Langmuir 2002, 18, 6887-6895
6887
Surface Rheological Properties of Native and S-Ovalbumin Are Correlated with the Development of an Intermolecular β-Sheet Network at the Air-Water Interface Anne Renault,† Ste´phane Pezennec,*,‡ Fabien Gauthier,§ Ve´ronique Vie´,† and Bernard Desbat| Groupe Matie` re Condense´ e et Mate´ riaux, UMR CNRS 6626, Universite´ Rennes 1, Campus de Beaulieu, Baˆ t. 11A, F-35042 Rennes CEDEX, France, Physico-Chimie et Technologie des Ovoproduits, UMR INRA-ENSAR 1055, 65, rue de Saint-Brieuc, CS 84215, F-35042 Rennes CEDEX, France, Laboratoire de Spectrome´ trie Physique, UMR CNRS 5588, BP87, Universite´ Joseph Fourier, F-38402 St. Martin D’He` res, France, and Laboratoire de Physico-Chimie Mole´ culaire, UMR CNRS 5803, Universite´ Bordeaux 1, F-33405 Talence CEDEX, France Received March 20, 2002. In Final Form: June 10, 2002 Polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS), ellipsometry, and shear elastic constant measurements were used to study the adsorption and the behavior of ovalbumin and S-ovalbumin at the air-water interface at different values of the subphase pH. Native and S-ovalbumin exhibited similar behaviors, with a maximum plateau value of the shear elastic constant near the isoelectric pH of the protein. However, higher surface concentration values were reached with S-ovalbumin in low net charge conditions, which suggest adsorption of aggregates or multilayer adsorption. For both proteins, the statistical analysis of PM-IRRAS spectra demonstrated that the aging of the interfacial film and the increase of the shear elastic constant were correlated with a significant increase in the relative contribution of intermolecular β-sheets in the amide I band with time. This increase was significantly faster at low pH values. At the same pH value and age of the interface, the relative contribution of intermolecular β-sheets was significantly higher for S-ovalbumin.
Introduction Due to their amphipathic properties, proteins adsorb from solutions to hydrophobic interfaces, either solid or fluid ones. Protein adsorption is essentially entropically driven, the dehydration of hydrophobic surfaces and hydrophobic exposed regions of the protein molecules being favored. Adsorption can also occur at hydrophilic (solid) surfaces if electrostatically favored.1 Numerous data are available about the kinetics and thermodynamics of protein adsorption, as provided by tensiometric, ellipsometric, and radiometric methods.2-8 Models for the indepth structure of the interfacial film have been studied * To whom correspondence should be addressed. Fax: +33 2 23 48 53 50. E-mail:
[email protected]. † Groupe Matie ` re Condense´e et Mate´riaux, UMR CNRS 6626, Universite´ Rennes 1. ‡ Physico-Chimie et Technologie des Ovoproduits, UMR INRAENSAR 1055. § Laboratoire de Spectrome ´ trie Physique, UMR CNRS 5588, BP87, Universite´ Joseph Fourier. | Laboratoire de Physico-Chimie Mole ´ culaire, UMR CNRS 5803, Universite´ Bordeaux 1. (1) Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1-15. (2) Miller, R.; Fainerman, V. B.; Makievski, A. V.; Kra¨gel, J.; Grigoriev, D. O.; Kazakov, V. N.; Sinyachenko, O. V. Adv. Colloid Interface Sci. 2000, 86, 39-82. (3) Norde, W. Cells Mater. 1995, 5, 97-112. (4) Xu, S.; Damodaran, S. J. Colloid Interface Sci. 1993, 159, 124133. (5) Dickinson, E. ; Murray, B. S. ; Stainsby, G. In Advances in Food Emulsions and Foams; Dickinson, E., Stainsby, G., Eds.; Elsevier Applied Science: London, 1988; p 123. (6) de Feijter, J. A. ; Benjamins, J. In Food Emulsions and Foams; Dickinson, E., Ed.; Royal Society of Chemistry: London, 1987; p 72. (7) Guzman, R. Z.; Carbonell, R. G.; Kilpatrick, P. K. J. Colloid Interface Sci. 1986, 114, 536-547. (8) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403-414.
