Effect of pH on Structural, Topographical, and Dynamic Characteristics

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Effect of pH on Structural, Topographical, and Dynamic Characteristics of Soy Globulin Films at the Air-Water Interface Cecilio Carrera Sańchez,† Sara E. Molina Ortiz,‡ Ma. Rosario Rodrı´guez Ninõ,† Ma. Cristina Anõn,‡ and Juan M. Rodrı´guez Patino*,† Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, c/. Prof. Garcıá Gonza´ lez, s/nu´ m. 41012-Sevilla, Spain, and Centro de Investigacio´ n y Desarrollo en Criotecnologıá de Alimentos (CIDCA), Facultad de Ciencias Exactas, Universidad Nacional de la Plata, Calle 47 y 116, 1900-La Plata, Argentina Received January 28, 2003. In Final Form: June 12, 2003 Structural and topographical characteristics of two major fractions of soy globulin from a soy protein isolate, β-conglycinin (a 7S globulin) and glycinin (a 11S globulin), including the effect of chemical reduction of glycinin with dithiothreitol (DTT), spread at the air-water interface at 20 °C and as a function of pH were determined from π-A isotherms coupled with Brewster angle microscopy. The structural characteristics of 7S and 11S globulin spread monolayers depend on film aging. We have observed a significant shift of the π-A isotherms toward higher molecular areas over time. The aging effect was due to unfolding of the protein at the interface. The monolayer structure was more expanded on the aqueous subphase at pH 2.0, and the opposite was observed at pH 5.0. The chemical reduction of glycinin with DTT produced a significant expansion of the monolayer structure. A change in the monolayer structure was observed at a surface pressure of 13.5-16.5 mN/m. At a microscopic level, the heterogeneous monolayer structures visualized at pH 5.0 or near to the monolayer collapse proved the existence of large regions of protein aggregates. Relative reflectivity increases with surface pressure and was a maximum at the monolayer collapse, with the maximum reflectivity for glycinin at every pH.

* To whom correspondence should be addressed. Fax: +34 95 4557134. Phone: +34 95 4556446. E-mail: [email protected]. † Universidad de Sevilla. ‡ Universidad Nacional de la Plata.

levels of lysine and low fat content,11 and an ability to improve texture12,13 has increased the use of soy proteins. Globulins account for about 50-90% of seed proteins. Globulins 7S and 11S are two major storage proteins in soybeans. The 11S globulin has a quaternary structure composed of 12 subunits with a molecular weight of about 360 kDa. It is a dimer of two identical hexamers. Three of these subunits in the hexamer are acidic and the other three are basic in nature. Each pair of acidic and basic subunits is linked via a disulfide bond. Native soy glycinin because of its compact tertiary structure, which is stabilized by disulfide cross-linking, has limited foaming7,8,14,15 and emulsifying7,8,16,17 properties. However, reduction of some disulfide bonds may improve their foaming and emulsifying ability by allowing greater conformational flexibility.18,19 The quaternary structure of 7S globulin is made up of three subunits, with a molecular weight of about 180 kDa. The 7S globulin is a glycoprotein which does not contain disulfide bonds. The quaternary structure of 7S and 11S is affected by environmental factors such as pH, ionic strengths, and temperature,7,8,20 with repercussions in functional prop-

(1) Dickinson, E.; McClements, D. J. Advances in Food Colloids; Blackie: Glasgow, 1995. (2) Dickinson, E. Colloids Surf., B 1999, 15, 161. (3) Walstra, P. Chem. Eng. Sci. 1993, 48, 333. (4) Clark, D. C.; Wilde, P. J. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; p 267. (5) Dalgleish, D. G. In Food Proteins and their Application; Damodaran, S., Paraf, A., Eds.; Marcel Dekker: New York; 1997; pp 199223. (6) Benjamins, J. Static and dynamic properties of proteins adsorbed at liquid interfaces. Ph.D. Thesis, University of Wageningen, Wageningen, The Nederlands, 2000. (7) Kinsella, J. E. J. Am. Oil Chem. Soc. 1979, 56, 242. (8) Utsumi, S.; Matsumura, Y.; Mori, T. In Food proteins ad their application; Damodaran, S., Paraf, A., Eds.; Marcel Dekker: New York, 1997; pp 257-291. (9) Zayas, J. F. Functionality of proteins in food; Springer-Verlag: Berlin, 1997.

(10) Hettiarachchy, N. S.; Kalapathy, U. In Functional properties of proteins and lipids; Whitaker, J. R., Fereidoon, S., Munguia, A. L., Yada, R. Y., Fuler, G., Eds.; American Chemical Society: Washington, DC, 1998; pp 80-95. (11) Riaz, N. M. Cereal Foods World 1999, 44, 88. (12) Renkema, J. M. S.; Knabben, J. H. M.; van Vliet, T. Food Hydrocolloids 2001, 15, 407. (13) van Vliet, T.; Martin, A. H.; Bos, M. A. Curr. Opin. Colloid Interface Sci. 2002, 7, 462. (14) Wagner, J. R.; Gue´guen, J. J. Agric. Food Chem. 1999, 47, 2173. (15) Yu, M. A.; Damodaran, S. J. Agric. Food Chem. 1991, 39, 1563. (16) Liu, M.; Lee, D.-S.; Damodaran, S. J. Agric. Food Chem. 1999, 47, 4970. (17) Wagner, J. R.; Gue´guen, J. J. Agric. Food Chem. 1999, 47, 2181. (18) German, J. B.; O’Neill, T. E.; Kinsella, J. E. J. Am. Oil Chem. Soc. 1985, 62, 1358. (19) Kim, S. H.; Kinsella, J. E. J. Food Sci. 1987, 52, 128.

