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Structural changes of soy proteins at the oil–water interface studied by fluorescence spectroscopy
Colloids and Surfaces B: Biointerfaces 93 (2012) 41–48
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Structural changes of soy proteins at the oil–water interface studied by fluorescence spectroscopy Maneephan Keerati-u-rai a,∗ , Matteo Miriani a,b , Stefania Iametti b , Francesco Bonomi b , Milena Corredig a a b
Department of Food Science, University of Guelph, Ontario, N1G 2W1, Canada Università degli Studi di Milano, Department of Food Science, Milano, Via Celoria 2, Italy
a r t i c l e
i n f o
Article history: Received 14 September 2011 Received in revised form 30 November 2011 Accepted 2 December 2011 Available online 13 December 2011 Keywords: Soy proteins Soy protein isolate Glycinin -conglycinin Soy protein emulsion Front face fluorescence Heat treatment
a b s t r a c t Fluorescence spectroscopy was used to acquire information on the structural changes of proteins at the oil/water interface in emulsions prepared by using soy protein isolate, glycinin, and -conglycinin rich fractions. Spectral changes occurring from differences in the exposure of tryptophan residues to the solvent were evaluated with respect to spectra of native, urea-denatured, and heat treated proteins. The fluorescence emission maxima of the emulsions showed a red shift with respect to those of native proteins, indicating that the tryptophan residues moved toward a more hydrophilic environment after adsorption at the interface. The heat-induced irreversible transitions were investigated using microcalorimetry. Fluorescence spectroscopy studies indicated that while the protein in solution underwent irreversible structural changes with heating at 75 and 95 ◦ C for 15 min, the interface-adsorbed proteins showed very little temperature-induced rearrangements. The smallest structural changes were observed in soy protein isolate, probably because of the higher extent of protein–protein interactions in this material, as compared to the -conglycinin and to the glycinin fractions. This work brings new evidence of structural changes of soy proteins upon adsorption at the oil water interface, and provides some insights on the possible protein exchange events that may occur between adsorbed and unadsorbed proteins in the presence of oil droplets. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Soy proteins are widely employed as ingredients in foods because of their nutritional and processing functionality. These proteins have the ability to adsorb at the oil–water or air–water interfaces [1,2], reducing interfacial tension and forming a thick layer which provides stability to the oil droplets. Soy protein isolate (SPI) is mostly constituted of glycinin and -conglycinin. Glycinin is a hexamer, composed of two trimers stacking on top of one another [3]. Its molecular structure consists of an acidic and a basic polypeptide linked by a disulfide bond, which gives structural rigidity to the protein. Soy glycinin is a very heterogeneous protein, with various subunit combinations and a molecular mass in the 300–380 kDa range. The structure is held together by hydrogen bonds and electrostatic interactions, and it can be dissociated using thermal treatment, pH, or chaotropic agents such as urea [4–6]. The second most abundant protein in SPI is -conglycinin, a glycoprotein present as a trimer with a molecular mass in the 150–200 kDa
∗ Corresponding author. Tel.: +1 519 824 4120x56101; fax: +1 519 824 6631. E-mail address: [email protected] (M. Keerati-u-rai). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.12.002