by X-ray and neutron reflectivity.9-13 Various techniques have been applied to the investigation of rheological properties of the interfacial film.14-17 Protein adsorption can contribute in several different ways to the stabilization of polyphasic, dispersed systems such as foams and emulsions, which may be a crucial technical goal, for example, in food system design. By reducing the interfacial tension, adsorbed proteins help to increase the interfacial area. Steric and electrostatic properties of adsorbed proteins can also participate in the stabilization of dispersed systems, for example, by hindering the coalescence of emulsion droplets. The interfacial films of adsorbed proteins also stabilize dispersed systems due to their rheological properties, which increase the resistance of the interfacial film to mechanical stress, for example, in a foam. It is now widely accepted that those crucial rheological properties of interfacial protein films are closely related (9) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M. J. Chem. Soc., Faraday Trans. 1995, 91, 2847-2854. (10) Horne, D. S.; Atkinson, P. J.; Dickinson, E.; Pinfield, V. J.; Richardson, R. M. Int. Dairy J. 1998, 8, 73-77. (11) Harzallah, B.; Aguie´-Be´ghin, V.; Douillard, R.; Bosio, L. Int. J. Biol. Macromol. 1998, 23, 73-84. (12) Puff, N.; Cagna, A.; Aguie´-Be´ghin, V.; Douillard, R. J. Colloid Interface Sci. 1998, 208, 405-414. (13) Marsh, R. J.; Jones, R. A. L.; Sferrazza, M.; Penfold, J. J. Colloid Interface Sci. 1999, 218, 347-349. (14) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 227-239. (15) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 240-250. (16) Benjamins, J.; Lucassen-Reynders, E. H. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; p 341. (17) Lucassen-Reynders, E. H.; Benjamins, J. In Food Emulsions and Foams. Interfaces, Interactions and Stability; Dickinson, E., Rodrı´guez Patino, J. M., Eds.; Royal Society of Chemistry: Cambridge, 1999; p 195.
10.1021/la0257586 CCC: $22.00 © 2002 American Chemical Society Published on Web 07/30/2002
6888
Langmuir, Vol. 18, No. 18, 2002
to the conformational changes underwent by adsorbed proteins, which allow them to develop intermolecular interactions. As briefly discussed later in this paper, data have been obtained by many groups about protein conformational changes occurring upon adsorption at solid surfaces or at the oil-water interface. Concerning the air-water interface, information about the structural properties of adsorbed proteins is scarce, essentially due to the lack of suitable investigation methods. The correlation between the conformational changes after adsorption and the macroscopic properties of the interfacial film4,12,18-21 is not fully understood. Recently, the potential of infrared reflection-absorption spectroscopy (IRRAS) in the study of protein conformational changes at the air-water interface has been described.22 To overcome the problem of water vapor absorption, polarization-modulated infrared reflectionabsorption spectroscopy (PM-IRRAS) has been developed.23,24 Because it provides a differential reflectivity signal, this technique selectively extracts the interfacial layer signal from the background generated by the subphase and the atmosphere and offers a large increase in sensitivity. PM-IRRAS has been successfully used to obtain detailed information about molecular conformation and orientation in fatty acid, phospholipid, peptide, or alcohol monolayers.25-29 The conformational changes in a macromolecular complex such as the photosystem II core complex at the gas-water interface have also been evidenced by PM-IRRAS.30 Ovalbumin is the major egg-white protein. It is a phosphorylated and glycosylated globular protein of 385 amino acids (45 kDa).31 Its secondary structure consists of approximately 30% R-helices and 30% β-sheets.32,33 Ovalbumin is a suitable model for the study of the adsorption of globular proteins, since a large set of data are available about essentially macroscopic aspects of its interfacial behavior (tensioactivity, adsorption kinetics, interfacial shear, and dilatational rheology).6,16,17,34,35 We recently combined ellipsometry, surface tension, and (18) Caessens, P. W. J. R.; De Jongh, H. H. J.; Norde, W.; Gruppen, H. Biochim. Biophys. Acta 1999, 1430, 73-83. (19) Razumovsky, L.; Damodaran, S. Langmuir 1999, 15, 13921399. (20) Hagolle, N.; Launay, B.; Relkin, P. Colloids Surf., B 1998, 10, 191-198. (21) Wu¨stneck, R.; Kra¨gel, J.; Miller, R.; Fainerman, V. B.; Wilde, P. J.; Sarker, D. K.; Clark, D. C. Food Hydrocolloids 1996, 10, 395-405. (22) Meinders, M. B. J.; van den Bosch, G. G. M.; De Jongh, H. H. J. Trends Food Sci. Technol. 2000, 11, 218-225. (23) Blaudez, D.; Buffeteau, T.; Cornut, J. C.; Desbat, B.; Escafre, N.; Pe´zolet, M.; Turlet, J.-M. Appl. Spectrosc. 1993, 47, 869-874. (24) Blaudez, D.; Turlet, J.-M.; Dufourcq, J.; Bard, D.; Buffeteau, T.; Desbat, B. J. Chem. Soc., Faraday Trans. 