Amaranth 7S Globulin Langmuir Films and Its Interaction with l-α

Oct 14, 2013 - In addition, the doughnut shape domains were also captured by AFM phase mode, as shown in Figure 10. Here we show the clearly the small...
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Amaranth 7S Globulin Langmuir Films and Its Interaction with L‑αDipalmitoilphosphatidilcholine at the Air−Fluid Interface Alcione Garcia-Gonzalez,†,‡ A. L. Flores-Vazquez,† A. P. Barba de la Rosa,§ E. A. Vazquez-Martinez,† and J. Ruiz-Garcia*,† †

Colloids and Interfaces Laboratory, Institute of Physics, Autonomous University of San Luis Potosi, Alvaro Obregon 64, 78000 San Luis Potosi, S.L.P., Mexico ‡ Universidad Autonoma de Nuevo León, UANL, Facultad de Ciencias Quimicas, Av. Universidad S/N, Cd. Universitaria, San Nicolas de los Garza, N.L. C.P. 66451, Mexico § Institute for Scientific and Technological Research at San Luis Potosí, Camino a la Presa San Jose s/n, Lomas 4a Seccion, 78231 San Luis Potosí, S.L.P., Mexico S Supporting Information *

ABSTRACT: Amaranth seeds are one of the more promising food ingredients, due to their high protein content, among which the most important are storage proteins known as globulins. However, little is known about the physicochemical of the globulin proteins. In this work, we study the physicochemical behavior of films made of amaranth 7S globulin and its interaction with a model membrane made of Lα-dipalmitoylphosphatidylcholine (L-α-DPPC) at the air− liquid interface. The study was done by means of Langmuir balance, Brewster angle microscopy (BAM), fluorescence microscopy, and atomic force microscopy (AFM). We found that isotherms of pure 7S globulin directly deposited on either water or buffer subphases behave similarly and globulin forms a condensed film made of globular and denature structures, which was confirmed by BAM observations. Good mixtures of the protein with L-α-DPPC are formed at low surface pressure. However, they phase separate from moderate to high surface pressure as observed by BAM. Isotherms detect the presence of the protein in the mixture with L-α-DPPC, but we were unable to detect it through BAM or AFM. We show that fluorescence microscopy is a very good technique to detect the presence of the protein when it is well-mixed within the LE phase of the lipid. AFM images clearly show the formation of protein mono- and multilayers, and in phase mode, we detected domains that are formed by protein and LE lipid phase, which were corroborated by fluorescence microscopy. We have shown that globulin 7S mix well with lipid phases, which could be important in food applications as stabilizers or emulsifiers, but we also show that they can phase separate with a moderate to high surface pressure.

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INTRODUCTION

shown that seed storage proteins are generally composed of four fractions. The globulins represent the main storage protein group in legumes. There are two major classes of these kind of proteins, namely, 7S and 11S fractions, which are classified according to their sedimentation coefficients, given in Svedbergs (S).5,6 7S globulins are composed of three main subunits called α (57−68 kDa), α′ (57−72 kDa), and β (42− 52 kDa). These are bound by noncovalent binding to form a trimer with a molecular weight that ranges from 170 to 200 kDa. Each subunit has one or two N-linked glycosyl groups. The trimer structure is stabilized in high ionic strength solutions.2 Globulins precipitate at pH between 4.5 and 6.8,7,8 but its maximum insolubility occurs at pH 4.5−6.0,

Amaranth (Amarantus hypochondriacus L.) is one of the few nongrass species with a great potential to become a good dietary source of a cereal-like grain crop. Amaranth seeds have a high protein content (17.9%) with a better balance of essential amino acids than those found in cereals and legumes.1,2 Therefore, amaranth proteins are one of the more promising food ingredients, capable of complementing or even substituting many cereal or legume proteins. Amaranth seeds have a high proportion of storage proteins known as globulins (22− 42%) and glutelins (14−18%), which can be extracted with acid or alkaline solutions.3 The structure and function of these proteins present a strong dependence with the ionic strength, pH, and temperature, but little is known about their physicochemical behavior, however. Storage proteins in seeds are of great importance for supplying the world’s protein requirements.4 It has been © 2013 American Chemical Society

Received: June 16, 2013 Revised: October 14, 2013 Published: October 14, 2013 14046

