Heat-Induced Aggregation of a Globular Egg-White Protein in

CSIRO Molecular Science, Bag 10, Clayton South, Victoria 3169, Australia. Received July 28, 2002. In Final Form: January 8, 2003. Aqueous solution con...
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Heat-Induced Aggregation of a Globular Egg-White Protein in Aqueous Solution: Investigation by Atomic Force Microscope Imaging and Surface Force Mapping Modalities Lidia V. Najbar, Robert F. Considine,† and Calum J. Drummond* Food Science Australia, Sneydes Road, Werribee, Victoria 3030, Australia, and CSIRO Molecular Science, Bag 10, Clayton South, Victoria 3169, Australia Received July 28, 2002. In Final Form: January 8, 2003 Aqueous solution conditions have been chosen so that a globular gelling food protein, ovalbumin, is adsorbed on a molecularly smooth muscovite mica surface. Atomic force microscope (AFM) fluid-tapping and electrical double layer imaging modes and force mapping have been used to characterize the heatinduced aggregation mechanism and surface properties of the protein. At a solution concentration of 0.5 µg/mL, single globular structures, which were consistent with the dimensions of ovalbumin monomers, were observed with the fluid-tapping imaging mode. Heat treatment caused protein aggregation and produced larger globular structures. At a solution concentration of 20 µg/mL, a protein film was formed and the electrical double layer mode of imaging revealed that the heat-induced globular aggregates were more heterogeneous than the native protein. The heterogeneity of the heat-treated ovalbumin film was also apparent from the effective film thickness maps derived from the force data obtained on approach of the AFM tip toward the surface covered with protein. The interfacial spring constant for heat-treated ovalbumin was moderately lower than for the native protein. This is indicative of a reduction of the internal cohesiveness of the molecular structure upon heating. The observation that there are regular multiple adhesion events, upon retraction of the AFM tip from the surface, for heat-induced ovalbumin aggregates suggests that the globular structures were composed of noncovalently linked monomeric units of ovalbumin.

Introduction The heat-induced gelling ability of various globular proteins plays an important role in the textural and waterholding properties of many food products.1 The sequence of reactions that lead to macroscopic gelation is unfolding, aggregation, and deposition of the aggregates into the gel matrix. In essence, it is the form of the network structure and intermolecular interactions between protein molecules that govern the final physical characteristics and properties of the gel.2 Manipulating the pH, the ionic strength, the protein concentration, and the denaturation procedure upon heating can influence the network structure formed. For instance, at pH values far from the isoelectric point, the translucent appearance and elastic properties of globular protein gels have been attributed to fine networks of strands interspersed within the gel network. Near the pH of the isoelectric point (usually in the pH range of 4-6), brittle curdlike gel microstructure has been attributed to random associations of aggregated protein particles.3,4 A typical example of particulate gel formation is manifested when an egg is boiled. Ovalbumin is a globular protein present in egg-white, which is predominantly responsible for the gelling phenomenon. At neutral and basic pH, the process of gelation of ovalbumin has been * To whom correspondence should be addressed. Present address: cap-XX Pty. Ltd., Units 9 & 10, 12 Mars Road, Lane Cove NSW 2066, Australia. E-mail: [email protected]. † Present address: Barwon Water, Victoria, Australia. (1) Heertje, I. Food Struct. 1993, 12, 343-364. (2) Hermansson, A.-M. In Protein Structure-Function Relationships in Foods; Yada, R. Y., Jackman, R. L., Smith, J. L., Eds.; Blackie Academic and Professional: Glasgow, 1994; pp 22-42. (3) Egelandsdal, B. J. Food Sci. 1980, 45, 570-573. (4) Hatta, H.; Kitabatake, N.; Doi, E. Agric. Biol. Chem. 1986, 50, 2083-2089.

