Spreading of oil from protein stabilised emulsions at air/water interfaces

Spreading of a drop of an emulsion made with milk proteins on air/water interfaces was studied. From an unheated emulsion, all oil molecules could spread onto the air/water interface, indicating that the protein layers around the oil globules in the emulsion droplet were not coherent enough to withstand the forces involved in spreading. Heat treatment (90 °C) of emulsions made with whey protein concentrate (WPC) or skim milk powder reduced the spreadability, probably because polymerisation of whey protein at the oil/water interface increased the coherence of the protein layer. Heat treatment of emulsions made with WPC and monoglycerides did not reduce spreadability, presumably because the presence of the monoglycerides at the oil/water interface prevented a substantial increase of coherence of the protein layer. Heat treatment of caseinate-stabilised emulsions had no effect on the spreadability. If proteins were already present at the air/water interface, oil did not spread if the surface tension (γ) was <60 mN/m. We introduced a new method to measure the rate at which oil molecules spread from the oil globules in the emulsion droplet by monitoring changes in γ at various positions in a ‘trough’. The spreading rates observed for the various systems agree very well with the values predicted by the theory. Spreading from oil globules in a drop of emulsion was faster than spreading from a single oil drop, possibly due to the greater surface tension gradient between the oil globule and the air/water interface or to the increased oil surface area. Heat treatment of an emulsion made with WPC did not affect the spreading rate. The method was not suitable for measuring the spreading rate at interfaces where surface active material is already present, because changes in γ then were caused by compression of the interfacial layer rather than by the spreading oil.     

Characterization of the emulsification properties of 2S albumins from sunflower seed

The ability of 2S albumins from sunflower seeds to stabilize oil-in-water emulsions has been investigated, demonstrating that one of the proteins (SFA8) effectively stabilizes emulsions, while another (SF-LTP) does not stabilize emulsions. The surface tension and surface dilation viscosity of these two proteins were measured, rationalizing the emulsifying ability of SFA8 in terms of its ability to form a strongly elastic monolayer at interfaces. The secondary structure changes that occur upon adsorption of SFA8 to the oil/water interface have also been studied by fluorescence, circular dichroism (CD), and Fourier-transform infrared (FT-IR) spectroscopy. It was found that the beta-sheet content of the protein increased upon adsorption at the expense of alpha-helix and random structure. Moreover, FT-IR measurements indicate the presence of intermolecular beta-sheet formation upon adsorption. Fluorescence studies with an oil-soluble fluorescence quencher indicate that the single tryptophan residue present in SFA8 may become located in the oil-phase of the emulsion. This residue is thought to be partially buried in the native protein, and these data suggest that changes in the polypeptide region flanking this residue may play an important role in the molecular rearrangement that occur on or following adsorption to the oil/water interface.     

Langmuir and Langmuir-Blodgett films of proline-rich N-terminal domain peptide of gamma-zein

The proline-rich N-Terminal domain peptides of γ-zein (VHLPPP)_n with n = 1 and 3 (peptides I and II) are shown to form stable Langmuir films at air/water interface and the films have been characterized using surface pressure–molecular area (π–A), surface potential–molecular area (ΔV–A) isotherms, respectively. The longer peptide sequence does not show dramatic increase in surface or interfacial properties suggesting that the minimum length of n = 1 is sufficient to achieve the necessary surface properties. Brewster angle micrographs also agreed with these results. The high surface-active nature of the peptide suggests a fairly non-polar character at air/water interface and at solid/air interface when coated expresses a high surface energy.

Additives such as isopropyl alcohol (IPA) and polyvinyl alcohol (PVA) with the peptides showed more homogenous films at the air/water interface and also improved mechanical and tensile properties. The organized assembly of peptide I at the air/water and solid/air interface suggests that even thin layer of the peptide could play an important role in coating the inner surface of protein body membrane in storage proteins. Composite films of such short peptides with biocompatible polymers may find applications as surface coatings and in biomaterials.     