Introduction The protein film structure is important from a practical point of view because it defines its utility. The precise role of protein structure and how its structural transformation in a food contributes to functional properties are not well understood and are the topic of much research.1,2 The physical principles governing the formation and stability of food colloids (foams and emulsions) are complex, especially if protein macromolecules are involved as emulsifiers.3,4 Although the study of food dispersed systems generated from proteins is dominated by research into milk proteins,5,6 there exists an increasing interest in the use of vegetable proteins from legumes7,8 for the formation and stabilization of food emulsions and foams. Soy proteins exhibit high functional properties compared to other plant proteins.8-10 The demand for safe, highquality, health foods with good nutritional value, high

10.1021/la034143l CCC: $25.00 © 2003 American Chemical Society Published on Web 07/10/2003

Soy Proteins at the Air-Water Interface

erties7,8,14,17,19-23 of these proteins. The strong pH dependence of the molecular conformation and the associated functional properties7,8,24,25 mean that the optimum functionality of soy proteins occurs at pH < 5, which limits their application as food ingredients. Thus, more research is required to resolve this and other issues related to the use of soy proteins in food formulations. In this work, we complement a previous study20 by investigating the structural characterization and topography of spread films of two major fractions of soy globulin from a soy protein isolate, β-conglycinin (a 7S globulin) and glycinin (a 11S globulin), including the chemical reduction of glycinin with 10 mM dithiothreitol (DTT), at the air-water interface as a function of pH and at 20 °C. With the exception of two preliminary studies,21,26 the structural characteristics of soy proteins at the air-water interface have not been systematically analyzed so far. The film structural characteristics were determined27-29 from π-A isotherms coupled with a microscopic, sensitive, effective, and noninvasive technique: Brewster angle microscopy (BAM). Materials and Methods Materials. To form the spread surface film, protein was spread in the form of a solution using water at pH 8 as a spreading solvent. The sample was stored at 4 °C, and all work was done without further purification. Samples for interfacial characteristics of soy protein films were prepared using Milli-Q ultrapure water and were buffered at pH 2.0 and 5.0. Analytical-grade acetic acid and sodium acetate for buffered solutions at pH 5.0 were used as supplied by Sigma (>95%) without further purification. HCl (analytical grade, Panreac) and KCl (analytical grade, Merck) were used for adjusting the pH to 2.0 and the ionic strength of the aqueous solutions, respectively. The ionic strength was 0.05 M in all the experiments. The isolation of β-conglycinin (fraction 7S) and glycinin (fraction 11S) soy globulins, solubility, and structural characterization (including scanning differential calorimetric analysis, polyacrylamide gel electrophoresis under native conditions, surface hydrophobicity, and fluorescence spectroscopy) of 7S and 11S globulins have been described elsewhere.20 Glycinin was reduced using 10 mM DTT (11S + 10 mM DTT) as described elsewhere.8,30 Surface Film Balance. Measurements of the surface pressure (π) versus average area per molecule (A) were performed on a fully automated Langmuir type film balance using a maximum area of 5.62 × 10-2 m2, as described elsewhere.31,32 The mean deviation was within (0.1 mN/m for surface pressure and (0.125 × 10-3 m2 per mg for area. The subphase temperature was controlled at 20 °C by water circulation from a thermostat, within an error range of (0.3 °C. The temperature was measured by a thermocouple located just below the air-water interface. Aliquots of aqueous solutions of soy protein fractions (1.3 × 10-4 to 1.5 × 10-4 mg/µL) at pH 8 were spread on the interface. (20) Molina, S.; Carrera, C.; Rodrı´guez Ninõ, Ma. R.; Anõń, C.; Rodrı´guez Patino, J. M. Colloids Surf., B, in press. (21) Wagner, J. R.; Gue´guen, J. J. Agric. Food Chem. 1995, 43, 1993. (22) Martin, A. H.; Grolle, K.; Bos, M. A.; Cohen Stuart, M. A.; van Vliet, T. J. Colloid Interface Sci. 2002, 254, 175. (23) Martin, A. H.; Bos, M. A.; van Vliet, T. Food Hydrocolloids 2002, 16, 63. (24) Petruccelli, S.; Anõń, M. C. J. Agric. Food Chem. 1996, 44, 3005. (25) Puppo, M. C.; Anõń, M. C. J. Food Sci. 1999, 64, 50. (26) Carrera, C.; Molina, S.; Rodrı´guez Ninõ, Ma. R.; Anõń, C.; Rodrı´guez Patino, J. M. Food Hydrocolloids, in press. (27) Rodrı´guez Patino, J. M.; Carrera, C. S.; Rodrı´guez Ninõ, Ma. R. Food Hydrocolloids 1999, 13, 401. (28) Rodrı´guez Patino, J. M.; Carrera, C. S.; Rodrı´guez Ninõ, Ma. R. Langmuir 1999, 15, 2484. (29) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, Ma. R.; Cejudo, M. J. Colloid Interface Sci. 2001, 242, 141. (30) Kim, S. H.; Kinsella, J. E. J. Agric. Food Chem. 1986, 34, 623. (31) Rodrı´guez Patino, J. M.; Ruı´z, M.; de la Fuente, J. J. Colloid Interface Science 1992, 154, 146. (32) Carrera, S. C.; Rodrı´guez Ninõ, Ma. R.; Rodrı´guez Patino, J. M. Colloids Surf., B 1999, 12, 175.

Langmuir, Vol. 19, No. 18, 2003 7479 To allow for spreading, adsorption, and rearrangements of the protein, 30 min was allowed to elapse before measurements were taken. The spreading method adopted in these experiments allows quantitative spreading of the proteins, as discussed elsewhere.26 The compression rate was 3.3 cm min-1, which is the highest value for which isotherms have been found to be reproducible in preliminary experiments with β-casein27 and globular proteins.29 Surface pressure-area isotherms were measured at different times for each monolayer in order to see the effect of the age of the film on its behavior. At each time, the film was subjected to three compression-expansion cycles at approximately 30 min, 70-90 min, and 225-400 min after spreading. Each isotherm was measured at least nine times using four new aliquots. All isotherms were recorded continuously by a device connected to the film balance and then analyzed off-line. Brewster Angle Microscope. A commercial Brewster angle microscope, BAM2, manufactured by NFT (Go¨ttingen, Germany) was used to study the topography of the monolayer. Further characteristics of the device and operational conditions were described elsewhere.27,28 The surface pressure measurements, area, and gray level as a function of time were carried out simultaneously by means of a device connected between the film balance and the Brewster angle microscope. These measurements were performed during continuous compression and expansion of the monolayer at a constant rate with different shutter speeds ranging from 1/50 to 1/500 s. A shutter speed of 1/50 s is appropriate for the precise analysis of soy protein films at the air-water interface at low surface pressures, but higher shutter speeds (1/250 or 1/500 s) are more appropriate for the analysis of soy protein films at higher surface pressures.26 The reflectivity at each point in the BAM image depends on the local thickness and film optical properties. These parameters can be measured by determining the light intensity at the camera and analyzing the polarization state of the reflected light. At the Brewster angle,33 I ) Cδ2, where I is the relative reflectivity, C is a constant, and δ is the relative film thickness.