range. This protein is composed of three different subunits; ␣, ␣ and  [7]. Studies using recombinant proteins have demonstrated that the three polypeptides have a similar core structure, and ␣, ␣ have acidic glycosilated extension regions [7,8]. Unlike glycinin, -conglycinin does not have disulfide bridges. Both these proteins are present in an aggregated form in solution [9]. It has been previously demonstrated that both major soy proteins adsorb at the oil–water interface [10,11]. When oil-in-water emulsions are prepared with SPI, the oil droplets show a negatively charged layer with a -potential of about −15 mV at neutral pH [12]. Furthermore, the proteins adsorb as a monolayer with a thickness corresponding to that of the average size of the protein oligomers, around 30–40 nm [12]. Although a number of studies have been published on the emulsifying properties of soy proteins and on the stability of the emulsions to various environmental and processing conditions [11,13,14], only a few of them addressed fundamental questions related to structural rearrangements at the interface. A recent study using fluorescence spectroscopy allowed deriving some hypotheses on the changes occurring during thermal treatment on glycinin and -conglycinin in solution, or after adsorption at interface [15]. In the present work, the changes occurring in SPI, glycinin, and -conglycinin proteins after adsorption
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at oil-in-water interfaces, as well as after thermal treatment of the solutions or emulsions, were followed using fluorescence spectroscopy. Fluorescence spectroscopy is a well established technique to observe protein conformational changes as well as to study the changes in protein-based matrices during processing or storage [16–19]. Front face fluorescence spectroscopy has been introduced to overcome some of the limitations related to the analysis of solid, turbid, viscous, and concentrated samples [20,21]. In proteins, the dominant fluorophore is the indole group of tryptophan, and its emission is very sensitive to solvent polarity. Hence, the fluorescence spectrum of a protein is determined by the chemical environment of the fluorescent amino acids, and changes in the tryptophan (Trp) spectrum can be used to identify conformational changes of proteins, occurring, for example, upon adsorption onto an oil–water interface. When Trp residues move from a more hydrophobic environment to a hydrophilic one, the emission maximum shifts to longer wavelengths (red shift), whereas the orientation toward a more hydrophobic location causes a blue shift. The intensity of these shifts is a function of both the extent of exposure, and of the number of involved residues [19]. Results obtained using micro-DSC suggests that small structural changes occur to soy proteins after adsorption at the oil/water interface [2,12]. In addition, only limited conformational changes have been reported for glycinin protein adsorbed at the air–water interface using infrared reflection–absorption spectroscopy [1]. The aim of this work was to study the structural changes of soy proteins when present at the oil–water interface in emulsions. The nature and extent of these changes was compared to changes induced on the soluble protein by physical (heat) or chemical (chaotropes) denaturation. Furthermore, to discriminate between structural changes occurring at the interface from those affecting the non-adsorbed proteins, a structural analysis was performed on both the cream and serum fractions, separated with centrifugation. Finally, structural characterizations of proteins in heated emulsions and of emulsions prepared from heated proteins allowed discriminating the structural changes of native, heat-denatured, and surface-denatured proteins.
2. Materials and methods 2.1. Protein preparation Soy protein isolate (SPI) was prepared by dispersing (1:10 ratio, w/v) defatted soy flakes (a gift from the Solae Company, St. Louis, MO, USA dispersion index 90) in 100 mM Tris–HCl buffer, pH 8.0. After stirring at room temperature for 1 h, the soluble fraction was separated by centrifugation at 12,000 × g for 30 min at 10 ◦ C (Beckman Coulter, Model J2-21, Fullerton, CA, USA). The centrifuged dispersion was then adjusted to pH 4.8 with 2 M HCl, and refrigerated at 4 ◦ C for 2 h. The precipitated protein was recovered by centrifugation as described above, washed with 8 volumes of 10 mM sodium acetate, pH 4.8, solubilized in ultrapure water (Barnstead International, E-pureTM D4641, Ames, IA, USA), and adjusted to pH 7.5 with 2 M NaOH. After overnight dialysis against water at 4 ◦ C, the material was freeze dried and stored at −20 ◦ C. Fractions rich in glycinin and -conglycinin were isolated as previously described [22] with minor modifications. Defatted soy flakes (The Solae Company) were suspended in ultrapure water in 1:15 ratio (w/v) and the pH adjusted to 8.0 with 2 M NaOH. After stirring the suspensions for 2 h at room temperature, the insoluble fractions were separated by centrifugation at 9000 × g for 30 min at 20 ◦ C. Sodium bisulfite (0.98 g/l) was added to the soluble fraction after adjusting the pH to 6.4 with 1 M HCl. After overnight