shown to be induced by specific heat treatment and to be preceded by the formation of aggregates of protein molecules.5,6 The heat-induced aggregates are somewhat unique, in that they are soluble and consequently appear as clear solutions.7-10 These aggregates are thought to be precursor intermediates that form the basic structure necessary for building up the gel network. Under gelling conditions, clear and elastic ovalbumin gels are formed in this pH range. Although a number of attempts have been made to characterize the structure and size of ovalbumin aggregates by various biophysical methods,6,8-12 little is known about the specific mechanism of assembly of these aggregates and, in particular, about the properties of the precursor aggregates. This is also the case at low pH (e.g., pH 3.0), where clear ovalbumin gels are formed upon heating under the conditions required for gelation. Developing a better understanding of the aggregation mechanism of globular proteins upon heat treatment is desirable because the process of aggregation governs gelation and subsequently the final properties of the formed gel. An improved understanding of how to control aggregation is needed for the rational design of food ingredients with desirable gelling or novel functional properties. (5) Hegg, P. O. J. Food Sci. 1982, 47, 1241-1244. (6) Doi, E.; Kitabatake, N. Food Hydrocolloids 1989, 3, 327-337. (7) Kato, A.; Nagase, Y.; Matsudomi, N.; Kobayashi, K. J. Agric. Chem. 1983, 47, 1829-1834. (8) Koseki, T.; Kitabatake, N.; Doi, E. Food Hydrocolloids 1989, 3, 123-134. (9) Koseki, T.; Kitabatake, N.; Fukuda, T.; Doi, E. Food Hydrocolloids 1989, 3, 135-148. (10) Tani, F.; Murata, M.; Higasa, T.; Goto, M.; Kitabatake, N.; Doi, E. J. Agric. Food Chem. 1995, 43, 2325-2331. (11) Kitabatke, N.; Doi, E. Food Rev. Int. 1993, 9 (4), 445-471. (12) Nemoto, N.; Koike, A.; Osaki, K.; Koseki, T.; Doi, E. Biopolymers 1993, 33, 551-559.

10.1021/la0263108 CCC: $25.00 © 2003 American Chemical Society Published on Web 02/19/2003

AFM Study of Ovalbumin

In this work, surface topographical information for globular protein structures and for protein films has been obtained in fluid-tapping13 and electrical double layer14,15 imaging modes, respectively. The force of interaction between the atomic force microscope (AFM) tip and the protein film has also been mapped. Aggregate dimensions and differences in the elastic properties of the protein films are reported. Structural information obtained from the AFM modalities has been compared with gel electrophoresis data. Results from the AFM and gel electrophoresis measurements have allowed us to suggest a mechanism for the aggregation of ovalbumin. Materials and Methods Preparation of Ovalbumin Solutions. Ovalbumin (albumin, chicken egg, A-5503, 98% purity) was purchased from SigmaAldrich Chemical Co. (St. Louis, MO). Protein solutions for both gel electrophoresis and AFM experiments were prepared in the same way. Ovalbumin (1.0% w/w) was prepared in Milli-Q water and stirred at room temperature for 0.5 h to dissolve. The solutions were diluted to protein concentrations of 0.5 and 20 µg/mL (to give the final ionic strength of 1 mM, pH 3.0). For heat treatment, 2 mL aliquots of prepared ovalbumin protein solution were placed in capped thin 0.5 mm glass tubes (0.78 mm diameter), heated at 80.0 °C ( 0.1 in a thermostatically controlled water bath for 30 min, and then cooled to room temperature (25 °C). Although the heat-treated solutions contained soluble protein aggregates (as confirmed by gel electrophoresis, see below), they appeared as clear solutions and no solution turbidity reminiscent of typical biological aggregation was observed. Equipment Preparation. All glassware and the AFM fluidtapping cell were rinsed with a cleaning concentrate (Biorad Laboratories, CA), further rinsed with copious amounts of Milli-Q water (measured conductivity of not less than 18.2 MΩ cm), and dried with nitrogen gas that had been passed through a hydrocarbon/moisture trap (model HMT200-2, R&D Separations, Rancho Cordova, CA). Gel Electrophoresis. The unheated and heat-treated protein solutions were subjected to three running conditions: nativePAGE (polyacrylamide gel electrophoresis), SDS (sodium dodecyl sulfate), and SDS-reducing (in the presence of β-mercaptoethanol). The solutions were diluted with appropriate sample buffer (approximately 15 µg/µL per well) and then loaded onto precast Gradipore 4-20% gels. The run time for these gels was 90 min at an initial voltage of 150 V and 50 mÅ (Ready Gel Cell system, Biorad Laboratories). A Gradipore stain solution was used to stain the gels overnight. The gels were destained and kept in a 6% (v/v) acetic acid solution. Atomic Force Microscopy. AFM measurements were performed with a Nanoscope III MultiMode atomic force microscope (Digital Instruments, Santa Barbara, CA) using an E-scanner (10 µm × 10 µm × 3 µm). Freshly cleaved muscovite mica was used as a substrate in all investigations. Commercially available silicon nitride oxide-sharpened tips (Nanoprobes, Digital Instruments) were used for all AFM work. Fresh tip and mica were used with every sample change due to potential contamination of the tip with continuous imaging. Given that the point of zero net charge (pzc) of mica and ovalbumin is around pH ) 2 and pH ) 4-5, respectively,16 it was anticipated that the protein and substrate were oppositely charged at pH ) 3, leading to electrostatic adsorption. Further, adsorption onto the silicon nitride tip (with a pzc of pH ) 5-7) was anticipated to be minimal due to electrostatic repulsion. (13) Hansma, P. K.; Cleveland, J. P.; Radmacher, M.; Walters, D. A.; Hillner, P. E.; Bezanilla, M.; Fritz, M.; Vie, D.; Hansma, H. G.; Prater, C. B.; Massie, J.; Fukunaga, L.; Gurley, J.; Elings, V. Appl. Phys. Lett. 1994, 64, 1738-1740. (14) Senden, T. J.; Drummond, C. J.; Kekicheff, P. Langmuir 1994, 10, 358-362. (15) Fleming, B. D.; Wanless, E. J. Microsc. Microanal. 2000, 6, 104112. (16) Budavari, S.; O’Neil, M. J.; Smith, A.; Heckelman, P. E.; Kinneary, J. F. The Merck Index; Merck Research Laboratories: Whitehouse Station, NJ, 1996.