1996, 92, 525-530. (25) Alonso, C.; Blaudez, D.; Desbat, B.; Artzner, F.; Berge, B.; Renault, A. Chem. Phys. Lett. 1998, 284, 446-451. (26) Castano, S.; Desbat, B.; Laguerre, M.; Dufourcq, J. Biochim. Biophys. Acta 1999, 1416, 176-194. (27) Castano, S.; Desbat, B.; Dufourcq, J. Biochim. Biophys. Acta 2000, 1463, 65-80. (28) Bellet-Amalric, E.; Blaudez, D.; Desbat, B.; Graner, F.; Gauthier, F.; Renault, A. Biochim. Biophys. Acta 2000, 1467, 131-143. (29) Ulrich, W. P.; Vogel, H. Biophys. J. 1999, 76, 1639-1647. (30) Gallant, J.; Desbat, B.; Vaknin, D.; Salesse, C. Biophys. J. 1998, 75, 2888-2899. (31) Nisbet, A. D.; Saundry, R. H.; Moir, A. J. G.; Fothergill, L. A.; Fothergill, J. E. Eur. J. Biochem. 1981, 115, 335-345. (32) Stein, P. E.; Leslie, A. G. W.; Finch, J. T.; Carrell, R. W. J. Mol. Biol. 1991, 221, 941-959. (33) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235-242. (34) Benjamins, J.; van Voorst Vader, F. Colloids Surf. 1992, 65, 161-174. (35) de Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759-1772.
Renault et al.
rheological measurements to show that the charge effects are determinant in the interfacial behavior of ovalbumin.36 S-Ovalbumin is a thermostable form of the protein37,38 which appears spontaneously during egg-white storage. S-Ovalbumin can also be produced from the native form by moderate heating in alkaline conditions. Only few data are available about the structural differences between native and S-ovalbumin.39,40 S-Ovalbumin has been proposed to be a conformer of the native form,41 the conformational differences involving only a few residues. Ovalbumin and its S form therefore are suitable models to study the impact of low-extent conformational modifications on the interfacial behavior. The purpose of this work was, by the means of complementary methods (PM-IRRAS, ellipsometry, and shear elastic constant measurements), to investigate the conformational changes of ovalbumin upon adsorption at the air-water interface in various charge conditions. Our aim was to identify the intermolecular interactions involved in the macroscopic interfacial rheological properties and, as a complement of our recent work,36 to compare S-ovalbumin and its native counterpart from both macroscopic and molecular points of view. The originality of our results is that ovalbumin conformational changes were correlated to the interfacial macroscopic rheological properties. Experimental Section Proteins. Diphosphorylated A1-ovalbumin was isolated by anion-exchange chromatography as already described.36 Briefly, the albumen from a single egg was diluted 10-fold in 50 mM Tris-HCl, pH 8.0 (buffer A), and gently stirred at 4 °C overnight. The precipitated material was discarded by centrifugation and filtration. Egg-white proteins were separated on a Q-Sepharose Fast Flow (Pharmacia, Saclay, France) column using linear NaClconcentration gradients in buffer A. Fractions corresponding to A1-ovalbumin were pooled, concentrated by precipitation with ammonium sulfate, dialyzed against ultrapure water, and lyophilized. For S-ovalbumin preparation, lyophilized A1-ovalbumin was solubilized in 50 mM sodium carbonate, pH 9.9, at a 10 g L-1 concentration, and heated at 55 °C for 24 h. The heated preparation was dialyzed against ultrapure water and used immediately or lyophilized. The upward shift in the denaturation temperature upon conversion into S-ovalbumin was checked by differential scanning calorimetry with a Pyris 1 system (PerkinElmer), using 5 g L-1 protein solutions in 50 mM Tris-HCl, pH 8.0, and a 5 °C min-1 heating rate (data not shown). Concentrations of purified ovalbumin and S-ovalbumin solutions were determined by absorption at 280 nm using a specific extinction coefficient E1%1cm ) 6.60 ( ) 2.93 × 104 M-1 cm-1).42 Buffers for PM-IRRAS, ellipsometry, and shear elastic constant measurements were designed to give a common ionic strength (according to the Henderson-Hasselbach equation), close to 60 mM: 70.5 mM sodium citrate-NaOH, pH 3.5; 33.5 mM sodium citrate-NaOH, pH 4.6; 50 mM sodium phosphate, pH 6.0; and 26.5 mM sodium carbonate-NaOH, pH 10.0. The corresponding D2O buffers were prepared using the same standard protonated chemicals as solutions in deuterium oxide D2O to give pD 3.9, pD 5.0, pD 6.4, and pD 10.4, as calculated from the direct pHmeter reading using the relation pD ) pH + 0.4. For all the (36) Pezennec, S.; Gauthier, F.; Alonso, C.; Graner, F.; Croguennec, T.; Brule, G.; Renault, A. Food Hydrocolloids 2000, 14, 463-472. (37) Smith, M. B.; Back, J. F. Aust. J. Biol. Sci. 1965, 18, 365-377. (38) Smith, M. B.; Back, J. F. Aust. J. Biol. Sci. 1968, 21, 539-548. (39) Kint, S.; Tomimatsu, Y. Biopolymers 1979, 18, 1073-1079. (40) Paolinelli, C.; Barteri, M.; Boffi, F.; Forastieri, F.; Gaudiano, M. C.; Della Longa, S.; Congiu Castellano, A. Z. Naturforsch. 1997, 52, 645-653. (41) Huntington, J. A.; Patston, P. A.; Gettins, P. G. W. Protein Sci. 1995, 4, 613-621. (42) Batra, P. P.; Roebuck, M. A.; Uetrecht, D. J. Protein Chem. 1990, 9, 37-44.