standing at 4 ◦ C, the protein suspension was centrifuged at 7000 × g for 20 min at 4 ◦ C. A glycinin-rich fraction was recovered in the precipitate, solubilized in ultrapure water, and adjusted to pH 7.5 with 2 M NaOH. Sodium chloride (0.25 M final concentration) was added to the remaining supernatant, which was adjusted to pH 5.0 with 1 M HCl and stirred for 1 h in an ice bath. After centrifugation at 9000 × g for 30 min at 4 ◦ C the supernatant was diluted with cold ultrapure water in 2:1 ratio (v/v) and adjusted to pH 4.8 with 2 M HCl. A -conglycinin rich fraction was recovered by centrifugation at 7000 × g for 20 min at 4 ◦ C. The precipitate was resolubilized with ultrapure water and adjusted to pH 7.5 with NaOH. Both isolated fractions (-conglycinin and glycinin rich fractions) were dialyzed overnight at 4 ◦ C against ultrapure water, and freeze-dried. The freeze dried proteins were stored at −20 ◦ C. Protein content was determined using the combustion method for nitrogen (Leco FP-528, Mississauga, ON, Canada), and using N × 6.25 for calculation (approved method 46-30 AACC, 2000). The purity of polypeptides was assessed by using SDS-PAGE. The glycinin-rich fraction was 90% pure, and -conglycinin about 86%. 2.2. Emulsion preparation Previous work demonstrated that 1% (w/v) SPI and conglycinin were necessary to prepare a stable emulsion of 10% soybean oil with a monomodal distribution of droplet size [2,12], whereas 2% protein (w/v) was needed to prepare an emulsion with glycinin [12]. Protein suspensions prepared in 50 mM sodium phosphate buffer, pH 7.4, were left overnight at 4 ◦ C to fully hydrate, and then were equilibrated at room temperature prior to homogenization. A pre-homogenization step with 10% soybean oil (w/w) (Sigma–Aldrich, St. Louis, MO, USA) was performed by using a shear dispersing unit (PowerGen 125, Fisher Scientific, Ottawa, ON, Canada) for approximately 1 min, and followed immediately by homogenization in a microfluidizer (110S model, Newton, MA, USA) for five passes with an input pressure of 300 kPa, corresponding to an overall pressure of 40 MPa. Emulsions were stored at 4 ◦ C, and analyzed the next day. 2.3. Heat treatment Thermal treatment of solutions and emulsions was carried out in a circulating water bath (Thermo Haake DC10, Sigma–Aldrich, St. Louis, MO, USA) at two different temperatures (75 ◦ C and 95 ◦ C) for 15 min. These temperatures were chosen based on the denaturation temperatures of -conglycinin and glycinin, as measured by microDSC [2]. Heating at 75 ◦ C for 15 min was performed for emulsions stabilized with a -conglycinin-rich fraction, while 95 ◦ C for 15 min was used for emulsions stabilized with a glycinin-rich fraction. Emulsions prepared with SPI were treated at either 75 or 95 ◦ C for 15 min. In addition to heating the emulsions after homogenization, emulsions were also prepared with heated protein dispersions. In this case, after cooling the heated protein solution in an ice bath, the protein solutions were immediately homogenized with 10% soy oil as reported above. 2.4. Microcalorimetry Protein samples were diluted to 5 mg/ml with 50 mM sodium phosphate buffer, pH 7.4, and analyzed by micro-calorimetry (VPDSC, MicroCal Inc., North Hampton, MA, USA) to verify the presence of thermal transitions in the original protein samples or to verify complete denaturation in heated samples. Both emulsions and cream samples were analyzed. The cream layer separated from the serum phase by centrifugation at 12,000 × g for 15 min (Brinkmann Instruments, Westbury, NY, USA) was carefully removed and dried on filter paper (Whatman # 1, Fisher Scientific, ON, Canada). The