Results Structural, Topographical, and Aging Characteristics of β-Conglycinin (Fraction 7S) Monolayers. Aqueous Subphase at pH 2.0. The π-A isotherms for β-conglycinin spread monolayers at the air-water interface at pH 2.0 and at 20 °C are shown in Figure 1A. These isotherms were plotted assuming that all spread protein molecules form the monolayer and no loss into the bulk aqueous phase takes place. There was a difference in the π-A isotherms as a function of time after protein spreading. It can be seen that there is a shift of the isotherms toward higher molecular areas as the spreading time increases. These experiments were repeated at the same waiting time after the protein spreading for an undisturbed monolayer. The π-A isotherms obtained under these conditions matched those obtained after consecutive compression-expansion of the same monolayer (Figure 1A). On the other hand, the rate of monolayer compression did not have a significant effect on the π-A isotherm (data not shown) and the isotherms were reproducible after repeated experiments using new aliquots of the protein spreading solution, for the same aging time. These results demonstrate that it is possible to measure reproducible π-A isotherms with the protocol adopted in this work as discussed elsewhere.26 β-Conglycinin monolayers at the air-water interface adopt two different condensation states and the collapse phase (Figure 1A). As for β-conglycinin monolayers at pH 8.0,26 the surface pressure at which this transition takes place also depends on the aging time (Figure 1A). For the third compression of the monolayer (at 340 min after the protein spreading), the transition toward a more con(33) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1992.

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Figure 1. The effect of aging time on (A) the surface pressurearea isotherm at the air-water interface (compression curve). Symbols: (0) 30 min after spreading, (4) 70 min after spreading, and (O) 340 min after spreading. The continuous line represents the isotherm compressional coefficient (κ). (B) Relative reflectivity as a function of surface pressure for β-conglycinin monolayers spread at the air-water interface. Symbols: (0) 30 min after spreading, shutter 1/50 s; (4) 70 min after spreading, shutter 1/250 s; and (O) 340 min after spreading, shutter 1/250 s. Temperature ) 20 °C. pH ) 2.0. I ) 0.05 M. The equilibrium surface pressure (πe) and the surface pressure at the transition between two condensation states (πt) are indicated by means of two arrows, respectively.

densed monolayer structure was observed at 15.3 mN/m, as confirmed by the isotherm compressional coefficient deduced from the slope of the π-A isotherm (κ ) -dπ/dA), which is included in Figure 1A as an example. The collapse was produced at a surface pressure higher than the equilibrium surface pressure, πe = 30.7 mN/m,20 which is indicated in Figure 1A by means of an arrow. This phenomenon indicates that the monolayer was in a metastable state at π > πe. However, for the amount of spread protein used in this work (in order to optimize the quantitative spreading of the protein), we were unable to observe the typical plateau27-29 for the protein collapse at lower molecular areas. Results of BAM, in particular the relative reflectivity (Figures 1B and 2) and topography (Figure 3) as a function of time and/or surface pressure obtained with β-conglycinin monolayers at pH 2.0, clearly show the same structural characteristics as those deduced from the π-A isotherms. It can be seen (Figure 2) that the relative reflectivity increases as the monolayer is compressed, passes through a maximum at the collapse point, and then decreases with the monolayer expansion. The plateau observed at the collapse point (Figure 2A) is due to a saturation of the camera (image completely white) at the lower shutter speed used in these experiments. The data shown in Figures 1B and 2 demonstrate the importance of the use of an appropriate shutter speed for the study of protein films over the overall range of the protein monolayer’s existence.26 The increase in reflected light intensity with surface pressure, and especially at the monolayer collapse, suggests an increase in the monolayer thickness from a more expanded to a more condensed structure, and a further

Carrera Sa´ nchez et al.

Figure 2. Time evolution of the relative reflectivity (O) and surface pressure (-) upon a compression-expansion cycle for β-conglycinin monolayers spread at the air-water interface at pH 2.0 and at 20 °C. (A) 30 min after spreading, shutter 1/50 s; (B) 70 min after spreading, shutter 1/250 s; and (C) 340 min after spreading, shutter 1/250 s.

increase as the monolayer collapse takes place. In fact, at the highest surface pressure I ) 6.03 × 10-6, which means that the monolayer thickness increased 1.2 times in relation to that at πe (at this surface pressure I ) 4.16 × 10-6). During the compression (Figures 1B and 2), the absence of significant reflectivity peaks proves that the thickness of the film is uniform over the interface (Figure 3A). The intensity peaks (Figure 2) at the end of the expansion and after the first compression (which were not representative of the overall topography of the monolayer) were due to the presence of large aggregates of collapsed protein (Figure 3B). BAM images for a collapsed film (Figure 3C) were practically the same as for other β-conglycinin structures. However, at the collapse point some folds were observed along the interface with different illumination (data not shown). At a microscopic level, the compression-expansion cycle was practically reversible within the time of the experiment because the I-time curve during the compression was practically the same as that during the expansion (Figure 2). Aqueous Subphase at pH 5.0. The π-A isotherms for β-conglycinin spread monolayers at the air-water interface at pH 5.0 and at 20 °C are shown in Figure 4A. As for pH 2.0, there was a shift of the isotherms toward higher molecular areas as film aging increased. In addition, the π-A isotherms obtained for an undisturbed monolayer matched those in Figure 4A. For the third compression of the monolayer (at 330 min after the protein spreading), a transition toward a more condensed monolayer structure was observed at 15.2 mN/m. The collapse was produced at a surface pressure higher than the equilibrium surface pressure, πe = 24.6 mN/m,20 which is indicated in Figure 4A by means of an arrow. However, the relative reflectivity (Figures 4B and 5) and topography (Figure 3) as a function


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Figure 3. Visualization of β-conglycinin monolayers spread at the air-water interface at 20 °C by Brewster angle microscopy. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm. Key: (A) BAM image at π ) 2.0 mN/m and at pH 2.0 (first compression), (B) BAM image at π ) 0 mN/m and at pH 2.0 (expansion and second and successive compressions), (C) BAM image at π ) 32.0 mN/m and at pH 2.0 (compression), (D) BAM image at π ) 0 mN/m and at pH 5.0 (compression), (E) BAM image at π ) 0 mN/m and at pH 5.0 (compression), (F) BAM image at π ) 20.0 mN/m and at pH 5.0 (compression), (G) BAM image at π ) 14.0 mN/m and at pH 5.0 (expansion), (H) BAM image at π ) 0 mN/m and at pH 5.0 (expansion), and (I) BAM image at π ) 0 mN/m and at pH 5.0 (expansion).