High-performance capillary electrophoresis for characterization of hapten—protein conjugates used for production of antibodies against soyasaponin I

Micellar electrokinetic capillary chromatography using sodium cholate as the micellar phase has been investigated for characterization of hapten—protein conjugates. Special focus has been placed on the hapten soyasaponin I which is a quantitatively dominating glycoside in seeds of several legumes including pea (Pisum sativum L.) and soybean [Glycine max (L.) Merr.]. Soyasaponin I has been isolated from pea and used as hapten for production of anti-saponin specific polyclonal antibodies. Soyasaponin I was coupled to Kunitz soybean trypsin inhibitor (KSTI) and bovine serum albumin. The degree of coupling was determined by high-performance capillary electrophoresis (HPCE). Capillaries dynamically coated with zwitterions were found to be efficient for reduction of interaction between the silica capillary surface and the proteins. The applicability of HPCE for determination of coupling density was confirmed by investigation of a model hapten (p-nitrophenyl-- -galactoside; PNPG) coupled to KSTI. The PNPG—KSTI conjugates were examined by both HPCE and by spectrophotometric determination of the PNPG density on KSTI. The HPCE method was shown to be efficient in studies of the formation of hapten—protein conjugates and to be more specific than alternative techniques applied for determination of coupling densities.     

Protein folding at emulsion oil/water interfaces

It has long been known that proteins change their conformation upon adsorption to emulsion oil/water interfaces. However, it is only recently that details of the specifics of these structural changes have emerged. The development of synchrotron radiation circular dichroism (SRCD), combined with advances in FTIR spectroscopy, has allowed the secondary and tertiary structure of proteins adsorbed at emulsion oil/water interfaces to be studied. SRCD in particular has provided quantitative information and has enabled new insights into the mechanisms and forces driving protein structure re-arrangement to be achieved.The extent of conformational re-arrangement of proteins at emulsion interfaces is influenced by several factors including; the inherit flexibility of the protein, the distribution of hydrophobic/hydrophilic domains within the protein sequence and the hydrophobicity of the oil phase. In general, proteins lose much of their tertiary structure upon adsorption to the oil/water interface and have considerable amounts of non-native secondary structure. Two key conformations have been identified in the structure of proteins at interfaces, intermolecular β-sheet and α-helix. The preferred conformation appears to be the α-helix which is the most compact amphipathic conformation at the oil/water interface. The polarity of the oil phase can have a considerable influence on the degree of protein conformational re-arrangement because it acts as a solvent for hydrophobic amino acids. The new conformation of proteins at interfaces also means that proteins undergo less heat induced re-arrangement at interfaces than in solution. Different conformations of proteins at interfaces impact on emulsification capability, emulsion stability and protein/emulsion digestion. Hence advances in the understanding of protein conformation at interfaces can help to identify suitable proteins and conditions for the preparation of emulsion based food products.     

Protein quality of irradiated Brazilian beans

Beans are a major source of dietary protein in Brazil. However, high losses due to insect infestation occur after each harvest. To combat these losses, radiation processing of beans offers promise as an alternative to chemical treatment, provided the nutritional quality of beans is not impaired by the radiation treatment. Conflicting results have been published about the effect of radiation on the biological value of legume proteins. Therefore, two varieties of Brazilian beans were studied: 1) Phaseolus vulgaris L., var. carioca and 2) Vigna unguiculata (L.) Walp, var. macaçar. The beans were irradiated with doses of 0, 0.5, 1.0, 2.5, 5.0 and 10 kGy. Since irradiated beans will be consumed after appropriate storage, the beans under study were stored for 6 months at ambient temperature. Protein quality was measured by a biological assay employing the nitrogen balance approach in weanling rats. The animals were fed with optimally cooked beans, which were the only source of protein (10%). Nitrogen contents of legumes, diets, animal urine and faeces were determined by Kjeldahl analysis. The indices for apparent protein quality: net protein utilisation, digestibility and biological value were not influenced by irradiation. Thus, radiation treatment of Brazilian beans offers considerable promise as an effective insect disinfection process, without impairing the biological quality of the valuable bean protein.     