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and partial precipitation begins between pH 6.0 and 6.8.2 Therefore, its behavior close to neutral can be highly affected by precipitation. It is known that hydrophobic areas of polypeptides are exposed outward uncovering all aromatic residues at acidic and alkaline solutions. It has been reported that amaranth globulins showed two hydrophobicity peaks, one at pH 5.5 and the other above pH 9.5.8 In addition, it has been proposed that these hydrophobic peaks observed in amaranth globulins may have been caused by a combination of changes in tertiary structure exposing the more hydrophobic core.9,10 On the other hand, the emulsion stability and fat-binding capacity of proteins can be predicted by using both hydrophobic and hydrophilic properties of proteins. Hydrophobic parts of proteins easily interact with oils, and its effective hydrophobicity moment gives an idea of the capacity of proteins in the stabilization of the protein−lipid interaction. Hydrophilic parts like to be in contact with water, while hydrophobic amino acid groups like to be in contact with lipid chains, making some proteins, such as globulins, good interfacial molecules; so they can be used as stabilizers or emulsifiers. In addition, amaranth globulins are heat-stable proteins and are expected to be helpful as good emulsifiers with a high nutritional value.2 For this reason, it is important to have a better understanding of their physicochemical behavior at interfaces and their interaction with fats such as lipids like L-DPPC. Studies of protein adsorption at the air−water interface, using the Langmuir trough technique, have given insights on their behavior,11 protein−lipid interactions,12 and protein conformation at a hydrophilic−hydrophobic interface.13 Other techniques have been used along the Langmuir trough and give more information on the protein film behavior at the air−water interface. For example, Brewster angle microscopy gives direct information on a mesoscale of the self-assembly patterns formed by proteins.14−16 At the molecular scale, X-ray diffraction experiments at grazing incidence angles have been very useful to determine protein molecular ordering of apolipoproteins.15 However, in food industries the main motivation of adsorption of proteins at fluid−fluid interfaces is the understanding of the adsorption mechanisms to improve their applications as emulsifiers and foaming systems.17 A step in this direction must come from a better understanding of the physicochemical behavior of proteins at interfaces and its interactions with lipids. For example, it is still largely unknown how the spatial structure of protein molecules strongly influences their essential properties, such as surface and biological activity. Furthermore, in an emulsion or a microemulsion it is not always clear if proteins are folded or unfolded, which is important for their surface activity but not much important as a source of nutrients. Therefore, surface and interfacial phenomena involving proteins are quite common and of great importance in nature and technology, and the formation of Langmuir films of proteins is an obvious way for their study, as well as for studying their interaction with biological model membranes.18 In particular, phospholipids,12 fatty acids,19 cholesterol,20 and protein monolayers16 are of great interest because these are some of the main structural elements of cell membranes. In addition, the study of adsorption phenomena, to form Gibbs monolayers, is also of great interest in biology, chemical technology, and biotechnology and offers new paths in the understanding of protein and polymer chemistry.13 For example, there have been a wide range of studies about pure protein interface adsorption as well as on a lipidic Langmuir

monolayer, which have yielded a good understanding of their diffusion kinetics from the bulk to the interface as well as their interaction with lipid monolayer domains or phases. Indeed, it is possible to study the protein penetration−interaction in a lipidic monolayer as a function of surface pressure; these studies provide information about the interactions, distribution, and structure of protein−lipid layer. 12,16 In Langmuir monolayer studies one measures pressure−area isotherms, Π(A,T) = γ0(T) − γ(A,T), where T is temperature, A is area/ molecule, and γ0 and γ are the surface tension of pure water and that when the monolayer is present, respectively. As mentioned above, there are optical techniques that can be also applied concomitantly to observe the monolayer organization at the air−water interface, such as polarized fluorescence microscopy (PFM)21,22 and Brewster angle microscopy (BAM).23 These optical techniques are quite sensitive for observing details in Langmuir monolayers, such as phase transitions, phase coexistence, and molecular tilting.19,24 Both techniques have been used to observe the formation of domain structures on monolayers penetrated by proteins,25−28 but their lateral resolution is limited by the accuracy of the optical microscope. The transfer of films from the air−water interface to a solid support, using the well-known Langmuir−Blodgett (LB) technique, can be utilized to study the microstructure of pure protein or films of mixtures protein−lipid: The microstructure of the film in the solid support can then be imaged by using electron microscopy29,30 or atomic force microscopy (AFM).31 For example, the former technique has been used to study the miscibility of proteins and lipids at the air−water interface.32 AFM has been used to study LB films of several systems such as DPPC,33 human proteins,34 and mixtures of DPPC with Nnitrosodiethylamine/bovine serum albumin.14 Nevertheless, it has been until recently that this microscope technique has been used for food protein monolayer penetration studies into lipid films,16 among other systems. In this work, we study the interfacial behavior of the pure protein globulin 7S and its mixture with L-dipalmitoilphosphatidilcoline (L-DPPC) at the air−water interface by isotherm measurement using the Langmuir trough. BAM images were obtained along the isotherms. The Langmuir−Blodgett technique was used to transfer films, which were analyzed by atomic force microscopy and fluorescence microscopy to get a better understanding of the interaction behavior between the protein and the lipid monolayer.

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EXPERIMENTAL SECTION To try to understand the behavior of globulin 7S at interfaces and its interaction with lipids (L-DPPC), experiments were conducted with two different subphase solutions: ultrapure water with pH ∼ 6.2 (checked before each experiment) and phosphate buffer solution 20 mM at pH 7.0. In addition, two different strategies to form the films were performed: (i) direct deposition of the protein on the air−liquid interface or on the lipid monolayer and (ii) injecting the protein solution below the air−liquid interface or the lipid monolayer. Materials and Subphase Solutions. The amaranth 7S globulin was extracted and purified from A. hypochondriacus L. seeds, cv. Nutrisol was used for 7S globulin fraction isolation according to Barba de la Rosa et al.3 Briefly, suspensions of defatted flour/extracting agents (1:10 w/v) were stirred for 1 h at room temperature and centrifuged at 9000 g for 20 min. The resulting pellet was resuspended in water, stirred for 1 h, and centrifuged, and the pellet was resuspended in 10 mM 14047

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Figure 1. (Left panel) Π vs A isotherms of globulin 7S films spread on a ultrapure water subphase and on a phosphate-buffered water subphase. (Right panel) BAM images corresponding to the position of surface pressure corresponding to the letters in the left panel. The size of the BAM images are 450 × 550 μm2.