Figure 4. The effect of aging time on (A) the surface pressurearea isotherm at the air-water interface (compression curve). Symbols: (0) 30 min after spreading, (4) 85 min after spreading, and (O) 330 min after spreading. (B) Relative reflectivity as a function of surface pressure for β-conglycinin monolayers spread at the air-water interface. Symbols: (0) 30 min after spreading, shutter 1/50 s; (4) 85 min after spreading, shutter 1/250 s; and (O) 330 min after spreading, shutter 1/250 s. Temperature ) 20 °C. pH ) 5.0. I ) 0.05 M.

of time and/or surface pressure show significant differences from those obtained with β-conglycinin monolayers at pH 2.0. The relative reflectivity increases as the monolayer is compressed, passes through a maximum at the collapse

point, and then decreases with the monolayer expansion (Figure 5). But, as for the π-A isotherms, at a microscopic level the reflectivity and topography of β-conglycinin monolayers at pH 5.0 depend on the monolayer aging. The topography and reflectivity during the compression and expansion of the monolayer depend on the monolayer history. Bright close-packed domains were observed during compression up to the monolayer collapse. Surprisingly, these microdomains (Figure 3D) were observed just after the monolayer spreading (at π ) 0 mN/m) alternating with some fractures of the monolayer (Figure 3E). These structures, like an ice-plate with numerous bright domains, which were in continuous formation/destruction, were characteristic for β-conglycinin monolayers at pH 5.0 during compression, but the brightness increased with the surface pressure (Figure 3F). The intensity peaks observed in the I-π plot during the monolayer compression (Figure 5A) prove the heterogeneity of the interface at a microscopic level. During the following expansion, after the collapse, the film undergoes the reverse evolution with quite similar morphologies observed during compression, including the presence of fractures in some regions of the monolayer (Figure 3G) in a short range of surface pressures, with 2D-foam formation at low pressures (Figure 3H,I). The presence of fractal structures at the maximum expansion of the monolayer was relevant (Figure 3I). However, the reflectivity peaks on expansion were present even at the maximum molecular area (at π ) 0 mN/m). Thus, it was evident that the condensed structure, which was attained during monolayer compression, was maintained during expansion, but with more intense reflectivity peaks than when the monolayer undergoes compression. Clearly, the compression-expansion process was not completely reversible within the time of the experiment due to the fact that the film adopted different states during the compression-expansion cycle. In fact, during the monolayer expansion there exists a relaxation time, during which

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Figure 5. Time evolution of the relative reflectivity (O) and surface pressure (-) upon a compression-expansion cycle for β-conglycinin monolayers spread at the air-water interface at pH 5.0 and at 20 °C. (A) 30 min after spreading, shutter 1/50 s; (B) 85 min after spreading, shutter 1/250 s; and (C) 330 min after spreading, shutter 1/250 s.

the monolayer structure attained at the end of compression was maintained. During successive compression-expansion cycles, the brightness of microdomains decreased and the reflectivity peaks as well (Figure 5B). Thus, during the third compression-expansion cycle the topography of the monolayer was practically homogeneous and the I-π plot was symmetrical during the compression and expansion of the monolayer (Figure 5C), with some reflectivity peaks due to the prevalence of regions of collapsed protein. That is, the hysteresis observed has a kinetic character. At the highest surface pressure I ) 5.06 × 10-6, which means that the monolayer thickness increases 1.42 times in relation to that at πe (at this surface pressure I ) 2.5 × 10-6). That is, the film thickness was higher as the monolayer collapsed. Structural, Topographical, and Aging Characteristics of Glycinin (Fraction 11S) Monolayers. Aqueous Subphase at pH 2.0. The π-A isotherms for glycinin spread monolayers at the air-water interface at pH 2.0 and at 20 °C are shown in Figure 6A. It can be seen that the π-A isotherms did not depend on film aging. The same data were obtained for an undisturbed monolayer at the same waiting time (data not shown). The relative reflectivity (Figure 6B) increased as the monolayer was compressed and was a maximum at the collapse point. At the highest surface pressure I ) 1.20 × 10-5, which means that the monolayer thickness increased 1.8 times in relation to that at πe (at this surface pressure I ) 3.75 × 10-6). The results of π-A isotherms (Figure 6A) indicated that glycinin monolayers at the air-water interface adopted two different condensation states and the collapse phase.


Figure 6. The effect of aging time on (A) the surface pressurearea isotherm for glycinin monolayers spread at the air-water interface (compression curve). Symbols: (0) 30 min after spreading, (4) 90 min after spreading, and (O) 345 min after spreading. (B) Relative reflectivity as a function of surface pressure for glycinin monolayers spread at the air-water interface. Symbols: (0) 30 min after spreading, shutter 1/50 s; (4) 90 min after spreading, shutter 1/250 s; and (O) 345 min after spreading, shutter 1/50 s. Temperature ) 20 °C. pH ) 2.0. I ) 0.05 M.