Development of an Intermolecular β-Sheet Network experiments, protein solutions were freshly prepared from lyophilized samples, directly in the final buffer. Ellipsometry and Surface Tension. The ellipsometric measurements were carried out with a conventional null ellipsometer using a He-Ne laser operating at 632.8 nm.43 The variation of the ellipsometric angle is a relevant probe for changes occurring at the interface. The ellipsometric angle (∆) and the surface pressure (Π) were recorded simultaneously. The surface pressure was measured with the Wilhelmy system. The sample trough was made out of Teflon and had a volume of 8.4 mL. The protein was diluted in the buffer and poured into the trough directly after preparation. All the experiments were done at room temperature (18.5-19.5 °C). Shear Elastic Constant Measurements. The rheometer setup44,45 uses the action of a very light float applying a rotational strain to the surface through a magnetic couple. Practically, at the center of a 48 mm diameter Teflon trough, a 10 mm diameter paraffin-coated aluminum disk floats at the air-water interface, surrounded by the surface whose rigidity is measured. The subphase is 5 mm deep. The float carries a small magnet, of known magnetic moment, and is kept centered in the trough by a permanent field B0 ) 6 × 10-5 T, parallel to the Earth’s field, created by a little solenoid located just above the float. A sinusoidal torque excitation is applied to the float in the 0.01-100 Hz frequency range, by an oscillating field perpendicular to the solenoid field, generated with a pair of Helmholtz coils. The permanent solenoid field acts as a restoring torque, equivalent to a surface having a 0.16 mN m-1 rigidity. The maximum rotation amplitude is typically in the 10-4 rad range. The device behaves like a simple harmonic oscillator. Sensitive angular detection of the float rotation is achieved using a mirror fixed on the magnet and reflecting a laser beam onto a differential photodiode. The amplitude and phase of the angular response are measured and considered to reflect directly the rotational strain of the surface. A first “blank” experiment is systematically performed on pure water. The frequency spectrum of the oscillator response is measured in the 0.01-100 Hz range. From the motion equation, the complex expression of the angular response of the float as a function of time is derived, and the complex amplitude term is fitted to the whole spectrum. The results provide the values of the equation parameters, especially the moment of inertia of the float. The same experiment is then performed on a stabilized film of adsorbed protein. The frequency spectrum exhibits a shift in the resonance frequency which depends on the value of the shear elastic constant µ. A fit of the amplitude equation to the experimental spectrum, identical to that described above, gives the value of µ. The time course of the interfacial rheological changes during adsorption and aging of the protein film can also be followed by measuring the oscillator response at a fixed frequency (typically 5 Hz), which enabled us to detect the rheological stabilization of the protein film before the measurement of frequency spectra. As far as possible, ellipsometric and shear elastic constant measurements were performed with the same solution and started at the same time. PM-IRRAS. The principle and experimental setup of PMIRRAS have been described in detail elsewhere.23,24 This technique combines Fourier transform infrared reflection spectroscopy with fast modulation of the polarization of the incident beam between parallel (p) and perpendicular (s) directions. The two-channel processing of the detected signal gives the differential reflectivity spectrum ∆R/R ) (Rp - Rs)/(Rp + Rs). To remove the contribution of water absorption, the spectra are divided by that of the subphase; they are expressed in PM-IRRAS units, which are dimensionless but not arbitrary. With an angle of incidence of 75°, transition moments in the interface plane give strong and upward-oriented bands, while transition moments perpendicular to the interface give weaker and downward-oriented bands. Spectroscopic measurements were performed in a circular Teflon trough (8 mL). Spectra were recorded with an 8 cm-1 resolution on a Nexus 870 spectrometer (Nicolet) equipped with (43) Berge, B.; Renault, A. Europhys. Lett. 1993, 21, 773-777. (44) Ve´nien-Bryan, C.; Lenne, P.-F.; Zakri, C.; Renault, A.; Brisson, A.; Legrand, J.-F.; Berge, B. Biophys. J. 1998, 74, 2649-2657. (45) Zakri, C.; Renault, A.; Berge, B. Physica B 1998, 248, 208-210.