Neurotransmitter Biomolecule Transfers Across Liquid/Liquid Interface Through A Thick Organic Membrane-Modified Electrode

Electrochemical studies at liquid/liquid interfaces (L/L, or soft interfaces) have disclosed a biomimetic model to mimic charge transfers at cytomembrane surface. Herein, we reported two neurotransmitter biomolecules of dopamine and adrenalone across the L/L interface by a thick organic membrane-modified electrode. This system comprised polarized electrode/oil and oil/water interfaces in series in which the electron transfer (ET) of redox 7,7,8,8-tetracyanoquinodimethane (TCNQ) at electrode/oil interface drove ion transfer (IT) of biomolecules at oil/water interface. This ET-IT coupled reaction overcame the limitation of polarized potential window at conventional single polarized L/L interface. The crucial design of a thick organic membrane could ensure the generated TCNQ anions maintained at electrode/oil interface during the voltammetry, which could not result in interruptions to biomolecule transfers. Through this system, their Gibbs transfer free energies were accurately determined (44.4 and 39.4 kJ mol^?1 for dopamine and adrealone, respectively). Moreover, facilitated biomolecule transfers were evaluated by crown ionophores where both facilitated numbers and constants were determined simultaneously. Owing to the simple electrochemical setup, this system would hold great potentials in future hydrophilic biomacromolecule transfers, such as DNA, peptides and proteins.     

Gelation and interfacial behaviour of vegetable proteins

Recent studies on gelation and interfacial properties of vegetable proteins are reviewed. Attention is focused on legume proteins, mainly soy proteins, and on wheat proteins. The rheological properties of vegetable protein gels as a function of heating time or temperature is discussed as well as the interfacial gelation upon adsorption of soy and wheat proteins at the air/water interface. It is shown that modification of proteins improves protein functionality and application.     

Electrophoretic studies for determining soy proteins–xanthan gum interactions in foams

The aim of this work was to elucidate soy proteins–xanthan gum interactions at a molecular level by studying protein composition at the air–water interface of foams and in the solutions used to make them and to see if the different properties of heat denatured protein were reflected in the proportions in which they were present at the interface or in the ability to interact with xanthan. To this end SDS-PAGE and densitometry was employed. Initial protein concentration and xanthan influenced the composition of proteins in the solutions used to make the foams. The increase in NSP concentration of solutions (0.5–6 wt.%) in the absence of xanthan promoted the formation of aggregates of low molecular weight (160 kDa), the association of A an B polypeptides and a decrease in and ′ subunits. As DSP concentration of solutions increased, an increase in the proportion of aggregates of high molecular weight (above 200 kDa) and B-polypeptide was observed. On addition of xanthan (0.025 and 0.05%) to protein solutions (0.5 and 2%), the formation of aggregates of high molecular weight was favoured for both NSP and DSP. In the absence of xanthan, no preferential adsorption of soy polypeptides was observed at the air–water interface of NSP foams. However in DSP foams, there was a preferential adsorption of B-polypeptides. Xanthan present in NSP foams (0.5 or 2%), caused an increase in the proportion of aggregates of high molecular weight at the interface as compared with the composition of solutions used to make the foams. An increase in proportion of AB-polypeptides (for 0.5% NSP and 0.025% xanthan) and B-polypeptides together with polypeptides of molecular weight lower than 14 kDa (for 0.5% NSP and 0.05% xanthan) was also observed at the interface in NSP foams. On the contrary, the presence of xanthan in DSP foams caused a decrease in the proportion of aggregates of high molecular weight and a concomitant increase in B-polypeptide. The B-11S polypeptide predominated the interface of DSP foams probably for its hydrophobicity and basic characteristics.     