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Tryptophan fluorescence emission spectra (ex , 280 nm; em , 300–450) were measured in a solid sample holder fitted to a spectrofluorometer (Shimadzu, RF5301, Mandel Scientific co. Inc., Mississauga, ON, Canada). Excitation and emission slits were set at 5 nm. Prior to analysis, emulsions were diluted 1:10 with 50 mM sodium phosphate buffer, pH 7.4. In the case of cream analysis, the cream layer blotted on filter paper, as described above, was washed with buffer to remove the excess protein, and re-suspended in buffer to its initial volume fraction (10% oil). The serum was obtained from centrifugation of emulsions at 10,000 × g for 20 min, removed using a needle and syringe, and filtered through a 0.22 m HPLC filter. Both cream and serum were diluted 1:1 with buffer immediately before fluorescence measurements. 2.6. Statistical analysis All experiments were conducted in triplicate (three separate emulsion preparations) and data are reported as averages and standard deviations. 3. Results and discussion 3.1. Spectroscopic features of urea-denatured, heat-denatured, and surface adsorbed soy proteins in whole emulsions Emission fluorescence spectra of proteins in solution and in the corresponding emulsions are shown in Fig. 1 along with those of the urea-denatured proteins. In the conditions used here, Trp residues are sole responsible of the observed signal, and contributions from other fluorescent amino acid residues may be considered negligible. Trp are found in both the acidic and basic polypeptides of glycinin. In general, the acidic subunit has more Trp than the basic subunit, which has one Trp, except for the B3 variant, which has two. There are more Trp residues (three to five) in each glycinin monomer than in -conglycinin [23]. In fact, only one Trp residue is found in the ␣ subunit, two in ␣ , and none in the  subunit of -conglycinin. The spectra of native proteins in Fig. 1 and the relative abundance of Trp residues discussed above highlight that structural information may only be derived by an analysis of the position of the fluorescence emission maximum, and not by the intensity of the emission, as pointed out by almost all studies in which fluorescence has been used to monitor loss or acquisition of protein structure [19]. Shifts of fluorescence emission to longer wavelengths (red shift) are indicating exposure of the fluorophores to a hydrophilic environment [15,19,24]. Previous reports have shown that the structure of soy proteins is lost upon treatment with chaotropes like urea or guanidine hydrochloride [15,25,26]. Treatment of the various protein solutions with urea results in a marked red shift of fluorescence
Fluorescence Intensity (A.U.)
A
300 250 200 150 100 50 900
Fluorescence Intensity (A.U.)
2.5. Fluorescence measurements
350
Fluorescence Intensity (A.U.)
cream was then resuspended in 50 mM sodium phosphate buffer, pH 7.4, to its initial volume fraction (10%). Samples were held at 20 ◦ C for 15 min prior to heating from 20 to 115 ◦ C at a 1 ◦ C/min scan rate, and then cooled to 20 ◦ C. Reference samples were either 50 mM sodium phosphate buffer, pH 7.4, or 10% soybean oil in emulsion prepared with 2% Tween 80 (Sigma–Aldrich, ON, Canada). Enthalpy changes (H) and denaturation temperature midpoint (Td ) were calculated using the OriginTM software version 7.0 (OriginLab, Northampton, MA, USA), as the area under the peak and the temperature at peak maximum, respectively.
43
B
800 700 600 500 400 300 200 100 500
C
400 300 200 100 0
320
340
360
380
400
Wavelength (nm) Fig. 1. Emission fluorescence spectra of (A) -conglycinin (10 mg/ml), (B) glycinin (20 mg/ml) and (C) soy protein isolate (10 mg/ml). Emission spectra (ex : 280 nm) were recorded on protein solutions in buffer (solid lines), in buffer containing 8 M urea (dotted lines), or on 10% soy oil emulsions (dashed lines). All samples were diluted ten-fold in respective buffer prior to measurements. Note the difference in the Y axis. Spectra are representative, for statistical analysis of maximum peak (see Table 1).