Langmuir, Vol. 19, No. 7, 2003 2881 For all AFM measurements, 40 µL of protein solution was carefully injected into the fluid cell using a 200 µL pipet tip through the inlet perpendicular to the cell, and both cell ends were sealed with plugs. Ninety degree angle images were also taken to diagnose possible artifacts or protein dragging. Images that remained the same with continuous up, down, and 90° angle scanning were considered as reproducible. Different areas of the mica were also scanned and captured to make sure that the images were representative. For all work, the images in the height mode were flattened and fitted to a third-order polynomial equation to remove the curvature induced by the systematic scanner bow. Tapping mode with simultaneous phase mode was used for obtaining images of isolated surface-adsorbed protein molecules/ aggregates in solution. Typical scan rates used were 1-2 Hz with scan sizes of up to 1 µm. Before imaging in tapping mode, the cantilever was tuned to the resonant frequency (typically 18-20 kHz). Electrical double layer mode with simultaneous deflection mode was used for obtaining images of surface-adsorbed protein films in solution. Before imaging in electrical double layer mode, the lateral scan axes were disengaged, and the force-separation curve of the tip versus sample was measured. As evident in the force measurements (see below), a repulsive force was found to exist between the tip and the protein sample. Therefore, the setpoint was adjusted to minimize the force of interaction between the tip and sample, thus limiting sample deformation. In force-volume mode (also referred to as force mapping), an array of regularly spaced force-separation curves are acquired across the sample surface, generating a family of force curves.17,18 Force-volume measurements were acquired immediately upon injection. In this work, 64 force-separation curves (8 × 8 matrix) were acquired over a 1 µm scan area of the ovalbumin film (20 µg/mL). For each approach and retract force curve acquired, AFM Analysis V2.0 software (Prof. D. Y. C. Chan, The University of Melbourne, Australia) was used to convert the z-deflection data into force (in nN) as a function of tip-sample separation (nm) using a measured spring constant of around 0.15 N/m. The method of added end mass of Cleveland et al.19 was used to determine the spring constant. Each force curve was then analyzed for film thickness, force on compression, and adhesion using spreadsheet operations.

Results and Discussion Gel Electrophoresis. A 1 mM ovalbumin solution (pH 3.0) was heat-treated for 30 min at 80 °C. Gel electrophoresis was used to characterize the formation of aggregates with increased heating time. The gel electrophoresis protein bands for native ovalbumin are presented in Figure 1. A major band at around 45 kDa corresponds to native ovalbumin (lane 2). Occasionally, a minor band was evident around 70 kDa, the origin of which is unknown and which probably reflects low analytical purity of the ovalbumin solution. The gel reveals that with increased heating time (after 1 min) high molecular weight aggregates (above 200 kDa) were formed. These aggregates were too large to enter the stacking gel and consequently they remained at the top of the gel (lane 3). Treatment of heat-treated ovalbumin samples with sodium dodecylsulfate (lane 4) revealed that the aggregates were dissociated into monomers, indicating that they were predominantly linked by noncovalent interactions. Atomic Force Microscopy. Topography. The surface topography of ovalbumin on mica at a concentration of 0.5 µg/mL was measured in both fluid-tapping and electrical double layer modes. Images obtained in the latter mode were poorly resolved, probably because the electrostatic (17) Radmacher, M.; Cleveland, J. P.; Fritz, M.; Hansma, H. G.; Hansma, P. K. Biophys. J. 1994, 66, 2159-2165. (18) Considine, R. F.; Dixon, D. R.; Drummond, C. J. Langmuir 2000, 16, 1323-1330. (19) Cleveland, J. P.; Manne, S.; Bocek, D.; Hansma, P. K. Rev. Sci. Instrum. 1993, 64, 403-405.