GmbH, Germany) microscope, with a spatial resolution of ca. 2 μm. The BAM laser was made incidence at the Brewster angle (53.13°) of the interface, and the analyzer was rotated for best contrast and to look for anisotropies of the film. Each BAM image size is 462 μm × 564 μm. The NIMA trough and BAM were placed together onto an optical table. The dipper speed for LB film preparation was 1 mm/min and was used in the upward stroke; the surface pressure was fixed to a predetermined value during the film extraction. Freshly cleaved mica was used as a substrate for the transferred films. Amaranth 7S Globulin Films. As mentioned above, two different methods were used to form the films at the air−water interface. In the first method, the amaranth 7S globulin solution was carefully deposited drop-by-drop with a microsyringe over the entire cleaned water or air−buffer interface, and after 4 or 6 h the monolayer was compressed; experimental isotherms did not show significant difference on these waiting times. In the second method, the protein solution was carefully injected below the air−water or air−buffer interface. Since the protein is partially soluble in water, in this method we waited 6 h before performing the first compression; this delay time will ensure protein diffusion and trapping by the interface, especially in the second method. In both methods 200 μL of amaranth 7S globulin solution was used. The area/molecule of Figures 1 and 2 is estimated using the average molecular weight of the protein of 185 kDa. Films of Mixtures of L -DPPC/7S Globulin. The incorporation of 7S globulin into the L-DPPC or the fluorescently doped L-DPPC monolayers16 was done in two ways: In the first case, the protein solution was spread drop-bydrop with a microsyringe onto the water or buffer subphases, and immediately 250 μL of L-DPPC was deposited on the already formed 7S globulin film. We also waited 4 h before the first compression process was performed to allow complete stabilization of the system. In the second method, the L-DPPC

phosphate buffer, 1 mM ethylenediaminetetraacetic acid (EDTA), pH = 7.5, containing also 0.1 M NaCl. A second extraction was done with the same buffer. The supernatants were mixed and named as 7S globulins. The quantification of protein was done using the protein assay (BIO-RAD, Lab Hercules, CA) and was obtained an average molecular weight of 185 kDa. The protein solution was used at a concentration of 0.9046 mg/mL. The phospholipid, L-DPPC (≥0.99%, SigmaAldrich Inc., USA), sample was used without further purification. L-DPPC was dissolved in chloroform (SigmaAldrich, USA, ≥99.99%) to prepare the spreading solution at a concentration 0.353 mg/mL. For the fluorescence microscopy experiments, a different solution was used and consisted of 98% of L-DPPC and 2% of NBD-hexadecylamine (≥95%, Molecular Probes Inc., Oregon, USA). Both compounds were dissolved in chloroform to prepare the spreading solution at the same final molecular concentration as the one used with pure L-DPPC experiments. The phosphate solution was prepared by dissolving mono and dibasic sodium phosphate salts, to obtain pH 7 buffer solutions. Ultrapure water (Nanopure-UV, Barnstead/Thermoline, 18.3 MΩ•cm of resistivity) was used throughout all preparations and for cleaning all instruments and glassware. Isotherm Langmuir and Brewster Angle Microscopy (BAM) Observations. All isotherms and transferred LB films were done on a computerized Nima LB trough (601-BAM, Nima Technology Ltd., England). To measure the surface pressure, Π(A,T) = γ0(T) − γ(A,T), a Wilhelmy plate was used. The temperature was kept constant at 25 ± 0.2 °C by a water recirculator bath (NESLAB, RTE-211, USA). The compression rate was always at 15 cm2/min. All experiments were carried out in a dust-free environment room cleaned with a laminar flow hood. BAM observations were made along the isotherm measurements with a BAM I-ELLI 2000 (Nanofilm Technologies 14048

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Figure 2. (Left panel) Π vs A isotherms of globulin 7S films obtained after the protein solution was injected below the air−water and air−buffer interfaces. (Right panel) BAM images corresponding to the position of surface pressure corresponding to the letters in left panel. The size of the BAM images are 450 × 550 μm2.

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RESULTS AND DISCUSSION To study the pure protein behavior and its interaction with LDPPC, isotherm measurements and BAM observations were performed along the isotherms. The microstructure of the protein films and its mixture with L-DPPC has been analyzed. The LB films were characterized by means of AFM and fluorescence microscopy. Isotherms and BAM Observations. Figures 1 and 2 show isotherms obtained for the pure protein films made of globulin 7S, deposited directly and below the interface, respectively, which indicate that films of globulin 7S can be readily formed at the air−liquid interface. When the protein was spread directly over either water or buffer subphases, the surface pressure has a small value. For reference we applied an small zero offset, and therefore the isotherm rose immediately upon compression; this behavior indicates that a large amount of protein was trapped on the interface. However, the protein is insensitive to the subphase: in other words, the isotherms on water and on buffer are basically identical (Figure 1). In contrast, when the protein solution was injected below either the air−water or the air−buffer interfaces the isotherms show a marked difference in surface pressure, mainly at the larger areas per molecule (Figure 2). However, they show a similar qualitative behavior (see Figure SI-1), indicating that this difference is due only to the amount of protein trapped by the interface; that is, in buffer there is more protein trapped at the interface than in water. This slightly different behavior must be due that 7S globulin is more soluble in buffer than in pure water; thus it diffuses longer in bulk buffer with much less aggregation than in water; after finishing each experiment, we found that more 7S globulin precipitated at the bottom of the trough in water than in buffer. This protein precipitation is not surprising since the pH used in these experiments are within the partial precipitation range