emission [15]. The observed red shift with denaturation by 8 M urea was highest for glycinin (18 nm, from 329 to 347), followed by conglycinin (10 nm, from 334 to 344) and by SPI (10 nm, from 332 to 342) (Fig. 1; Table 1). The emission maximum wavelengths for all the spectra collected in this work are summarized in Table 1, for ease of comparison. In the case of -conglycinin, the Trp residues are part of the solvent-exposed N-terminal extended domains of the ␣ and ␣ subunits (Trp63 and Trp63, Trp100, respectively). Hence, the smaller red shift recorded for -conglycinin in the presence of Urea, compared to glycinin. In the case of SPI, the shift in the spectrum resembled that of the -conglycinin in solution. This behavior may be caused by the relative ratio of the two proteins in the SPI, by the presence of aggregates of the various proteins, and/or by differences in processing history, which may result in a lower
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Table 1 Position of the emission maximum in fluorescence spectra. Values are the average of three separate experiments. The only sample showing a blue shift with respect to native proteins is highlighted in bold.a Sample
Fluorescence emission maximum (nm) -Conglycinin Unheated
Native protein Urea-treated protein Heated protein Unheated emulsions Cream from unheated emulsions Serum of unheated emulsions Cream from heated emulsions Serum from heated emulsions Cream from emulsions prepared with heated protein Serum from emulsions prepared with heated protein
◦
75 C
Unheated
334.2 ± 0.8 344.0 ± 0.0
Soy protein isolate ◦
95 C
Unheated
329.1 ± 0.2 347.2 ± 0.1 335.6 ± 0.0
338.0 ± 0.3 341.2 ± 0.3 327.3 ± 3.0 343.1 332.0 343.4 332.2
± ± ± ±
333.8 ± 0.2
0.3 0.7 0.3 0.6
75 ◦ C
95 ◦ C
333.7 ± 0.2
335.6 ± 0.4
332.4 ± 0.2 342.1 ± 0.2
335.8 ± 0.6 340.9 ± 0.2 329.7 ± 0.2
334.2 ± 0.1 340.1 ± 0.6 331.8 ± 0.4 341.2 334.9 339.4 336.1
± ± ± ±
0.4 0.2 0.2 0.2
343.3 333.3 340.2 332.2
± ± ± ±
0.2 0.4 0.6 0.2
341.8 337.6 339.6 353.2
± ± ± ±
0.2 0.4 0.6 1.7
1.8e-3
Heat Capacity (cal/oC)
A 1.2e-3
6.0e-4
0.0
Heat Capacity (cal/oC)
4.8e-3
B
3.6e-3
2.4e-3
1.2e-3
Fluorescence Intensity (A.U.)
Value are the average of emission spectra ± standard deviation of three replicate experiments.
Fluorescence Intensity (A.U.)
a
Glycinin
Heat Capacity (cal/oC)
1.8e-3
C
1.2e-3
6.0e-4
0.0
Fluorescence Intensity (A.U.)
0.0
350
250 200 150 100 50 900 0
40
60
80
o
100
120
Temperature ( C) Fig. 2. Thermal transitions measured using microcalorimetry for 10% oil-in-water emulsions (solid line) and corresponding solutions (dotted lines) of -conglycinin 10 mg/ml (A), glycinin 20 mg/ml (B) and soy protein isolate 10 mg/ml (C).
B
800 700 600 500 400 300 200 100 500
C
450 400 350 300 250 200 150 100 50 0
20
A
300
320
340
360
380
400
Wavelength (nm) Fig. 3. Intrinsic fluorescence spectra of (A) -conglycinin (10 mg/ml), (B) glycinin (20 mg/ml) and (C) soy protein isolate (10 mg/ml). Emission spectra were recorded for the whole emulsions (solid line), the adsorbed proteins in the cream phase (broken line), and the unadsorbed protein in the continuous phase (centrifugal serum) (dotted line). The maximum wavelength values are summarized in Table 1.
A
1.5e-3
Heat Capacity (Cal/oC)
Fluorescence Intensity (A.U.)
M. Keerati-u-rai et al. / Colloids and Surfaces B: Biointerfaces 93 (2012) 41–48
1.0e-3
5.0e-4
0.0
Fluorescence Intensity (A.U.)
Heat Capacity (Cal/oC)
4.0e-3 B
3.0e-3 2.0e-3 1.0e-3 0.0
Fluorescence Intensity (A.U.)