The transition toward a more condensed monolayer structure was observed at 13.5 mN/m, as confirmed by the isotherm compressional coefficient deduced from the slope of the π-A isotherm (data not shown). Glycinin monolayers collapsed at π > πe, πe ) 27.7 mN/m,20 which is indicated in Figure 6A by means of an arrow. Thus, the glycinin monolayer is in a metastable state at π > πe. BAM images (Figure 7) corroborated that only a homogeneous phase was present during the compression of glycinin up to the monolayer collapse (Figure 7A). That is, the topographies of glycinin monolayers with different states of condensation were practically indistinguishable. However, at the collapse point (at π > πe) some folds and protein aggregates were observed over the interface with different illumination (Figure 7B,C). Differences in the image contrast were an indication that collapsed residues of glycinin monolayers were aligned, on average, parallel to the barrier movement. Finally, after the expansion, the monolayer undergoes breakup of the collapse structure and some aggregated protein domains are present at the end of the expansion, at lower surface pressures (Figure 7D), but with a gel-like topography different from that observed for aggregates of β-conglycinin residues (Figure 3B). The absence of reflectivity peaks (Figure 6B) during the compression-expansion cycles proved the homogeneity in thickness and isotropy in the plane vertical to the molecular chain, as observed from BAM images (Figure 7). At a microscopic level, the compression-expansion cycle was reversible. That is, the reflectivity of the monolayer did not depend on the aging time (data not shown). Aqueous Subphase at pH 5.0. The π-A isotherms for glycinin spread monolayers at the air-water interface at pH 5.0 and at 20 °C are shown in Figure 8A. It can be seen that the π-A isotherms did depend on the film aging, with a shift of the isotherms toward higher molecular


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Figure 7. Visualization of glycinin monolayers spread at the air-water interface at 20 °C by Brewster angle microscopy. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm. Key: (A) BAM image at π ) 15.0 mN/m and at pH 2.0 (compression), (B) BAM image at π ) 30.0 mN/m and at pH 2.0 (compression), (C) BAM image at π ) 36.0 mN/m and at pH 2.0 (compression), (D) BAM image at π ) 1.0 mN/m and at pH 2.0 (expansion), (E) BAM image at π ) 10.0 mN/m and at pH 5.0 (compression), (F) BAM image at π ) 45.0 mN/m and at pH 5.0 (compression), (G) BAM image at π ) 36.0 mN/m and at pH 5.0 (compression), (H) BAM image at π ) 42.0 mN/m and at pH 5.0 (compression), and (I) BAM image at π ) 45.0 mN/m and at pH 5.0 (compression).

Figure 8. The effect of aging time on (A) the surface pressurearea isotherm for glycinin monolayers spread at the air-water interface (compression curve). Symbols: (0) 30 min after spreading, (4) 80 min after spreading, and (O) 345 min after spreading. (B) Relative reflectivity as a function of surface pressure for glycinin monolayers spread at the air-water interface. Symbols: (0) 30 min after spreading, shutter 1/50 s; (4) 80 min after spreading, shutter 1/250 s; and (O) 345 min after spreading, shutter 1/250 s. Temperature ) 20 °C. pH ) 5.0. I ) 0.05 M.

area as aging time increased. In addition, the π-A isotherms obtained for an undisturbed monolayer at the same waiting time matched those in Figure 8A. A transition toward a more condensed monolayer structure

was observed at 14.5 mN/m (Figure 8A). Glycinin monolayers collapsed at π > πe, πe ) 24.9 mN/m.20 At the highest surface pressure I ) 1.23 × 10-5, which means that the monolayer thickness increased 1.8 times in relation to that at πe (at this surface pressure I ) 3.96 × 10-6). BAM images (Figure 7) corroborated that a heterogeneous phase was present during the compression of glycinin up to the monolayer collapse, with numerous flickering microdomains (Figure 7E) which increased in brightness as the monolayer was compressed up to the collapse (Figure 7F). That is, the topography of glycinin monolayers with different states of condensation was practically indistinguishable. However, at π > πe glycinin collapsed via a fracturing mechanism in which the monolayer cracks cooperatively over large length scales giving some regions of collapsed proteins separated by other regions with a lower condensation state, observed over the interface from differences in illumination (Figure 7G-I). A difference in the image contrast was an indication that collapsed residues of glycinin monolayers were aligned, on average, parallel to the barrier movement. A shear plane can be distinguished during the monolayer compression (and during the expansion as well) between two regions of collapsed protein, as indicated in Figure 7G by means of two arrows. Finally, after the expansion, the monolayer undergoes breakup of the collapse structure and some aggregated protein domains were present at the end of the expansion, at the lower surface pressures (data not shown). The absence of reflectivity peaks (Figure 8B) during the compression-expansion cycles proved the homogeneity in thickness and isotropy in the plane vertical to the molecular chain, as observed from BAM images (Figure 7). At a microscopic level, the compression-expansion cycle was reversible. That is, the reflectivity and topography of the monolayer did not depend on the aging time (data not shown).

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Figure 9. The effect of aging time on (A) the surface pressurearea isotherm for glycinin + 10 mM DTT monolayers spread at the air-water interface (compression curve). Symbols: (0) 30 min after spreading, (4) 120 min after spreading, and (O) 225 min after spreading. (B) Relative reflectivity as a function of surface pressure for glycinin monolayers spread at the airwater interface. Symbols: (0) 30 min after spreading, shutter 1/50 s; (4) 120 min after spreading, shutter 1/250 s; and (O) 225 min after spreading, shutter 1/250 s. Temperature ) 20 °C. pH ) 2.0. I ) 0.05 M.

Structural, Topographical, and Aging Characteristics of Reduced Glycinin (Fraction 11S + 10 mM DTT) Monolayers. Aqueous Subphase at pH 2.0. The effect of 10 mM dithiothreitol on structural and topo-