was spread drop-by-drop on the subphase with a microsyringe. We waited 20 min before carefully injecting the protein solution in the bulk, below the already formed L-DPPC Langmuir monolayer. The compression started after 6 h to allow protein diffusion to the interface. In both methods, 200 μL of amaranth 7S globulin solution were used. All experiments were run in duplicate, and no significant differences were found among them. AFM Observations. Transferred films of both pure protein and its mixture with L-DPPC were observed with a NanoScope IIIa AFM (Digital Instruments, California, USA) using a Jscanner. The tapping mode was selected to obtain topographic, deflection, or phase contrast images using 125 μm long Si3N4 tips with a nominal resonance frequency of 300 kHz and a spring constant of 40 N/m. During scanning, the oscillation frequency was typically set 0.1−3.0 kHz below resonance. Low scan rates (typically between 0.5 and 1 Hz) were chosen and images were analyzed by using the Digital Instruments software. First, AFM images were obtained at low magnification, but once a proper area was found, the scanning was changed to a higher resolution. AFM images presented here are height images unless indicated otherwise. Fluorescence Observations. The transferred films of mixture of doped L-DPPC with 7S globulin were observed with a confocal microscope (Carl Zeiss, LSM 510 META). For the acquisition of the images an EC Plan 40× n.a. 0.75 objective was used. The samples were illuminated with a laser with a wavelength λ = 488 nm; the light line was filtered by HFT filters with 488/561 nm, and a pinhole opening of 560 μm was used. Films were supported on mica, as in the case of the AFM LB films, and they were placed directly on the support of the microscope. 14049

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Figure 3. (Left panel) Π vs A isotherms of L-DPPC monolayer spread on globulin 7S films at the air−water interface and air−buffer interface. (Right panel) BAM images corresponding to the positions of surface pressure corresponding to the letters in the left panel. The size of the BAM images are 450 × 550 μm2.

easier at interfaces than 11S globulin.16 This result must be due to the fact that 7S globulin is more soluble than 11S globulin in both subphases. Globulin 7S/L-DPPC Films. Figure 3 shows isotherms of the L-DPPC and 7S globulin when the protein was dispersed on the air−water and air−buffer interfaces, and afterward an LDPPC solution was dispersed over it. Since the protein is water-soluble, we waited 4 h to allow equilibration of the formed film at the interface before compression. Note that we are using the area per molecule of the L-DPPC molecule as a reference.16 Upon compression, the surface pressure increased immediately, but both isotherms and BAM images do not show the typical LE−LC phase transition plateau or any molecular tilted domains of L-DPPC. This behavior indicates a good mixture of the protein with L-DPPC and the formation of a quite condensed, but compressible film as indicated by the slope of the isotherm. At low surface pressures, minor differences were detected between the isotherms of the films at the air−water and the air−buffer interfaces, in agreement with BAM observations (Figure 3a and d); in fact, BAM images showed the formation of tiny white domains. However, as the compression proceeds, the moderate compressibility of the films might be due to a smooth and continuous collapse of the film, since large three-dimensional structures are formed at higher surface pressures, as shown by BAM images (Figure 3c and f). However, at high surface pressure, the large white domains formed on water differ from those formed on buffer; this is, the white domains formed on water showed irregular borders, while those formed in buffer formed straight and smooth borders, see Figure 3c and f, respectively, where these large aggregates seem to be made of protein molecules. Therefore, the formation of these large domains might be due to demixing of the L-DPPC molecules from the protein molecules, as the surface pressure increases. The formation of smoother borders on buffer than on

mentioned in the Introduction section, and the protein should show a bigger precipitation amount in the water subphase because its pH is within the partial precipitation pH range. It is worth noting that the amount of protein at the interface in the injection experiment in buffer is close to that of the experiments where the protein was deposited directly on the interface. Since the isotherms are close and behave quite similarly, we assume that there is not much difference in the surface behavior of the protein film obtained by either method. Two slight slope changes can be observed along the isotherms at surface pressures around 7 and 22 mN/m for the air−water interface, and in the buffer subphase these slope changes occur at about 13 and 24 mN/m; see Figure 1. Slope changes could suggest a phase transition in Langmuir monolayers, but BAM observations suggest that there are no phase changes in these cases. In fact, BAM images show that, during compression there is a smooth single phase in both subphase, as shown in Figure 1a−c (water) and d−f (buffer), with small three-dimensional aggregates. However, protein conformational changes could occur during isotherm compression and might not be detected by BAM. These aggregates are slightly bigger in water than in buffer as expected due that the pH in water is in the partial precipitation range. Therefore, globulins are dispersed better at the air−buffer interface than at the air−water interface based on the size of the threedimensional protein aggregates observed by BAM. Isotherms also show that films are highly compressible, which indicates that the phases observed by BAM corresponds to a liquidexpanded-like (LE) phase. Throughout the film compression on both subphases, it is also possible that continuous collapse might have occurred, especially at higher surface pressures on water using both film formation techniques, and the three-dimensional aggregates observed by BAM are evidence for the continuous collapse. In general, it is important to mention that 7S globulin gets trapped 14050

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Figure 4. (Left panel) Π vs A isotherms of a Langmuir monolayer of L-α-DPPC mixed with globulin 7S, when globulin 7S was injected below the monolayer of L-α-DPPC. L-α-DPPC, at the air−water and air−buffer interfaces. (Right panel) BAM images corresponding to the position of surface pressure corresponding to the letters in left panel. The L-DPPC area/molecule is used as a reference. The size of the BAM images are 450 × 550 μm2.