Effects of spray drying on physicochemical properties of milk protein-stabilised emulsions

The effect of spray drying and reconstitution has been studied for oil-in-water emulsions (20.6% maltodextrin, 20% soybean oil, 2.4% protein, 0.13 M NaCl, pH 6.7) with varying ratios of sodium caseinate and whey protein, but with equal size distribution (d₃₂=0.77 μm). When the concentration of sodium caseinate in the emulsion was high enough to entirely cover the oil–water interface, the particle size distribution was hardly affected by spray drying and reconstitution. However, for emulsions of which the total protein consisted of more than 70% whey protein, spray drying resulted in a strong increase of the droplet size distribution. The adsorbed amount of protein ranged from 3 mg m⁻² for casein-stabilised emulsions to 4 mg m⁻² for whey protein-stabilised emulsions with a maximum of 4.2 mg m⁻² for emulsions containing 80% whey protein on total protein, which means that for all these emulsions about one quarter of the available protein was adsorbed at the oil–water interface. The adsorbed amount of protein was hardly affected by spray drying. After emulsion preparation casein proteins adsorbed preferentially at the oil–water interface. As a result of spray drying, the relative amount of β-lactoglobulin in the adsorbed layer increased strongly at the expense of _s1-casein and β-casein. Percentages of _s2-casein and κ-casein in the adsorbed layer remained largely unchanged. The changes in the protein composition of the adsorbed layer as a result of spray drying and reconstitution were the largest when beforehand hardly any whey protein was present in the adsorbed layer and hardly any sodium caseinate in the aqueous phase. Apparently, during spray drying conditions have been such that β-lactoglobulin could unfold, aggregate, and react with other cystein-containing proteins changing the particle size distribution of the emulsions and the composition of the adsorbed layer. It seemed, however, that non-adsorbed sodium caseinate in some way was able to protect the adsorbed casein proteins from being displaced by aggregating whey protein.     

Interaction and incorporation of ovalbumin with stearic acid monolayer: Langmuir-Blodgett film formation and deposition

In this communication, the surface activity of the ovalbumin (OVA) at the air/water interface was studied to establish the nature of the interaction with the stearic acid (SA) monolayer, based on Langmuir–Blodgett (LB) technique. The interaction was monitored by studying the time (t) variation of surface pressure (π) at constant area (A). The growth of π with time indicates a positive association between the SA and the OVA molecules. The surface compressibility analysis has been performed to specify the phase transition of OVA–SA mixed monolayer. Incorporation/association of OVA within the SA monolayer led to noteworthy changes in surface compressibility and was surface pressure as well as protein concentration dependent. Both the hydrophobic and the Vander wall type interactions are found to be responsible for the association. The quenching of tyrosine band in tryptophan excitation spectrum is observed in steady-state fluorescence spectroscopy. This suggests that the tyrosine is the probable binding site with SA. Due to incorporation of SA, the energy transfer from tyrosine to tryptophan is hindered. At higher pressure, OVA tend to squeeze out from the SA monolayer. The high-resolution field emission scanning electron microscope (FE-SEM) image confirms this observation. Aggregated protein structure observed at high pressure indicates unfolding of protein.     

Emulsification of caraway essential oil in water by lecithin and β-lactoglobulin: emulsion stability and properties of the formed oil–aqueous interface

The stability and droplet size of protein and lipid stabilised emulsions of caraway essential oil as well as the amount of protein on the emulsion droplets have been investigated. The amount of added protein (β-lactoglobulin) and lipid (phosphatidylcholine from soybean (sb-PC)) were varied and the results compared with those obtained with emulsions of a purified olive oil. In general, emulsions with triglyceride oil proved to be more stable compared with those made with caraway essential oil as the dispersed phase. However, the stability of the emulsions can be improved considerably by adding sb-PC. An increase in the protein concentration also promoted emulsion stability. We will also present how ellipsometry can be used to study the adsorption of the lipid from the oil and the protein from the aqueous phase at the oil–water interface. Independently of the used concentration, close to monolayer coverage of sb-PC was observed at the caraway oil–aqueous interface. On the other hand, at the olive oil–aqueous interface, the presence of only a small amount of sb-PC lead to an exponential increase of the layer thickness with time beyond monolayer coverage. The amounts of β-lactoglobulin adsorbed at the caraway oil–aqueous interface and at the olive oil–aqueous interface were similar, corresponding roughly to a protein monolayer coverage.     