Heat Capacity (Cal/oC)
2.0e-3 C
1.5e-3 1.0e-3 5.0e-4 0.0 0
20
40
60
80
100
120
o
Temperature ( C) Fig. 4. Thermal transitions measured using microcalorimetry for (A) -conglycinin, (B) glycinin and (C) soy protein isolate solutions in 50 mM sodium phosphate, pH 7.4. Unheated (solid line), heated at 75 ◦ C (dashed lines) and at 95 ◦ C (dotted lines) for 15 min.
susceptibility of the glycinin aggregates in the SPI to denaturation by chaotropes. Fig. 1 also shows the fluorescence spectra of the various proteins when present in the emulsions (both in solution and adsorbed at the interface). Compared to their corresponding solutions, the spectra also showed a red shift in all cases, although to a smaller extent than in the presence of urea, suggesting an increased exposure to the aqueous phase of the Trp residues after adsorption of the proteins at the oil–water interface, in agreement with previous reports [15]. If Trp residues were present in the portion of the proteins interacting with the oil interface, a blue shift would be expected. However, the fluorescence spectrum of -conglycinin in emulsion showed a red shift of 4 nm (from 334 to 338) compared to that of the protein in solution, whereas glycinin showed a red shift of 7 nm (from 329 to 336). It has been previously proposed [15] that the Trp residues located in the extended region of the -conglycinin subunits protrude in the aqueous phase, whereas the Trp-free core hydrophobic region of -conglycinin may act as
45
350
A 300 250 200 150 100 50 900
B
800 700 600 500 400 300 200 100 0 450
C
400 350 300 250 200 150 100 50 0
320
340
360
380
400
Wavelength (nm) Fig. 5. Emission fluorescence spectra of (A) -conglycinin (10 mg/ml), (B) glycinin (20 mg/ml) and (C) soy protein isolate (10 mg/ml). Emission spectra (ex : 280 nm) were recorded for unheated protein solutions (solid line), heated protein at 75 ◦ C for 15 min (dotted line), and heated protein solution at 95 ◦ C for 15 min (dashed line). All samples were diluted ten-fold in buffer prior to the measurement.
an anchor at the interface [15,27]. It is also established that the ␣ and ␣ subunits show better emulsifying properties than the  subunit of -conglycinin because of the extension regions [27]. In the case of the emulsions prepared with SPI, a very modest red shift was shown (2 nm, from 332 to 334 nm, see Table 1), again suggesting the presence of extensive intermolecular interactions in SPI, in agreement with recent microcalorimetry studies, where small thermal transitions were observed for soy proteins once adsorbed at the interface compared to the original proteins in solution [2]. In the frame of these studies, it must be noted that it was previously demonstrated that the microfluidization of the proteins in the absence of oil did not affect their emission fluorescence spectra [15].
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Fluorescence Intensity (A.U.)
Fluorescence Intensity (A.U.)
46
600
A
B
500 400 300 200 100 400 0
C
D
300
200
100
0
320
340
360
380
Wavelength (nm)
400
320
340
360
380
400
Wavelength (nm)
Fig. 6. Emission fluorescence spectra of (A) -conglycinin heated at 75 ◦ C for 15 min, (B) glycinin heated at 95 ◦ C for 15 min and (C and D) soy protein isolate heated at 75 ◦ C (C) and 95 ◦ C (D) for 15 min. Spectra were collected for cream from unheated emulsion (solid line); cream from heated emulsion (dashed line); cream from emulsion prepared with heated solution (dotted line). The maximum wavelength values are summarized in Table 1.