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Figure 1. Gel electrophoresis patterns of native ovalbumin before heat treatment (lane 2), after heat treatment (80 °C, 30 min) (lane 3), and after dispersion of heat-treated ovalbumin with SDS (lane 4). Molecular weight markers are shown in lane 1: 200 kDa, myosin; 97 kDa, phosphorylase b; 66 kDa, serum albumin; 45 kDa, ovalbumin; 31 kDa, carbonic anhydrase.

Figure 3. The vertical height (A) and half-height (B) diameter distribution for native (white) and heat-treated (80 °C, 30 min, dashed) ovalbumin (pH 3.0) at 0.5 µg/mL. In both cases, each histogram corresponds to a total of 50 heights measured over two different areas of the mica over a 1 µm × 1 µm scan.

Figure 2. A tapping mode height image of native (A) and heattreated (80 °C, 30 min) ovalbumin (B) at pH 3.0 (0.5 µg/mL). The height z-range is 10 nm.

attraction of the tip and mica resulted in high deformation forces at the mica/protein/tip interface. Height images obtained in fluid-tapping mode are shown for native (Figure 2A) and heat-treated (Figure 2B) ovalbumin. Both the native and heat-treated protein structures appear globular and compact, which is consistent with the globular conformation observed by X-ray crystallography.20 On

comparison of the native and heated ovalbumin, the heattreated ovalbumin appears to be distributed in substantially larger structures than the native ovalbumin. The dimensions of the ovalbumin structures of Figure 2 have been quantified, using section analysis, in terms of the vertical height (Figure 3A) and half-height diameter (Figure 3B). The vertical height corresponds to the maximum vertical distance measured from the top of the protein surface to neighboring mica. This value corresponds to a lower boundary for the maximum height of the protein structure. The half-height horizontal diameter corresponds to the measured diameter of the protein structure at half the vertical height. This measurement has been used to describe the lateral dimension of the protein structure, rather than the horizontal diameter at zero vertical height, to minimize the influence of tipinduced broadening.21 Image quantification must be applied with caution, since a variety of factors may influence the measured heights.22-24 Nonetheless, image quantification has been applied in the present study for the purpose of comparison with established molecular dimension data (crystallography). The vertical height for the native ovalbumin structures at 0.5 µg/mL was measured to be 1.5-2.5 nm, and the average half-height diameter was in the range of 5.5-6 nm (Figure 3). The dimensions of monomeric ovalbumin (20) Stein, P. E.; Leslie, A. G. W.; Finch, J. T.; Carrel, R. W. J. Mol. Biol. 1991, 221, 941-959. (21) Villarrubia, J. S. J. Res. Natl. Inst. Stand. Technol. 1997, 102, 425. (22) Weisenborn, A. L.; Khorsandi, M.; Kasas, S.; Gotzos, V.; Butt, H.-J. Nanotechnology 1993, 4, 106-113. (23) Yang, J.; Mou, J.; Shao, Z. Biochim. Biophys. Acta 1994, 1199, 105-114. (24) Morozov, V. N.; Morozova, T. Y.; Kallenbach, N. R. Int. J. Mass Spectrom. 1998, 178, 143-159.