the interfaces (Figure 3). At the start of the compression, the surface pressure on both interfaces were identical up to about 7 mN/m and 70 Å2/molecule, where they started to depart from each other, increasing the surface pressure of the isotherm on buffer faster than that on water, until finishing with the same superficial pressure Π ∼ 45 mN/m, but different molecular areas. The molecular areas were different by about 15 Å2/ molecule ∼35 Å2/mol at the air−buffer interface and ∼20 Å2/ mol at the air−water interface. Again, isotherms do not show the first-order LC-LE phase transition characteristic of the LDPPC Langmuir monolayers. BAM images, on other hand, show the presence of small domains that have internal tilt structure almost from the onset of compression, and become more noticeable as the surface pressure increases, as shown in Figure 4a−f. Therefore, the injection of the protein below the monolayer of the L-DPPC in both interfaces generates the condensation of the lipidic monolayer in each case, more on buffer than on water since the L-DPPC domains are bigger in buffer than in water at low surface pressures and causes the disappearance of the flat region of the LE-LC phase transition,36 typical of this type of monolayer. The latter can be explained because now we have a mixture and not a pure component, and the presence of the protein can help to nucleate the LC domains, especially in buffer, where we can observe the formation of domain doublets and triplets, which seems to be attached to each other at a center point (see Figure 4d); this domain behavior does not occur on pure water. However, BAM images in Figure 4a−f seem not to have enough resolution to observe the presence of the protein. The domains observed in Figure 4a−f corresponds to the LC phase of L-DPPC surrounded by the phase LE. This is clear since these domains exhibit optical anisotropy or internal structure, which must correspond to a tilted LC phase formed by the tilt of the phospholipid molecules tails with

water might indicate that this demixing process is easier on buffer than on water. This is, the L-DPPC molecules find it easier to diffuse out from the protein-rich domains in buffer than in water, due to in water the protein is more denatured trapping the L-DPPC molecules, making their diffusion-out of the protein-rich domains more difficult; this will cause the formation of instabilities causing the formation of irregular borders on the protein-rich domains. Similar behavior was found on both subphases with mixtures of globulin 11S and LDPPC films.16 The isotherms were qualitatively similar, but the isotherm on the buffer subphase showed a surface pressure slightly greater than the isotherm obtained on pure water. This again indicates that more material is trapped on buffer than on water, due mainly to the greater tendency of the protein to aggregate and precipitate to the bottom of the trough in water than in buffer, so less protein can reach the interface on the water subphase than on the buffer subphase. 7S globulin/L-DPPC isotherms show also a small shift at the takeoff area with respect to isotherms of pure L-DPPC in both interfaces; this behavior is a similar to that observed in 11S globulin/L-DPPC isotherms.16 The film formed by the mixture shows a fracture collapse at a surface pressure lower than monolayers of pure L-DPPC in both subphases for both 7S and 11S globulin16 mixed with LDPPC. Figure 4 shows the behavior of the 7S globulin/L-DPPC mixtures when the protein was injected below the already formed L-DPPC monolayer. Initially, the L-DPPC monolayer behaved as a pure lipid monolayer in the G-LE phase coexistence. However, with time it went into a pure-like LE phase and shows a small surface pressure. Isotherms were zerooffset; thus the surface pressure increased as soon as the film is compressed, but with a smaller isotherm slope as in the case when the protein and the L-DPPC were dispersed directly on 14051

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Figure 5. AFM image films of pure 7S globulin from both subphases, deionized water (a, b) and phosphate buffer (c, d).

The smaller granules in both subphases have about the estimated size of the protein but on water seem to be more denatured, where the protein size in water was 35.155 nm. It is known that the major axis of the unit cell of the adzuki bean protein has a length of 11.976 nm,35 and it has been proposed that the protein exists as a trimer of a size around 36 nm. Since the sizes of the globules observed on both subphases are uniform in size, about 34.598 ± 0.489 nm, we assume that these tiny globules correspond to single protein trimers. Furthermore, it is interesting to note the formation of fibers on both interfaces, where these fibers are formed by aligned globulin proteins on what appears to be denatured proteins. A possible explanation to why the protein molecules on water are somewhat more denatured than in buffer is because the pH used was between 5.5 and 6.0, which is within the range of the maximum and partial points of precipitation, while that in buffer is not. In addition, in buffer the presence of salt favors the protein stabilization; this causes a larger aggregation of 7S globulin in water, to form larger protein clusters or even its denaturing, while in buffer the system is already outside the critical pH for partial and/or total precipitation. Furthermore, the observed formation of fibers could be interesting in finding conditions to enhance this fiber formation, since protein fibers are important for the food industry. The profiles shown in

respect to the interface. The LC domains start joining together to form a continuous LC phase at about 20 mN/m. On buffer and high surface pressures, the domains maintain their shape, but they seem to be surrounded by thin white lines that we assume correspond to protein; see Figure 4f. In water, however, no presence of the protein was detected even at high surface pressures. In addition, the air−buffer interface shows a greater surface concentration of LC domains with a more marked anisotropy with respect to those found at the air−water interface. AFM Observations. Langmuir−Blodgett films for each of the different experiments were prepared, at a predetermined surface pressure, and analyzed by means of AFM to try to get insight on the morphology of the films at the microscopic level. Figure 5 shows images of transferred films of pure 7S globulin from both subphases, deionized water and phosphate buffer, at a lateral surface pressure of 4 mN/m. First of all, it is observed that films transferred on both cases form mostly a multilayer instead of a single monolayer; it can easily be observed the formation of a grainy surface with different height levels; white are at the highest, while dark are at the lowest level. Second of all, the granules formed on water are not as well-defined from those formed on buffer. 14052