Phase behavior and surface tensions of amphiphilic fluorinated random copolymer aqueous solutions

Phase behavior and surface tension of aqueous solutions of fluorinated random copolymers [perfluoroalkylacrylate]–[poly(ethyleneoxide)methacrylate], [C_mRf-acrylate]-[EO_n-methacrylate] with fluroalkyl carbon number m = 8, 6, 4, 2 and number of ethyleneoxide unit, n = 9 and 4.5 were investigated as a function of composition and different combinations of m and n. Isotropic solutions are formed at lower temperatures over wide concentration range of copolymer but at higher temperature phase separation occurs. The cloud point of copolymer decreases with decreasing n as well as m, and also with decreasing the number of poly(ethyleneoxide)methacrylate chain per perfluorinatedalkylacrylate chain, suggesting that the copolymers become more hydrophobic on decreasing m and n. Equilibrium and dynamic surface tension measurements show that copolymers become increasingly surface active as m as well as n decrease but the adsorption at the air–water interface is very slow due to bulkiness of the molecules. No clear evidence of the formation of micellar aggregates could be obtained from surface tension–composition curves.     

The effect of sucrose on the thermodynamic properties of ovalbumin and sodium caseinate in bulk solution and at air–water interface

This paper presents a study of the effect of sucrose on the molecular parameters and thermodynamic properties in a bulk aqueous medium and at the air–water interface for two proteins differing both in nature and structure, that is Na-caseinate and ovalbumin. To get more insight into the molecular nature of the effect of sucrose, mixing calorimetry, light scattering and tensiometry measurements have been made under different pHs (7.0 and 5.5) and temperatures (20–55°C) at an ionic strength of 0.005 mol dm⁻³. Combined temperature dependencies of light scattering and mixing calorimetry testify to hydrogen bonding (sucrose-protein and/or sucrose-water) as being the primary basis of the effect of sucrose on the molecular and thermodynamic properties of the proteins in the bulk and at interface of an aqueous medium. At pH 7.0, in the case of ovalbumin, the interaction with sucrose causes an increase in the protein hydrophilicity in the bulk aqueous medium followed by a decrease in the protein surface activity, whilst for Na-caseinate, there is an increase in the protein hydrophobicity due to Na-caseinate micelle dissociation and, consequently, to an increase in the protein surface activity. Lowering the pH to 5.5, accompanied by a strengthening of the competition between less charged proteins and sucrose for water molecules, induces a rise in the protein hydrophobic aggregation in the bulk. The special features of the latter process are probably mainly responsible for the changes in the surface activity of the proteins under influence of sucrose at pH 5.5.     

Partition and water/oil adsorption of some surfactants

Adsorption isotherms have been determined at the water/oil interface for five biphasic systems involving surfactants (non-ionic and ionic) present in both phases at partition equilibrium. The systems studied were polyoxyethylene(23)lauryl ether (Brij35) in water/hexane and four ionic surfactants, hexadecyltrimethylammonium bromide (CTAB), and a series of three tetraalkylammonium dodecylsulfate (TEADS, TPADS, and TBADS) in water/CH 2Cl 2. Interfacial tension measurements performed at the water/air and water/oil interfaces provided all the necessary information for the determination of the adsorption parameters by taking partition into account. These measurements also allowed the comparison of the adsorption properties at both interfaces which showed an increase of the adsorption equilibrium constant and a decrease of the maximum surface concentration at the water/oil interface compared to water/air. The values of the critical aggregation concentration showed, in all cases, that only the surfactant dissolved in the aqueous phase contribute to the decrease of the water/oil interfacial tension. In the case of the four ionic surfactants, the critical aggregation concentration obtained in biphasic conditions were lowered because of the formation of mixed surfactant-CH 2Cl 2 aggregates.     