AFM Study of Ovalbumin

measured by X-ray crystallography20 data suggest a molecule of 2.9 nm × 3.5 nm × 7.2 nm. The measured dimensions are generally consistent with monomeric ovalbumin. The dimensions of the heat-treated ovalbumin at 0.5 µg/mL were vertical heights of 3.5-5.0 nm and half-height diameters of 14-17 nm, both dimensions being substantially larger than the corresponding dimensions for native ovalbumin. These results suggest that the surface properties of ovalbumin have been modified upon heating, causing the molecules to assemble into larger aggregates. This is in agreement with gel electrophoresis results (Figure 1), where high molecular weight aggregates were observed upon heating ovalbumin solutions. In direct contrast to AFM imaging in the presence of low protein concentrations (0.5 µg/mL), tapping mode proved less successful than electrical double layer mode at resolving protein structures in the presence of high protein concentration solutions (20 µg/mL). In fluidtapping mode, the images were dominated by streaks parallel to the fast scan axis (X), consistent with temporary dampening of the cantilever oscillatory motion brought about by adhesion of the cantilever with the protein film. Height images of native (Figure 4A) and heat-treated (Figure 4B) ovalbumin (20 µg/mL) obtained in electrical double layer mode are shown. As a result of the high coverage of the mica, a measure of the vertical height (and consequently the half-height diameter) of the protein structures was not possible by section analysis. In an attempt to measure the thickness of the film, the contact force of the AFM tip was temporarily increased within a 200 × 200 nm2 area of the previous 1 × 1 µm2 scan. Then the contact force was returned to electrical double layer mode. It was anticipated that the protein film would be displaced in the region of high contact force, permitting the measurement of the vertical height from the bare mica to the surface of a nearby region of undisrupted protein film. Images obtained in this way show large protein accumulation at either side of the previous 200 × 200 nm2 scan (see Figure 4A inset) consistent with film disruption during the high contact force. However, the surface topography of the area scanned at high force appears to have the same topography and relative height as the surrounding areas of undisrupted film. This observation suggests that rapid redeposition of the protein occurred on the uncovered mica before a complete scan could be obtained. As an alternative approach, estimates of vertical height (in terms of film thickness) were obtained from force curve measurements upon AFM tip-film approach (see below). Unlike the native ovalbumin film, the heat-treated ovalbumin appears to have formed a relatively heterogeneous film (Figure 4B) which was largely composed of globular structures. Upon close inspection of the images (see 200 × 200 nm2 scan, inset Figure 4B), many of the globules appeared to be assembled into curl-like elongated structures. Certainly, electron microscopy and light scattering results at neutral pH have shown heat-induced ovalbumin aggregates to assemble into linear globular string-of-bead structures.6,8,10-12,25 This would be the first AFM study to support such a hypothesis. Forces of Interaction. The force of interaction (nN) between ovalbumin (20 µg/mL) and the AFM tip was measured as function of separation from hard-wall contact (nm). In this work, separation values have been referenced to the region of linear compliance in the raw deflection (25) Shirai, N.; Tani, F.; Higasa, T.; Yasumoto, K. J. Biochem. 1997, 121, 787-797.

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Figure 4. Electrical double layer mode images of native (A) and heat-treated (80 °C, 30 min) ovalbumin (B) at 20 µg/mL (pH 3.0). The inset in panel A is a 200 nm zoom of the scratched protein film (see the text for more detail). The inset in panel B is a 200 nm zoom in which the arrows point to observed linearly linked globular aggregates.

versus displacement data. On comparison, the slopes of linear compliance before and after protein addition were found to be essentially equivalent. Therefore, it is likely that the definition of zero separation used in this work corresponds to the tip-mica (hard-wall) separation. Quantitative analysis of force measurements procured in the AFM usually requires normalization of the force axis by the radius of the force probe (in this case, the AFM tip). However, approximating the tip geometry can introduce substantial quantitative error at very short separations,26 and this has been avoided in the present study by reporting the force axis in terms of the force (nN) rather than force/ radius (mN/m). Force-separation curves were aquired in an 8 × 8 matrix over a 1 µm scan area (i.e., 1 force curve/0.016 um2) of the ovalbumin film (at 20 µg/mL, pH 3.0). Example force curves for native ovalbumin are presented in Figure 5A; the labels 1A, 2C, 3B, and 8F correspond to the (26) Drummond, C. J.; Senden, T. J. Colloids Surf., A 1994, 87, 217234.

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Figure 5. Panel A shows examples of force-separation curves acquired in a 1 × 1 µm2 scan area of the ovalbumin film (at 20 µg/mL, pH 3.0). The labels 1A, 2C, 3B, and 8F correspond to the locations within the 8 × 8 matrix (1-8 × A-H). A representative force-separation curve (taken at location 1B) showing data within 15 nm of zero separation is shown in panel B. See the text for a detailed description of steps 1-7. The screening length of the repulsive force shown in the inset of panel B is approximately 10 Å.