graphical characteristics of glycinin films at the air-water interface at 20 °C and at pH 2.0 is shown in Figures 9 and 10. (i) As for untreated glycinin, the structural characteristics of glycinin + 10 mM DTT spread monolayers did not depend on film aging (Figure 9A). (ii) These monolayers adopt two different condensation states at the air-water interface (the transition between these states was observed at π = 14.3 mN/m) and the collapse phase (Figure 8A). The glycinin + 10 mM DTT monolayer is in a metastable state at π > 30.0 mN/m, which is the value of the equilibrium surface pressure at 20 °C.20 (iii) The relative reflectivity (Figure 9B) increased as the monolayer was compressed and was a maximum at the collapse point. At the highest surface pressure I ) 6.01 × 10-6, which means that the thickness of the glycinin + 10 mM DTT monolayer increased 1.25 times in relation to that at πe (at this surface pressure I ) 3.87 × 10-6). (iv) The reflectivity peaks observed during the monolayer compression (Figure 9B) reflected the fact that the thickness of the film was not uniform over the interface. In this regard, the glycinin + 10 mM DTT monolayer behaved in a different way from β-conglycinin and unreduced glycinin monolayers. In fact, the peaks of lower intensity observed in the glycinin + 10 mM DTT monolayer were not due to the aggregation of the protein at the interface, as for β-conglycinin, but were due to the existence of some fringes or holes of low thickness. (v) At a microscopic level, BAM images corroborated that a homogeneous phase was present only during the first compression of the glycinin + 10 mM DTT monolayer at low surface pressures (Figure 10A). At higher surface pressures, the presence of large dark holes (Figure 10B) or fringes (Figure 10C) which merge as the monolayer is compressed was observed over the interface, among a homogeneous bright region of collapsed protein (Figure 10D). The existence of these structures was characteristic

Figure 10. Visualization of monolayers of glycinin reduced with DTT spread at the air-water interface at 20 °C by Brewster angle microscopy. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm. Key: (A) BAM image at π ) 1.5 mN/m and at pH 2.0 (compression), (B) BAM image at π ) 12.0 mN/m and at pH 2.0 (compression), (C) BAM image at π ) 22.0 mN/m and at pH 2.0 (compression), (D) BAM image at π ) 23.0 mN/m and at pH 2.0 (compression), (E) BAM image at π ) 0.0 mN/m and at pH 2.0 (expansion), (F) BAM image at π ) 30.0 mN/m and at pH 5.0 (compression), (G) BAM image at π ) 33.0 mN/m and at pH 5.0 (compression), (H) BAM image at π ) 34.0 mN/m and at pH 5.0 (compression), and (I) BAM image at π ) 27.0 mN/m and at pH 5.0 (expansion).


Langmuir, Vol. 19, No. 18, 2003 7485

The absence of significant reflectivity peaks observed during the monolayer compression (Figure 11B) and topography (Figure 10F,G) reflected the almost uniform film over the interface. The absence of large dark holes was a difference of the topography of this protein at pH 5.0 in relation to that observed at pH 2.0 (Figure 10B,C). Another topographical characteristic of the glycinin + 10 mM DTT monolayer at pH 5.0 was the presence of numerous short fractures in the monolayer (Figure 10H). The existence of these small fractures led to large fractures defining a plane of shear between regions with different thicknesses during the monolayer expansion (Figure 10I). Discussion

Figure 11. The effect of aging time on (A) the surface pressurearea isotherm for glycinin + 10 mM DTT monolayers spread at the air-water interface (compression curve). Symbols: (0) 30 min after spreading and (O) 405 min after spreading. (B) Relative reflectivity as a function of surface pressure for glycinin monolayers spread at the air-water interface. Symbols: (0) 30 min after spreading, shutter 1/50 s; and (O) 405 min after spreading, shutter 1/250 s. Temperature ) 20 °C. pH ) 5.0. I ) 0.05 M.

for the glycinin + 10 mM DTT monolayer. These dark regions explain the existence of large reflectivity peaks of low intensity during the monolayer compression (Figure 9B). During the expansion after the collapse point (or for successive compressions), the holes of low protein density were compressed by regions of condensed proteins and a plane of shear between two regions of collapsed protein was defined, as indicated in Figure 10D by means of two arrows. These images proved that the regions with different states of condensation in the glycinin + 10 mM DTT monolayer were mutually immiscible, even at the highest surface pressures, close to the protein collapse. During the expansion of the monolayer, at the lower surface pressures, some collapsed residues maintained their aggregated structure (Figure 10E), given some intensity peaks during successive compressions (Figure 9B). (vi) Finally, at a microscopic level, the compressionexpansion cycle was practically reversible and the I-time curve did not depend on the aging time (data not shown). Aqueous Subphase at pH 5.0. The effect of 10 mM dithiothreitol on structural and topographical characteristics of glycinin films at the air-water interface at 20 °C and at pH 5.0 is shown in Figures 10 and 11. The structural characteristics of glycinin + 10 mM DTT spread monolayers did depend on the film aging (Figure 11A), with a more expanded structure as the time after the protein spreading increased. A change in the monolayer structure, with two different condensation states, was observed at π = 16.5 mN/m. The glycinin + 10 mM DTT monolayer was in a metastable state at π > 27.8 mN/m, which is the value of the equilibrium surface pressure at 20 °C.20 At the highest surface pressure I ) 6.03 × 10-6, which means that the thickness of the glycinin + 10 mM DTT monolayer increased 1.3 times in relation to that at πe (at this surface pressure I ) 3.87 × 10-6).

In this paper, we have sought to demonstrate that the structural and topographical characteristics of soy globulin (β-conglycinin and glycinin, including reduced glycinin) films spread at the air-water interface depend on the molecular structure of the protein and the pH of the aqueous phase. In a previous study, we have analyzed the β-conglycinin (fraction 7S) and glycinin (fraction 11S) structural characterization, hydrophobicity, and solubility as a function of pH.20 It was concluded that β-conglycinin would be in native conditions at pH 8.0; then it would be unfolded progressively at a decreasing pH. This conformational change is associated with a minor denaturation enthalpy, changes of thermal stability, and different exposure of the aromatic residues such as tryptophan and tyrosine. The first residue would be shifted to pH 2.0 as a consequence of the conformational change, to a more nonpolar region, giving rise to a greater exposure of tyrosine residues. At pH 5.0, the protein would produce aggregates due to its proximity to the isoelectric point. The glycinin fraction also undergoes structural changes due to the pH. In this case, the protein would be partially denatured and aggregated at pH 5.0, whereas at higher acidic pH it would be completely denatured and dissociated. Then, the conformational change at acidic pH would be paralleled by a greater exposure of tryptophan residues to a more polar region. The glycinin fraction treated with 10 mM DTT would be partially denatured at pH 8.0 and 5.0 and totally denatured at pH 2.0. The reduction process also produces an increase of the surface hydrophobicity of the molecule and an exposure of apolar groups.19 On the other hand, some differences were observed between surface activity of adsorbed protein and equilibrium spreading pressure for 7S, 11S, and 11S + 10 mM DTT. Poor surface activity was observed for pH 5.0. All these structural characteristics of the protein and their implication in interfacial characteristics of adsorbed and spread films at equilibrium also have an important effect on structural and topographical characteristics of spread films, as we have observed for pH 2.0 and 5.0 in the previous sections and for pH 8.0.26 Effect of the Aging of the Monolayer. The results of π-A isotherms for β-conglycinin at pH 5.0 (Figure 4A) and 8.026 and glycinin at pH 5.0 (Figure 8A) spread monolayers indicate that the structural characteristics of these systems depend on film aging. That is, the protein film changed significantly over time. In fact, for these soy globulins we have observed a significant shift of the π-A isotherms toward higher molecular areas after spreading. On the other hand, these results also show that the π-A isotherms registered after different aging times are practically parallel to each other. That is, the merging of these isotherms into one practically identical isotherm