3.2. Thermal transitions of the adsorbed proteins in emulsions Microcalorimetry has been employed to study the thermal transitions of soy proteins emulsion, this approach allows to appreciate thermodynamically relevant transitions associated to changes in the structural features of adsorbed proteins [9]. Homogenization of -conglycinin and glycinin in the absence of oil did not alter the DSC profile of either protein, confirming once again that the treatment “per se” did not promote major structural changes [9,15]. Two main thermal transition peaks were observed in both solutions and emulsions, at 69 ◦ C and 84 ◦ C (Fig. 2), that essentially correspond to the reported thermal transitions for -conglycinin and glycinin, respectively. In the -conglycinin containing emulsion, a second peak at 95 ◦ C was evident, that was absent in the DSC tracings obtained for the protein in solution. The appearance of a transition at high temperature may be consequent to rearrangements of the supramolecular structure of this protein at the interface, which could have led to a fraction of the protein transitioning at a higher temperature [12]. In the case of emulsions prepared with SPI, the transitions were in full agreement to those of the emulsions prepared with individual fractions, suggesting the absence of intermolecular interactions involving both proteins. 3.3. Structural characterization of the proteins present in individual phases of emulsions It is important to note that results shown in Figs. 1 and 2 were obtained with emulsion samples containing both absorbed and unadsorbed proteins. To distinguish between the structural changes occurring at the interface and those involving the protein in solution, fluorescence measurements were carried out on
emulsions after separation of the cream and serum phases. As shown by the fluorescence emission spectra in Fig. 3 and by the data in Table 1, the shift in the maximum emission wavelength in emulsions may be ascribed to contributions from both the protein adsorbed at the interface in the cream phase and from the unadsorbed protein present in the serum phase. The observed shifts were larger for the proteins at the interface than for the unadsorbed protein in the serum (Fig. 3); although, in all cases, the extent of the spectral changes was much lower than those observed for proteins treated with 8 M urea (Fig. 1; Table 1). As reported in Table 1, the fluorescence spectrum of -conglycinin on the oil droplet was red shifted 7 nm with respect to that of the native protein, compared to a 7 nm blue-shift of the protein in the serum phase versus the protein in buffer. In the case of glycinin, red shifts were 12 nm (cream versus original solution) and 1 nm (serum versus original solution). SPI samples showed a red shift of 8 nm (cream versus solution), and no shifts were noticeable in the proteins remaining in the serum phase. This latter observation once again suggested that the glycinin present in SPI contributes less to the observed changes than the glycinin in the glycinin-rich fraction. The 7 nm blue shift (from 334 to 327 nm) recorded for the conglycinin present in the serum, may come from an exchange between the adsorbed and unadsorbed proteins occurring at the interface, followed by aggregation of the protein released from the droplet surface. The aggregation itself may shift tryptophan residues to a more hydrophobic environment, as reported for other food proteins [19,24,28]. It is important to note that any interpretation of these spectroscopic shifts to assess the amounts of individual protein forms in the system is improper, as the data cannot provide relative figures for the various protein forms (i.e.,
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900 Fluorescene Intensity (A.U)
800
47
A
B
C
D
700 600 500 400 300 200 100
Fluorescence Intensity (A.U)
0 500
400
300
200
100
0
320
340
360
380
Wavelength (nm)
400
320
340
360
380
400
Wavelength (nm)
Fig. 7. Emission fluorescence spectra of (A) -conglycinin heated at 75 ◦ C for 15 min, (B) glycinin heated at 95 ◦ C for 15 min and (C and D) soy protein isolate heated at 75 ◦ C (C) and 95 ◦ C (D) for 15 min. Spectra of centrifugal serum from unheated emulsions (solid line), heated protein solutions (dotted line), serum from heated emulsion (dashed line) and serum from emulsions prepared with heated solutions (grey line). The maximum wavelength values are summarized in Table 1.