locations within the 8 × 8 matrix (1-8 × A-H). A representative force-separation curve (taken at location 1B) shown for data within 15 nm of zero separation has been provided in Figure 5B. Initially the AFM tip and ovalbumin film are separated at long range (1), and zero force of interaction exists between the tip and sample. On approach, an exponential repulsive force is encountered (2) followed by the onset of steep repulsion. Typically the steep repulsion proceeds by either single or multiple steps (3) until the AFM tip and sample are in hard-wall contact (5). As the piezo movement is reversed, the AFM tip moves through zero force (6) and is caught in an adhesive well (7); often subsequent adhesive events occur (8) until the AFM tip and the protein film are out of contact (9). The region of steep repulsion (location 2) proceeding by a step (3) is consistent with a pseudo-compliance of the AFM tip and the protein surface followed by collapse of the protein film. The final steep repulsion prior to hard-wall contact (location 4) is consistent with depletion of the collapsed protein film from the intervening gap between the AFM tip and the mica. The adhesive events (locations 7 and 8 of Figure 5B) are consistent with the breakage of multiple bonds in a protein bridge of the AFM tip and mica. The general profile of the force-separation data is consistent with earlier reports of the force of interaction between globular proteins absorbed at surfaces in aqueous solution.27

Najbar et al.

The origin of the exponential repulsive force on approach (region 2 of Figure 5B) may be due to electrical double layer overlap and as such can be characterized by a screening length. It has already been shown that aqueous solutions of globular proteins behave as asymmetric electrolytes and that the measured screening lengths can be considerably shorter than the classical Debye length.27,28 The corresponding screening length of the repulsive force shown in the inset of Figure 5B is approximately 10 Å. Both X-ray crystallography and AFM topography measurements suggest monomeric ovalbumin dimensions of at least 2 nm in the shortest dimension. Therefore, it is unlikely that free ovalbumin aggregates in solution contribute significantly to the electrical double layer. In light of this, a more reasonable notion of the electrical double layer is one of an asymmetric electrolyte consisting of free amino acid side chains of the protein film extending into solution. The electrical double layer was not always apparent, with a proportion of curves only exhibiting region 4 (linear compliance of the AFM tip and the protein surface) prior to hard-wall contact. For native ovalbumin, 61% of force-separation curves on approach exhibited typical electrical double layer behavior (per above), while such behavior was only apparent in 26% of the curves for heat-treated ovalbumin. The existence of regions with an apparent absence of an electrical double layer may be a reflection of regions of low surface charge density. That is, the effect of heat treatment on the ovalbumin surface can be described as inducing a higher proportion of low surface charge density domains. All force-separation curves on approach were analyzed for characteristic parameters describing the effective thickness and the force of compression of the protein film. When the AFM tip and the protein film are in “pseudo” linear compliance (region 4 in Figure 5B), the protein surface and the cantilever can be considered as two springs in series. As shown by Ducker et al.,29 the effective spring constant (Kp) of the deformable surface (in this case, protein) can be expressed as

Kp )

(

Ks

Ks -1 Km

) ( )

Ks

Ch -1 Cp

)

(1)

where Ks is the spring constant of the cantilever, Km is the measured stiffness of the surface, and Ch and Cp are the cantilever deflection per unit sample translation against the solid surface (mica) and the protein film, respectively. The effective spring constant of the protein film is a measure of film compressibility, since it corresponds to the resistance of the film to deformation. In addition, the separation that corresponds to the onset of linear compliance of the AFM tip and protein surface can be considered a measure of the effective protein film thickness. Surface maps of the effective thickness and interfacial spring constant of both native and heat-treated ovalbumin are presented in Figure 6. The values of the effective film thickness of the native protein (Figure 6A) were measured to be approximately 1.5-2.5 nm, indicative of a single layer of monomeric ovalbumin. The effective film thickness values of the heat-treated ovalbumin were in the range of 2-4 nm (Figure 6B), consistent with the presence of (27) Nylander, T.; Kekicheff, P.; Ninham, B. W. J. Colloid Interface Sci. 1994, 164, 136-150. (28) Marra, J.; Hair, M. J. J. Colloid Interface Sci. 1989, 128, 511522. (29) Ducker, W. A.; Xu, Z.; Israelachvili, J. N. Langmuir 1994, 10, 3279-3287.