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would be possible by the multiplication of the molecular area in the first π-A isotherm by a constant factor. In addition, we have also observed that the maximum surface pressure increased with aging time. This behavior is opposite to that observed with partial soluble lipids at the interface32 or for nonquantitative protein spreading.34,35 These results demonstrate that the aging effect observed in soy globulin monolayers is due to unfolding of the protein at the interface. However, we do not reject the possibility that a diffusional loss of aggregated protein takes place during the spreading process, which afterward is incorporated at the interface. This aging effect is characteristic of globular protein monolayers.35-37 It has been suggested36 that for globular protein a two-stage unfolding mechanism is operative, which includes a rapid unfolding of the protein after spreading, followed by a slow unfolding due to the rearrangement of the spread molecules at the interface. The π-A isotherms for glycinin at pH 8.026 and glycinin + 10 mM DTT at pH 8.026 and pH 5.0 (Figure 11A) spread monolayers registered after different aging times were not parallel to one another. On the other hand, the maximum surface pressure was practically independent of aging time. These results demonstrate that the aging effect observed in these monolayers is also due to unfolding of the protein at the interface, with significant changes in the tertiary (and probably in the secondary) structure of the protein. But we must reject the possibility of a loss of protein during the spreading process, because both the surface pressure at the minimum area and the molecular area at the beginning of the compression did not depend on the aging time. However, for glycinin (Figure 6A) and glycinin + 10 mM DTT (Figure 9A) spread monolayers at pH 2.0 the π-A isotherms are practically independent of film aging. These results can be explained by the conformational change that takes place in glycinin at the highest acidic pH together with a greater exposure of the hydrophilic residues in a more polar region and/or by the fact that the molecular conformational changes that take place upon the reduction of glycinin with DTT20 enhance the molecular hydrophobicity of the protein. Finally, for soy globulins at every pH the I-π plot does not depend on the aging of the monolayer, but only depends on the surface pressure (ref 26 and Figures 1B, 4B, 6B, 8B, and 11B). These results corroborate the idea28 that the I-π curve could reflect the surface equation of state of the spread material at the air-water interface. This master curve is characteristic for any material spread at the air-water interface. Structural and Topographical Characteristics of Soy Globulin Monolayers. The results for β-conglycinin, glycinin, and glycinin + 10 mM DTT indicate that a change in the slope of the π-A isotherms was produced (ref 26 and Figures 1A, 4A, 6A, 8A, and 11A) in the range of 13.5-16.5 mN/m, depending on the protein and pH, with the maximum values of the surface pressure at the transition point for soy globulins at pH 8.0 and for glycinin + DTT at pH 5.0. This behavior was not due to a change in the monolayer structure, as observed for surfactants and insoluble lipids,38 but was due to a change in the degree of condensation of protein molecules at the interface. The

change in the state of condensation of the monolayer was confirmed by the evolution of the reflectivity of the monolayer with the surface pressure (ref 26 and Figures 1B, 4B, 6B, 8B, and 11B). In fact, we have observed a step increase in the value of the reflectivity in the regime of surface pressures corresponding to the transition between the two condensation states of the monolayer. As for most globular proteins,6,29,39,40 at low surface pressures most amino acid residues in soy globulin molecules adopt a loop conformation at the air-water interface. But the loop conformation is more condensed at higher surface pressures and is displaced toward the bulk phase at the collapse point. The progressive unfolding of β-conglycinin and glycinin as the pH decreases and the associated conformational changes in the molecule have significant repercussions in the structural and topographical characteristics of the monolayer. The most condensed monolayer structure (as deduced by the translation of the π-A isotherm toward lower molecular areas) was observed at pH 5.0 as both proteins would be partially denatured and aggregated, close to the isoelectric point. The aggregation of these proteins at the air-water interface at pH 5.0 was confirmed at a microscopic level by BAM images (Figures 3, 7, and 10). The same phenomenon at the isoelectric point was observed for β-casein.41 Conversely, the most expanded monolayer structure was observed at pH 2.0, as the proteins would be completely denatured and dissociated.20 On the other hand, for glycinin + 10 mM DTT the π-A isotherms at pH 8.026 and at pH 2.0 (Figure 9A) are practically coincident. Then, the conformational change due to denaturation of glycinin by DTT would be similar to that produced by pH. The monolayer structure at constant pH also depends on the protein: (i) At pH 2.0, the π-A isotherm was the same no matter what the protein (β-conglycinin, glycinin, and glycinin + 10 mM DTT). That is, at the higher acidic pH β-conglycinin and glycinin molecules would be completely denatured and, thus, the effect of DTT on the glycinin monolayer structure is null. (ii) At pH 5.0, the π-A isotherms were displaced toward higher molecular areas from β-conglycinin to glycinin to glycinin + 10 mM DTT. That is, the reduction of SS/SH bonds in native glycinin by DTT produces a significant expansion of the monolayer structure. When the SS bridge is cleaved using DTT,30,42 the unfolding of glycinin at the air-water interface is facilitated, which can increase the number of points of contact of the protein with the interface, in agreement with the monolayer expansion. (iii) At pH 8.0, as β-conglycinin and glycinin are in a native state, the monolayer structure was more condensed for glycinin than for β-conglycinin. However, the treatment of glycinin with 10 mM DTT produced an expansion of the monolayer structure, even toward molecular areas higher than that for β-conglycinin. Thus, the same reasoning as above can be applied here. The more condensed structure of glycinin under native conditions should be associated with differences in ternary structure, which for glycinin is stabilized by hydrophobic and electrostatic interactions besides the disulfide bond between the acidic and basic polypeptides,43 among the differences in molecular mass.