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Figure 6. AFM images of the films performed when the protein 7S globulin was injected into deionized water (a, b) and phosphate buffer subphases (c, d).

better possible raw material than 11S globulin for the food industry. Films Formed by Mixtures 7S Globulin and L-DPPC. Figure 7 shows AFM images of the samples obtained when 7S globulin was deposited directly on the air−water or air−buffer interfaces, and immediately the L-DPPC solution was dispersed on top of it. In the first case (see Figure 7a,c), it is observed the formation of fractal-like structures surrounded by a smooth film. However, the height profile indicates a low roughness of the film. This suggests that practically all of the protein is unfolded, but note that the fractal−like domains have a nucleation center much higher than the rest of the domain. In fact, the fractal domains have about the same high as the surrounding phase, which might indicate that the domains correspond to the L-DPPC LC phase and the continuous phase to the LE phase. It is known that impurities can act as nucleation sites for crystal growth out of a liquid phase; therefore we think that the nucleation center is an aggregate of protein molecules (see Figure 3b). On buffer the situation is clearly different; there are a large number of small domains, but their edges are not well-defined. Furthermore, there is no clear evidence of globules corresponding to globulin, except that again seems to be aggregates of proteins (white dots) in some of the larger domains that might have acted as nucleation sites. The lack of

Figure 5c,d show measurements of the heights of the small globules as well of the nanofibers; the observed heights of both type of structures are ≤2.4 nm. Figure 6 shows the case of the films formed when the protein was injected into the subphase, where we can observe that the protein forms quite good films. However, on water the protein seems to be unfolded since the film is quite smooth, which resembles the formation of a polymer melt, see Figure 6a,b; the height profile shows a very smooth film with a few granules and fibers, but these fibers seem to come from denaturized proteins since they are not formed by a string of globules. In contrast, the protein film obtained on buffer showed again a good density of tiny globular structures, easily observed at all scales, uniform in size that corresponds to the trimer size, see Figure 6c,d. There are also fiber formations, and although they have few globules along the fibers, they also seem to come from denatured protein, since the relative height of the fibers is smaller than the expected size of the protein and their smoothness suggests that the proteins that form fibers are unfolded. In general, this result shows that 7S globulin has a higher tendency to unfold forming fibers while 11S globulin does not under the same conditions.16 The observed fiber formation is highly desirable in proteins; thus 7S globulin can be seen as a 14053

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Figure 7. AFM images when 7S globulin was deposited directly on the air−water (a, b) or air−buffer interfaces (c, d) and immediately when the LDPPC solution was dispersed on top of it.

phase do not seem to have very different mechanical properties, but there are tiny specks which are made of different material, which based on their size must correspond to individual globulin proteins. However, the number of these protein clusters is very low, which indicates that most of the protein must be unfolded and very well-mixed with the LE phase of the lipid monolayer. This must be the reason why we do not see much evidence of the presence of the globulin protein even by BAM. The AFM images obtained when the protein was injected in the buffer subphase, below the L-DPPC monolayer, also agrees quite well with the observations by BAM, see Figure 8b. This is, we observed the nucleation of small domains but in some cases the domains are joined as in those observed by BAM (see Figure 4d). Unexpectedly, the phase image of this domains show that they have different mechanical properties; that is, in phase mode many domains have a doughnut-like shape, while in height mode these domains are continuous. We discuss this type of domains in the next section. Fluorescence Microscopy Observations. In our search to try to find out where the protein is located within the film, we performed experiments where we doped the L-DPPC solution with 2% of the fluorescent NDB-hexadecylamine

evidence on the presence of the protein suggests that the protein is most likely unfolded and well-mixed within the LE phase of the lipid film. In addition, the fact that the LC domains are not well-defined on both interfaces might be due that unfolded protein in the LE phase does not allow a good quasilong-range organization of the LC phase domains, changing the line tension between the LC and LE phases of the lipid monolayer. When the protein solution was injected in the subphase, below the already deposited L-DPPC monolayer, the AFM images show the formation of rounded domains surrounded by a continuous phase; see Figure 8. This behavior is also in good agreement with BAM observations, where there is the formation of rounded domains at the interface (see Figure 4). The only difference is the scale size of the domains; the domains observed by AFM are much below the resolution of the BAM. Since upon compression the domains observed by BAM become larger and developed anisotropy, these domains must correspond to the LC phase of the L-DPPC monolayer; the profile in Figure 8 shows a height difference of about 0.5 nm in good agreement with the difference in height between coexisting LC and LE phases. The phase mode image reveals that this is the case; since both the domains and the continuous 14054

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Figure 8. AFM images when the protein solution was injected into deionized water (a) and phosphate buffer subphases (b), below the already deposited L-DPPC monolayer. The profile and the phase mode image (c) correspond to image a.