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in agreement with the more heterogeneous surface topography of the heat-treated ovalbumin apparent in the electrical double layer imaging results (see Figure 4B). The overlap of values in film thickness implies that a proportion of the heat-treated molecules maintain their monomer dimensions upon heating. The maps of the interfacial spring constant of native ovalbumin (Figure 6C) reveal broad regions of values in the range of 0.015-0.04 N/m, while the spring constant map of heat-treated ovalbumin (Figure 6D) is dominated by values in the range of 0.005-0.02 N/m. The reduction of Kp upon heating is consistent with a weakening of intramolecular interactions within the protein network, resulting in an increased susceptibility to deformation. This is not surprising as in general, when proteins are heat-treated above their denaturation temperature (which is 76 °C for ovalbumin at pH 3.0 from our differential scanning measurements), their internal hydrophobic interactions become weak and this often results in some unfolding of the molecules and their expansion or “softening”.30 The force-separation curves on retraction (see Figure 5) contain multiple adhesion events that occur beyond separations commonly associated with the adhesion between the AFM tip and mica (typically less than a few nanometers31). The adhesion events were found to occur predominantly within the first 10-20 nm of zero separation on retraction, although occasional events as far as 68 nm were observed (less than 1% of curves exhibit this kind of feature). The multiple adhesion events are consistent with ovalbumin bridging the AFM tip and the underlying mica; similar conclusions have been drawn from a variety of AFM adhesion measurements of biopolymeric and polyelectrolyte materials.32,33 The mode of adhesion is probably via van der Waals, electrostatic, and hydrogen-bond interactions formed during contact between the AFM tip and protein. The adhesion events at very long range may actually correspond to unwinding of a single ovalbumin molecule. For comparison, a rough estimate of the length of a completely extended molecule of ovalbumin is 132 nm. The characteristic separations at which regular adhesion events occur at short range can be correlated with a monomer length of a polymeric bridge between the AFM tip and the surface.34,35 The detachment frequency for the first 15 nm from zero separation of both native and heat-treated ovalbumin on retraction is shown in Figure 7. For the native protein (Figure 7A), adhesion events generally occurred at regular 2-3 nm intervals, indicative of a polymeric bridge composed of monomeric units each of 2-3 nm in length. This is consistent with the shortest dimension of monomeric ovalbumin from fluid-tapping at 0.5 µg/mL (Figure 3A) and the effective film thickness obtained at 20 µg/mL (Figure 6A). An interesting point to note is that similar regular detachments of 2-3 nm were also observed for the heat-treated protein (Figure 7B). This is in contrast to the dimensions observed by fluid-tapping and electrical double layer modes, where larger globular structures were observed upon heat treatment. It is possible, therefore, that the Figure 6. Surface maps of the effective film height (A,B) and interfacial spring constant (C,D) of native (A,C) and heat-treated ((B,D) 80 °C, 30 min) ovalbumin (pH 3.0) at 20 µg/mL. Each surface map was constructed from 64 force curve measurements in a 8 × 8 matrix taken over a 1 × 1 µm2 scan.

larger globular structures, as shown in the gel electrophoresis and fluid-tapping measurements. On inspection, the film thickness of heat-treated ovalbumin is relatively heterogeneous as compared to the native protein. This is

(30) Basiuk, V. A. In Biopolymers at interfaces; Surfactant Science Series, Vol. 75; Marcel Dekker: New York, 1998; pp 55-84. (31) Senden, T. J.; Drummond, C. J. Colloids Surf., A 1995, 94, 2951. (32) Dammer, U.; Popescu, O.; Wagner, P.; Anselmetti, D.; Guntherodt, H.-J.; Misevic, G. N. Science 1995, 267, 1173-1175. (33) Chatellier, X.; Senden, T. J.; Joanny, J.-F.; Di Meglio, J. M. Europhys. Lett. 1998, 41, 303-308. (34) Haupt, B. J.; Ennis, J.; Sevick, E. M. Langmuir 1999, 15, 38863892. (35) Ortiz, C.; Hadziioannou, G. Macromolecules 1999, 32, 780-787.

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Figure 7. The detachment frequency on retraction for the first 15 nm from zero separation for native (A) and heat-treated (B) ovalbumin (80 °C, 30 min), pH 3.0, 20 µg/mL. The arrows point to regular successive adhesion events in the order of 2-3 nm.