(34) MacRitchie, F. Adv. Colloid Interface Sci. 1986, 25, 341. (35) Kra¨gel, J.; Grigoriev, D. O.; Makievski, A. V.; Miller, R.; Fainerman, V. B.; Wilde, P. J.; Wu¨stneck, R. Colloids Surf., B 1999, 12, 391. (36) Tronin, A.; Dubrovsky, T.; Dubrovskaya, S.; Radicchi, G.; Nicolini, C. Langmuir 1996, 12, 3272. (37) Garofalakis, G.; Murray, B. S. Colloids Surf., B 1999, 12, 231. (38) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, Ma. R. Colloids Surf., B 1999, 15, 235.

(39) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427. (40) Phillips, M. C.; Evans, M. T. A.; Graham, D. E.; Oldani, D. Colloid Polym. Sci. 1975, 253, 424. (41) Rodrı´guez Ninõ, Ma. R.; Carrera, C.; Rodrı´guez Patino, J. M. Colloids Surf., B 1999, 12, 161. (42) Wolf, W. J. J. Agric. Food Chem. 1993, 41, 168. (43) Peng, I. C.; Quass, D. W.; Dayton, W. R.; Allen, C. E. Cereal Chem. 1984, 61, 480.


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monolayer structure is produced at the isoelectric point of the protein; (ii) the maximum surface pressure at the minimum molecular area (Figure 12B) follows the trend glycinin > reduced glycinin > β-conglycinin, which corroborates that the maximum interactions between protein residues at the air-water interface are related to the tertiary structure of the protein and the molecular mass. In the native state (at pH 8.0), the maximum interactions between protein residues coincide with the more condensed molecular structure and higher molecular mass of glycinin (Figure 2B). However, these differences are lower as the proteins are denatured (at pH 2.0). The fact that these interactions are higher for glycinin than for β-conglycinin at every pH strengthens the hypothesis that the molecular mass of the proteins has an effect on the structural characteristics of the protein monolayer. Reduced glycinin behavior is halfway between that of β-conglycinin and that of untreated glycinin. Moreover, the monolayer thickness in the more condensed state was observed for glycinin monolayers (Figure 12C). Surprisingly, for native β-conglycinin and glycinin the maximum thickness of the monolayer was practically independent of the pH. For reduced glycinin, the monolayer thickness was similar to that for untreated glycinin at pH 8.0 but was minimum on acidic subphases.

Figure 12. The effect of pH on (A) molecular area at which the surface pressure begins to be registered (A0, open symbols) and limiting area (Alim, closed symbols), (B) maximum surface pressure attained at the minimum area (πmax), and (C) maximum reflectivity attained at the minimum area for (0) β-conglycinin, (O) glycinin, and (4) glycinin + 10 mM DTT monolayers spread at the air-water interface at 20 °C.

The SH/SS groups in glycinin have an important role in the formation of intermolecular disulfide bonds, giving a more condensed film at the air-water interface, and preventing the aggregation of the protein at the interface, in relation to β-conglycinin. (iv) The above conclusion is supported by the fact that at every pH the surface pressure and the reflectivity (thickness) of the monolayer in the most condensed state were higher for glycinin than for β-conglycinin. The reflectivity of glycinin + 10 mM DTT is halfway between those for untreated soy globulins. There exist different structural (A0, Alim, and the maximum surface pressure attained at the minimum area, πmax) and topographical (the maximum reflectivity attained at the minimum area, Imax) parameters of the soy globulin monolayers (β-conglycinin, glycinin, and glycinin + 10 mM DTT) that summarize the above conclusions (Figure 12). A0 is the molecular area at which the surface pressure begins to be registered. If there are no changes in the structure of the monolayer, higher values for this parameter are associated with a film expansion. The limiting area (Alim) is the molecular area obtained by extrapolation to π ) 0 of the straight part of the isotherm. A decrease in the limiting area indicates a more condensed monolayer structure or even the existence of a relaxation phenomenon associated with monolayer molecular loss. It can be seen that: (i) the minima values of A0 and Alim (Figure 12A) are produced at pH 5.0 (glycinin at pH 8.0 is an exception), which confirm that the most condensed

Conclusions This study shows that the molecular structure and molecular mass of soy globulins (fractions 7S and 11S), including the reduction of 11S with 10 mM DTT, have an effect on structural and topographical characteristics of the spread films at the air-water interface, depending on the pH of the aqueous phase in a complicated way. These interfacial characteristics of soy globulins also depend on film aging. The aging effect was due to unfolding of the protein at the interface. The more condensed monolayer structure was observed for soy globulins at pH 5.0 and was more expanded for glycinin + 10 mM DTT at every pH. At a microscopic level, soy globulin monolayers are more homogeneous at a pH far away from the isoelectric point of the protein. Close to the isoelectric point (at pH 5.0), numerous bright closed-packed domains due to the interfacial aggregation of the protein, which were in continuous formation/destruction, are characteristic for the topography of the monolayer during the compression up to the collapse. At every pH, the collapse of soy globulin monolayers is produced at surface pressures higher than the equilibrium surface pressure, via a fracturing mechanism in which the monolayer cracks over large scales at pH 5.0. As for milk proteins, two condensation states were observed for soy globulin films. However, significant differences were observed in the structural, topographical, and dynamic characteristics between milk protein26,28 and soy globulin films at the air-water interface. The rate of change of structural characteristics with the aging time was higher for soy globulin than for milk proteins.37 Moreover, the pH exerts a higher influence on structural characteristics of soy globulin than for milk proteins.26,28 Acknowledgment. This research was supported by CYTED through Project XI.17, for CICYT (Spain) and ANPCyT (Argentine) through Grants AGL2001-3843-C0201 and PICT98 09-04265, respectively. LA034143L

Effect of pH on Structural, Topographical, and Dynamic Characteristics

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