Sample preparation for metalloprotein analysis: A case study using horse chestnuts

In the present work, 11 different procedures for protein and metalloprotein extraction from horse chestnuts (Aescullus hippocastanum L.) in natura were tested. After each extraction, total protein was determined and, after protein separation through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), those metals belonging to the protein structure were mapped by synchrotron radiation X-ray fluorescence (SRXRF). After mapping the elements (Cr, Fe and Mn) in the protein bands (ca. 33 and 23.7 kDa), their concentrations were determined using atomic absorption spectrometry (ET AAS).

Good results were obtained for protein extraction using a combination of grinding and sonication. However, this strategy was not suitable to preserve metal ions in the protein structure. In fact, there was 42% decrease on Mn concentration using this procedure, compared to that performed with sample agitation in water (taken as reference). On the other hand, when grinding and agitation with an extracting buffer was used, there was a 530% increase of Mn concentration, when compared to the reference procedure.

These results indicate agreement between metal identification and determination in proteins as well as the great influence of the extraction procedure (i.e., the sample preparation step) for preserving metals in the protein structures.     

Calorimetric comparison of the interactions between salivary proteins and Streptococcus mutans with and without antigen I/II

Antigen I/II can be found on streptococcal cell surfaces and is involved in their interaction with salivary proteins. In this paper, we determine the adsorption enthalpies of salivary proteins to Streptococcus mutans LT11 and S. mutans IB03987 with and without antigen I/II, respectively, using isothermal titration calorimetry. In addition, protein adsorption to the cell surfaces was determined spectrophotometrically. S. mutans LT11 with antigen I/II, yielded a much higher, exothermic adsorption enthalpy at pH 6.8 (ranging from −2073 × 10⁻⁹ to −31707 × 10⁻⁹ μJ per bacterium) when mixed with saliva than did S. mutans IB03987 (−165 × 10⁻⁹ to −1107 × 10⁻⁹ μJ per bacterium) at all bacterial concentrations studied (5 × 10⁹, 5 × 10⁸, and 5 × 10⁷ ml⁻¹), largest effects per bacterium being observed for the lowest concentration. However, the enthalpy of salivary protein adsorption to S. mutans LT11 became smaller at pH 5.8. Adsorption isotherms for the S. mutans LT11 showed considerable protein adsorption at pH 6.8 (1.2–2.1 mg/m²), that decreased only slightly at pH 5.8 (1.1–1.6 mg/m²), with the largest amount adsorbed at the lowest bacterial concentration. This suggests that the protein(s) in the saliva with the strongest affinity for antigen I/II is (are) readily depleted from saliva. In conclusion, antigen I/II surface proteins on S. mutans play a determinant role in adsorption of salivary proteins through the creation of enthalpically favorable adsorption sites.     

Determination,by dynamic surface-tension analysis,of the molar mass of proteins denatured in guanidine thiocyanate