Langmuir, Vol. 18, No. 18, 2002 6889 a liquid-nitrogen-cooled photovoltaic HgCdTe detector (SAT, France). For each spectrum, 400 scans were recorded (6-7 min). All spectra were recorded over the 800-4000 cm-1 region. The infrared beam probed a surface of about 1 cm2. The spectrum of the bare air-buffer interface was systematically recorded and used as the reference to divide further spectra. Statistical Analyses of D2O Spectra. Data Preprocessing. The statistical analyses were performed on spectra recorded in D2O. After baseline correction, spectra were truncated between 1800 and 1500 cm-1 in order to enclose the amide I and amide II spectral bands. Spectra were then normalized in order to correct them for overall intensity variations, according to the multiplicative scattering correction procedure.46 Briefly, each spectrum was expressed as a linear function of the overall average spectrum, by the means of linear regression, and then corrected for the corresponding intercept and factor terms. Therefore, subsequent statistical treatments did not take the global intensity variations into account and consisted only of the analysis of the spectral shape variations. Normalization and subsequent statistical analyses were performed using the GNU software package “R”.47 Principal Component Analysis (PCA). PCA is a multivariate statistical procedure which allows one to reduce data with a minimum loss of information by eliminating their redundancy, detect correlations between variables, and identify meaningful underlying variables.48 Briefly, in the general case of a data set consisting of several variables measured for different observations (or individuals), PCA leads to new variables, which are mutually orthogonal (uncorrelated) linear combinations of the original variables. These new variables are called principal components. Each principal component is defined by the coefficients in the linear combination of the original variables (variable loadings). Successive principal components exhibit decreasing values of variance; that is, they account for decreasing amounts of the initial “information”. Usually, only the first few principal components account together for the major part of the original variance and are considered in the subsequent analyses. Then, the initial data set can be described with a reduced number of nonredundant variables. Principal components thus provide a new coordinate system which allows one to draw similarity maps where similar observations occupy neighboring positions. Mathematically, principal components are simply defined as the eigenvectors of the data variance-covariance matrix. The associated eigenvalues reflect the proportion of variance that is accounted for by the different eigenvectors. The scores of the observations on principal components, which are used as new coordinates to draw similarity maps, are simply obtained by multiplying the data matrix by the eigenvector matrix. When PCA is applied to spectral data, observations are spectra and the original variables are the intensities measured at each wavenumber. Obviously, spectral data are highly redundant (the intensity at the ith wavenumber is highly correlated to the intensity at wavenumber i+1). PCA eliminates this redundancy. While in the initial data set any spectrum is defined by intensities at wavenumbers 1, 2, ..., n - 1, n, n generally being large (hundreds or thousands of data points), after PCA the same spectrum is defined by its coordinates (scores) on a small number (two in the present work) of new variables (principal components). Principal components, which are linear combinations of the original variables, hence are homologous to spectra and can be represented as such. They show the spectral patterns which are the most discriminating for the spectra.49 The scores of individual spectra on principal components are computed by one matrix multiplication. They are used to draw factorial maps, with axes defined by the principal components, where each spectrum is represented as a point. This method has been applied, for example, (46) Geladi, P.; McDougall, D.; Martens, H. Appl. Spectrosc. 1985, 39, 491-500. (47) Ihaka, R.; Gentleman, R. J. Comput. Graph. Stat. 1996, 5, 299314. (48) Jolliffe, I. T. Principal Component Analysis; Springer: New York, 1986. (49) Bertrand, D.; Scotter, C. N. G. Appl. Spectrosc. 1992, 46, 14201425.
6890
Langmuir, Vol. 18, No. 18, 2002
Renault et al.
Table 1. Fitting of a Linear Model to the Scores on Principal Component 1a
Table 2. Fitting of a Linear Model to the Scores on Principal Component 2a
(a) Analysis of Variance
(a) Analysis of Variance p value 10-16
