. The interactions between protein molecules and a periodically curved lipid bilayer in these systems still remain to be elucidated. However, our data show that the composition of the lipid might influence the stability of the enzyme, that is the introduction of the zwitterionic phosphatidylcholine leads to an increase in the long-term stability of glucose oxidase. This can probably be assigned both to the differences in the polar interface of the lipid bilayer and the changes in structure of the cubic phase. The properties of biosensors constructed from cubic phases containing glucose oxidase and ceruloplasmin were compared. Both enzymes have about the same molecular weight, but different electrochemical reactions were used for monitoring the enzyme activity. We have also studied the diffusion of a substrate molecule, glucose, in the cubic phase by means of holographic laser interferometry, nuclear magnetic resonance (NMR) and chronoamperometry to obtain more information on the cubic phase as a support for enzyme immobilisation.

Binding of Sodium Dodecyl Sulphate and Dodecyl Trimethyl Ammonium Chloride to β-Lactoglobulin: A Calorimetric Study

Abstract Thermally induced unfolding of β -lactoglobulin in the presence of surfactants was studied by differential scanning calorimetry (DSC). Anionic surfactants, sodium dodecyl sulphate (SDS), and cationics, dodecyl trimethyl ammonium chloride (DOTAC), were used. In a solution containing a 1:1 molar ratio of SDS and β -lactoglobulin, the protein was stabilised as evidenced by a substantial increase in the unfolding temperature. Further increase of the SDS concentration causes unfolding of the protein. As opposed to these effects of the anionic surfactant, a slight decrease in the unfolding temperature was observed in the presence of DOTAC under similar conditions. An increase of the DOTAC/ β -lactoglobulin molar ratios above 1:1 causes precipitation of the protein. The cationic surfactant could be fairly easily removed from a mixed β -lactoglobulin/DOTAC solution by dialysis. The anionic surfactant appeared to interact more strongly with the protein and the 1:1 molar interaction with SDS was impossible to separate by dialysis. The experimental findings are discussed in terms of possible binding sites for the surfactant and connected to micelle formation of the surfactants which is related in a predictable scheme to temperature and ionic strength effects.

The behaviour of protein preparations from blue-green algae (Spirulina platensis strain Pacifica) at the air/water interface

Abstract The surface tension of a protein sample isolated from the blue-green algae (cyanobacteria) Spirulina platensis strain Pacifica was studied using the Wilhelmy plate method. The isolated material was characterised by determining the protein and lipid content, SDS-PAGE electrophoresis, isoelectric focusing, and visible spectroscopy. The protein is capable of reducing the interfacial tension at the aqueous/air interface already at relatively lower bulk concentrations compared to common food proteins. The surface tension of the protein preparation seems to be quite independent of pH, which indicates that electrostatic interactions are of minor importance for the interfacial behaviour. We have also separated out fractions with different interfacial properties by centrifugation. When the protein was spread at the air/aqueous interface, the pressure area isotherm somewhat resembles those recorded for lipids, with a higher collapse pressure than usually observed for proteins. The interfacial behaviour of extracted lipids confirms that remaining traces of lipids in protein powder have only a minor influence on the surface activity of Spirulina protein. The surface-active components are likely to be protein and/or protein-pigment complexes rather than individual protein molecules.