see Figure 9b. However, note that in this case most LC lipid domains do not have a small black protein center as in the case where the lipids were deposited on the protein film. However, it can be noticed that small and quite dark protein domains are located in the continuous bright LE phase, and some of them show a doughnut-like shape, where the center of the doughnut is fluorescent, indicating that is made off lipid LE phase. More interesting is the presence of elongated gray “shades” within the LE phase, not as dark as the bigger LC domains. These shades must correspond to unfolded protein, which form a network within the LE lipid phase connecting the darker and bigger LC L-DPPC domains. In addition, the doughnut shape domains were also captured by AFM phase mode, as shown in Figure 10. Here we show the clearly the small domains with a doughnut shape encircled. Figure 10b and c shows AFM images in height and in phase contrast mode. Note that the domain encircled in part b shows the doughnut shape in phase contrast mode, where the mechanical properties of these domains are similar to those of the LE phase that surrounds the domains: This will explain the presence of a softer core in the domains.

molecule. It is known that this molecule likes to mix well in the LE phase and much less in the LC domains in Langmuir monolayers.24 If the protein is located within the LE phase as well as forming domains or aggregates within it, we expect these protein aggregates to show a lack of fluorescence. Figure 9 shows false color images of the experiment, when the protein was directly deposited on buffer and the lipid solution was immediately deposited on it. The transferred LB film was prepared at Π = 12 mN/m. One can easily observe two lipid phases in coexistence; the dark domains correspond to the LC phase surrounded by a continuous bright LE phase. Remarkably, the dark domains show a small black dot at the center and a number of bright lines radiating from the center to the edge of the domain. We think that the black dot is a small protein domain that served as a nucleation site for the LC lipid domain, similar to those observed in Figure 7. The bright lines must be due to the fluorescent dye, which is a bulky molecule and it is acting as an impurity in the LC domain, and therefore is concentrating in the line defects of the domains to eventually diffuse out of the LC domains.24 However, the amount of protein at the center of the LC lipid domains is very small; therefore we believe that the rest of the protein is within the LE phase of the L-DPPC Langmuir film. Thus we conclude that most of the protein is probably denature and that allows it to mix very well with the LE phase; this is in good agreement with BAM and AFM observations. On the other hand, when the 7S globulin was injected below the monolayer, it is again clearly observed the formation of dark LC lipid domains surrounded by a continuous bright LE phase;

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CONCLUSIONS Amaranth 7S globulin forms films in buffer and ultrapure water subphases. These films tend to form multilayer protein aggregates on both air−water and air−buffer interfaces as confirmed by BAM and AFM images, in contrast to 11S globulin that under some condition do form well-organized monolayers.16 However, 7S globulin films show a tendency to form fibers easier than 11S globulin. Protein aggregates suggest 14055

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Figure 9. Fluorescence microscopy images of the protein directly deposited on buffer (a) and the lipid solution immediately deposited on it (b). Images are 446.7 × 446.7 μm2.

that 7S globulins are partially soluble in water and buffer. 7S globulin films show the same physicochemical behavior when it was either spread on or injected below the air−buffer or air− water interfaces, as determined by isotherm and BAM observations. In addition, at the microscopic scale, AFM images show similar behavior as well. When L-DPPC is added to 7S globulin films, isotherms show a small difference, and 7S globulin have a phase separation from the lipid monolayer at high surface pressures, similar to that observed in 11S globulin. However, the shape of the protein rich-domains is different in water than in buffer; in water the protein-rich domains show irregular borders, while in buffer they show straight borders and often very sharp angles. This behavior is consistent with an easier diffusion of the lipid molecules out the protein-rich domain on buffer than in water, where the irregular borders might be Mullins−Sekerka instabilities formed during the diffusion of the lipid molecules out of the protein-rich domain. Neither BAM nor AFM images show clearly the presence of the protein, except when protein aggregates act as nucleation centers for the LC domains. When the protein is injected below the already formed L-DPPC monolayer, the isotherms show a similar behavior on both

Figure 10. Images showing when the protein solution was injected into phosphate buffer subphases, below the already deposited L-DPPC monolayer. Fluorescence microscopy (a), AFM images in height (b), and the same AFM images in phase contrast mode (c). Images are 446.7 × 446.7 μm2.

subphases at low surface pressures, which deviates from each other from moderate to high surface pressures. BAM images also show that on water the L-DPPC LC domains, in the 14056