A drop-based dynamic surface-tension detector (DSTD) has been used to study the dynamic surface tension behavior of proteins denatured in guanidine thiocyanate (GndSCN). The dynamic surface tension at the air–liquid interface is obtained by measuring the internal pressure of drops that grow and detach at a specified rate. In the method the sample of interest is injected and subsequently flows to the DSTD-sensing capillary tip. For this work, a novel DSTD calibration procedure utilizing two distinct mobile phases is applied. Here, the mobile phases are aqueous with different constituents, for example GndSCN and phosphate buffer, either added or omitted. The dual-mobile phase calibration procedure gives the analyst the capability of making protein measurements in a GndSCN–phosphate buffer mobile phase, while measuring a calibration standard in another mobile phase, such as water, in which the surface tension of the calibration standard is readily available. Results are presented with drop volumes of either 2 L (i.e. 2-s drops) or 7 L (i.e. 7-s drops) for proteins varying in molar mass from 12,000 to 330,000 g mol^–1. We demonstrate that the DSTD can be used to determine the molar mass of proteins denatured in GndSCN. The method applies a regime where the denatured protein is detected by surface-active properties, and selectivity with regard to molar mass is contained in the dynamic component of the DSTD signal. The dynamic surface pressure signals of the denatured proteins suggest that diffusion plays a large role in the kinetics of the surface activity. The limit of detection for the denatured proteins studied ranged from 3 mg L^–1 to 14 mg L^–1. The DSTD, coupled with the novel dual-mobile phase calibration procedure, can be used to investigate the fundamental properties of proteins. Insight into the behavior at the air–liquid interface for native and denatured proteins is achieved; this is a novel tool for studying protein denaturation, complementary to other common approaches such as spectroscopy and calorimetry. Furthermore, the reported method could be widely applied to the study of effects on the interfacial properties of proteins after a variety of chemical and physical modifications that are possible with the dual-mobile phase calibration procedure.     

Higher order structure of proteins solubilized in AOT reverse micelles

The higher order structure of proteins solubilized in an bis(2-ethylhexyl) sulfosuccinate sodium (AOT) reverse micellar system was investigated. From circular dichroic (CD) measurement, CD spectra of cytochrome c, which is solubilized at the interface of reverse micelles, markedly changed on going from buffer solution to the reverse micellar solution, and the ellipticity values in the far- and near-UV regions decreased with decreasing the water content (W₀: molar ratio of water to AOT), indicating that the secondary and tertiary structures of cytochrome c changed with the water content. The ellipticity of ribonuclease A, which is solubilized in the center of micellar water pool, in the near-UV region was dependent on W₀ and became minimum when W₀ of ca. 8 while the ellipticity in the far-UV region was almost constant, indicating that the tertiary structure of ribonuclease A was affected by the water content, but the secondary structure was conserved. The degree of curvature of the micellar interface appears to influence the protein structure because the reverse micelle size is linearly proportional to the W₀ value. As evidence of this, when the micelle size was comparable to the protein's dimensions, the structures were more affected by the water content. Judging from the dependence of the factor influencing the protein structure on the protein species, the location of solubilized protein in reverse micelles is significantly related to whether the protein structure in the system is affected by the micellar interface. In the cases of cytochrome c and lysozyme, the ellipticity against W₀ was dependent on the AOT concentration. In contrast, ribonuclease A gave very similar ellipticity values whatever the AOT concentration. In the n-hexane micellar system, cytochrome c exhibited lower ellipticity values and ribonuclease A in the lower W₀ range (W₀ < ca. 8) higher ellipticity values. These results indicated that the interaction between the protein and the micellar interface is a dominant factor influencing the protein structure in reverse micelles, and that it is governed by the location of solubilized proteins and the state of the micellar interface.     

Admittance measurements on protein layers adsorbed at the Pt/solution interface: effect of d.c. potential and a.c. field

The effect of changes in the electric field on the structure of protein layers adsorbed at the Pt electrode/solution interface has been investigated by means of admittance measurements. The measurements have been performed over a wide range of d.c. potentials, at different frequencies and amplitudes of the a.c. electric field, and at various pH values. The proteins used, cytochrome C and serum albumin, differ considerably with respect to their molecular masses, points of zero charge and structure stabilities. In contrast to serum albumin, cytochrome C has a relatively strong electric dipole moment. Nevertheless, the results for the two proteins are very similar. Both proteins stay adsorbed at the interface over the d.c. potential range studied and at every pH, irrespective of any electrostatic repulsion. Apparently, for both proteins factors other than electrostatic interactions are dominant in their final binding to the Pt/solution interface. In line with this, no indications were obtained that the orientation of adsorbed cytochrome C molecules is modulated by low-frequency (200–1000 Hz) reversal of the electric field of the interface. A strong a.c. field leads to irreversible structural changes in the adsorption layer for both proteins.