large globular structures may be composed of monomeric ovalbumin units. This would be consistent with the disassociation of the heat-formed aggregates into monomeric ovalbumin upon addition of SDS observed by gel electrophoresis (see Figure 1), implicating aggregates of noncovalently linked monomeric ovalbumin units. Aggregation Mechanism of Gel Precursors. Various models of the heat-induced aggregation of ovalbumin have been proposed.6,8,10-12,25 In the majority of cases, these were based on estimates of aggregate size (e.g., using light scattering and gel permeation chromatography) and structural information (transmission electron microscopy). Building upon the earlier work, a model of heat-induced aggregation is now proposed utilizing dimensional and molecular weight measurements of ovalbumin, combined with the new information regarding the modification of surface forces and mechanical properties upon heat treatment. The proposed model of this aggregation is depicted in Figure 8. Upon heat treatment of ovalbumin, a reduction of the intramolecular interactions of monomeric protein is invoked, as indicated by a moderate reduction in the interfacial spring constant from surface force experiments. This effect is probably a consequence of partial unfolding of ovalbumin upon heat treatment, which has been well documented.36 The compact globular form of ovalbumin observed by our AFM studies would be consistent with a structural state termed the “molten (36) Hagolle, N.; Launay, B.; Relkin, P. Colloids Surf., B 1998, 10, 191-198.

Najbar et al.

Figure 8. A proposed model for heat-induced aggregation of ovalbumin gel precursors. The small round black regions correspond to exposed low charge or hydrophobic regions on the protein surface. For a detailed description, refer to the text.

globule state”.10,37,38 This structural form has previously been reported for globular proteins and ovalbumin in acidic conditions.8,39 In this form, the protein exists in a compact globule similar to that of the native form, with its secondary structure mostly intact but with fluctuating tertiary structure. The resulting secondary structure of ovalbumin is almost the same at acidic and neutral pHs, but the side chains in the molecules are more flexible at lower pH,40 that is, the protein is partially unfolded. Although in general the structure and adsorption properties of the molten globule state at surfaces have not been examined before, this partially unfolded (though compact) state may explain why the heat-treated molecules could be tethered to longer distances compared to the native ovalbumin. In this work, a reduction in the electric double layer surface forces between the monomers of ovalbumin and the AFM tip upon heating can be interpreted as the effect of partial unfolding leading to the exposure of regions of low charge density. These newly exposed surface regions lead to increased attraction between the monomers, which link in a noncovalent manner to form higher molecular weight globular aggregates. These globular aggregates are composed of many monomers, which are noncovalently (37) Kuwajima, K. Proteins: Struct., Funct., Genet. 1989, 6, 87-103. (38) Dolgikh, D. A.; Gilmanshin, R. I.; Brazhnikov, E. V.; Bychkova, V. E.; Semisotnov, G. V.; Yu, S.; Venyaminov, G. V.; Ptitsyn, O. B. FEBS Lett. 1981, 136, 311-315. (39) Tatsumi, E.; Hirose, M. J. Biochem. 1997, 122, 300-308. (40) Mine Y.; Noutomi, Y.; Haga, N. J. Agric. Food Chem. 1991, 39, 443-446

AFM Study of Ovalbumin

bound, the globules further linking into linear structures as evidenced by electrical double layer mode imaging. The force-separation data and gel electrophoresis suggest that the inherent monomeric dimensions of ovalbumin within the globules are retained. It has been suggested that gelation probably arises from subtle surface contacts between a small number of exposed hydrophobic residues of heat-treated ovalbumin.41 It is probably the surfaceexposed hydrophobic regions that give rise to attraction between the higher molecular weight globules allowing them to assemble into what appear to be linear structures. Conclusions The application of three different AFM modes, viz., fluidtapping, electrical double layer, and force-volume modes, was required to probe the topography, heat-induced aggregation mechanism, and mechanical properties of ovalbumin at the molecular level. This multimode imaging approach has shown that fluid-tapping was more suitable (41) Egensdal, B. Int. J. Pept. Res. 1986, 28, 560-568.

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for imaging single globular structures but electrical double layer imaging was more suitable when a film covering the entire mica surface was formed. Film coverage was also essential for force-volume measurements. The results from this study suggest that the heat-induced precursor aggregates are globular and compact and are composed of noncovalently linked monomers of ovalbumin. Acknowledgment. L.N. gratefully acknowledges the support of Dr. John Pearce (formerly Food Science Australia), Dr. Geoff Smithers (Food Science Australia), and Dr. Regina Stockmann (Food Science Australia). L.N. also thanks Dr. Erica Wanless (University of Newcastle) and Dr. Mirjana Prica (Food Science Australia) for assistance in the initial AFM investigations. R.C. gratefully acknowledges the support and friendship of S. Gillies. R.C. was the recipient of an Australian Post-Graduate Research Award. We also thank the University of South Australia and the Cooperative Research Centre for Water Quality and Treatment for support. LA0263108