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(8) Marcone, M. F.; Yada, Y. Study of The Change Profile and Covalent Subunit Association of the Oligomeric Seed Globulin from Amaranthus hypochondriacus. J. Agric. Food Chem. 1992, 40, 385−389. (9) Tsutsui, T.; Li-Chang, E.; Nakai, S. A Simple Fluorometric Method for Fat-Binding Capacity as an Index of Hydrophobicity of Proteins. J. Food Sci. 1986, 51, 1268−1272. (10) Matsudomi, N.; Mori, H.; Kato, A.; Kobayashi, K. Emulsifying and Foaming Properties of Heat Denatured Soybean 11S Globulins in Relation to Their Surface Hydrophobicity. Agric. Biol. Chem. 1985, 49, 915−919. (11) Sánchez-González, S.; Ruiz-García, J.; Gálvez-Ruiz, M. J. Langmuir−Blodgett Films of Biopolymers: A Method to Obtain Protein Multilayers. J. Colloid Interface Sci. 2003, 267, 286−293. (12) Wang, X.; Zhang, Y.; Wu, J.; Cui, G.; Wang, M.; Li, J.; Brezesinski, G. Dynamical and Morphological Studies on the Adsorption and Penetration of Human Serum Albumin Into Phospholipid Monolayers at the Air/Water Interface. Colloids Surf., B 2002, 23, 339−347. (13) Proteins at liquid interfaces; Elsevier: Amsterdam, 1998; Vol. 7. (14) Valencia-Rivera, D. E.; Básaca-Loya, A.; Burboa, M. G.; Gutiérrez-Millán, L. E.; Cadena-Nava, R. D.; Ruiz-García, J.; Valdez, M. A. Interaction of N-nitrosodiethylamine/Bovine Serum Albumin Complexes with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Monolayers at the Air-Water Interface. J. Colloid Interface Sci. 2007, 316, 238−249. (15) Xicotencaltl-Cortes, J.; Mas-Oliva, J.; Castillo, R. Phase Transitions of Phospholipids Monolayers Penetrated with Apolipoproteins. J. Phys. Chem. B 2004, 108, 7307−7315. (16) Garcia-Gonzalez, A.; Flores-Vazquez, A., L; Maldonado, E.; Barba de la Rosa, A. P.; Ruiz-Garcia, J. Globulin 11S and Its Mixture With L-Dipalmitoylphosphatidylcholine at the Air/Liquid Interface. J. Phys. Chem. B 2009, 113, 16547−16556. (17) Möbius, D.; Miller, R.; Nylander, T. Protein at Liquid Interfaces. In Studies of Interface Science, Vol. 7; Möbius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998. (18) Möhwald, H. Phospholipid and Phospholipid-Protein Monolayers at the Air/Water Interface. Annu. Rev. Phys. Chem. 1990, 41, 441−476. (19) Ruiz-Garcia, J.; Qiu, X.; Tsao, M. W.; Marshall, G.; Knobler, C. M.; Overbeck, G. A.; Möbius, D. Splay Stripe Textures in Langmuir Monolayers. J. Phys. Chem. B 1993, 97, 6955−6957. (20) Cadena-Nava, R. D.; Martin-Mirones, J. M.; Vazquez-Martinez, E. A.; Roca, J. A.; Ruiz-García, J. Direct Observations of Phase Changes in Langmuir Films of Cholesterol. Rev. Mex. Fis. 2006, 52, 32−40. (21) Lösche, M.; Sackmann, E.; Möhwald, H. A Fluorescence Microscopic Study Concerning the Phase Diagram of Phospholipids. Ber. Bunsenges Phys. Chem. 1983, 87, 848−852. (22) Weis, R. M.; McConnell, H. M. Two-Dimensional Chiral Crystals of Phospholipid. Nature (London) 1984, 310, 47−49. (23) Henón, S.; Meunier, J. Microscope at the Brewster Angle: Direct Observation of First-Order Phase Transitions in Monolayers. Rev. Sci. Instrum. 1991, 62, 36−39. (24) Qiu, X.; Ruiz-Garcia, J.; Stine, K. J.; Knobler, C. M.; Selinger, J. V. Direct Observation of Domain Structure in Condensed Monolayer Phases. Phys. Rev. Lett. 1991, 67, 703−706. (25) Rodríguez-Patino, J. M.; Carrera Sánchez, C.; Molina Ortiz, S. E.; Rodríguez Niño, M. R.; Añoń , M. C. Adsorption of Soy Globulin Films at the Air−Water Interface. Ind. Eng. Chem. Res. 2004, 43, 1681−1689. (26) Kamilya, T.; Pal, P.; Talapatra, G. B. Interaction of Ovalbumin with Phospholipids Langmuir-Blodgett Film. J. Phys. Chem. B 2007, 111, 1199−1205. (27) Rodríguez-Patino, J. M.; Carrera-Sánchez, C.; CejudoFernández, M.; Rodríguez-Niño, M. R. Protein Displacement by Monoglyceride at the Air−Water Interface Evaluated by Surface Shear Rheology Combined with Brewster Angle Microscopy. J. Phys. Chem. B 2007, 111, 8305−8313.

presence of the protein, nucleate isolated while on buffer the LC are often associated. However, BAM images do not show clear evidence of the presence of the protein at the interface, but AFM show at higher resolution the presence of small protein domains, normally found as nucleation centers for the LC lipid phase growth, but the amount of these protein domains does not account for all of the protein that would shift the isotherms of the L-DPPC monolayer. Fluorescence experiments gave further information on the presence of the protein, which in some cases acted as a nucleation center for the lipid LC domains but was mostly found as denatured in the LE phase. Here we demonstrate that fluorescence microscopy is a powerful tool to detect protein within the LE phase of lipids. Finally, both fluorescence and AFM microscopies show the formation of doughnut-like shape protein domains that have LE phase at the center.

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ASSOCIATED CONTENT

S Supporting Information *

Reproducibility of the surface pressure vs area/molecule isotherms for all experimental conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 011-52-444-826-2362 to 65 ext. 121. Fax: 813-3874. Email: jaime@ifisica.uaslp.mx. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank E. Maldonado for help in protein purification. This work was supported by CONACYT Grant 131862, PIFIPROMEP, and UASLP-Fondos Concurrentes. A.L.F.-V. acknowledges CONACYT for postdoctoral support. A.G.-G. acknowledges CONACYT for a Ph.D. scholarship. Finally, authors thank M. C. José Bante Guera of CINVESTAV-IPN Unidad Merida for technical support with the confocal microscope.

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