in solution, at the interface, denatured and aggregated, denatured and non-aggregated). 3.4. Thermal treatment of proteins and emulsions, and properties of emulsion stabilized by heat-treated proteins The structural changes occurring to the proteins after heating solutions and emulsions were also studied, also in consideration of the practical relevance of thermal processes. The various samples were heated either at 75 or 95 ◦ C for 15 min, in the attempt to selectively denature individual protein fractions (see Fig. 2). Changes in the thermal transitions of the protein solutions before and after these thermal treatments are shown in Fig. 4, and reflect the differences in denaturation temperature between -conglycinin and glycinin. In particular, -conglycinin was fully denatured (no residual thermal transition) after having been heated at 75 ◦ C for 15 min, whereas glycinin lost its thermal transition after having been heated at 95 ◦ C for 15 min. This selective denaturation also occurred in the SPI samples, since the denaturation peak of conglycinin disappeared after heating at 75 ◦ C and that of glycinin was no longer present after heating at 95 ◦ C. Intrinsic fluorescence spectra for the various protein solutions before and after heating are shown Fig. 5. There were no differences (see also Table 1) in the maximum emission wavelength for -conglycinin after heating at 75 ◦ C for 15 min (Fig. 5A) compared to the unheated protein, in spite of the full loss of the thermal transition peak after this temperature (Fig. 4A). On the other hand, it has been reported that heating -conglycinin at 75 ◦ C increases the overall surface hydrophobicity of the protein and causes the formation of soluble aggregates upon cooling via hydrophobic interactions [15,29]. Also, it has been shown that heat
dissociated -conglycinin could partially rearrange into non-native trimers after cooling [15,30]. It is important to note that the spectra of -conglycinin reflect changes in the extended region of the protein, and changes in the core structure of the protein cannot be observed by fluorescence spectroscopy, due to the lack of fluorophores in the protein structural core domain. There was a 5 nm red shift after heating glycinin at 95 ◦ C for 15 min (Fig. 5B; Table 1), in agreement with earlier studies [15]. The red shift may indicate either a dissociation of the native quaternary structure (or of some undescribed supramacromolecular aggregates) or the formation of a more open and solvent-accessible tertiary structure. In the case of SPI solutions, there was a small shift (2 nm) in the fluorescence spectrum after heating at 75 ◦ C for 15 min and a 4 nm red shift after heating at 95 ◦ C for 15 min (Fig. 5C; Table 1). The higher shift at the higher temperature is in line with the behavior of glycinin, as discussed above. Fluorescence measurements were also performed on the cream and serum phases of emulsions subjected to heating, either at 75 ◦ C or 95 ◦ C for 15 min, and on emulsions prepared from heated proteins. The fluorescence spectra of the protein present at the interface (cream) and in the unadsorbed phase (serum) are shown in Figs. 6 and 7, respectively, whereas figures for maximum emission wavelengths are summarized in Table 1. Fig. 6 compares the differences in the cream phases in heated and unheated emulsions prepared with the various proteins before and after heating. In the case of -conglycinin (Fig. 6A), spectra of the proteins in the various cream sample were almost overlapping. There were no statistically significant changes also in the emission maximum of glycinin when adsorbed at the interface before or after heating at 95 ◦ C (Fig. 6B; Table 1), suggesting that adsorption prevented further structural rearrangements of glycinin at
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interface. The fluorescence spectra of proteins in SPI-stabilized cream showed a red shift of 3 nm after heating at 75 ◦ C (Fig. 6C), but no further changes after heating at 95 ◦ C (Fig. 6D). It could be hypothesized that in the case of SPI, the red shift is attributable to the structural change occurring to -conglycinin, as there is no further change at the higher temperature. In the case of SPI-based emulsions prepared with heated protein solutions, no significant change was observed for the emission maximum (Fig. 6C and D; Table 1) after heating at either 75 or 95 ◦ C. Fig. 7 compares the fluorescence emission spectra of the serum phases for the various emulsions. In the case of -conglycinin, the original, unheated protein solution showed a peak at 334 nm, with very little change after heating (Fig. 6A) or when a heated solution was used to prepare the emulsion. It has been previously demonstrated that under these conditions, a higher amount of protein adsorbs at the interface [12]. The unadsorbed fraction in heated glycinin-stabilized emulsions showed a red shift at 335 nm (heated after homogenization) or 336 nm (heated prior to homogenization, Fig. 7B). These figures are significantly higher than those for the native protein (329 nm) or for the unadsorbed protein in the unheated emulsion (330 nm, Fig. 6 and Table 1), and close to what observed when the protein was heated in solution. In the case of SPI-stabilized emulsions, while very little change was noted in the unadsorbed fractions after heating the emulsions at 75 ◦ C (Fig. 7C), or when emulsions were prepared from proteins heated at 75 ◦ C, in agreement with results obtained with -conglycinin (Fig. 7A). After heating the emulsion at 95 ◦ C for 15 min, the unadsorbed proteins showed a red shift similar to that described for the glycinin-based system (Fig. 7D). The largest fluorescence shift (17 nm, from 336 to 353 nm, Table 1) was observed when heated SPI were used. However, the fluorescence intensity in these samples was very low, because of the low residual protein present in the serum phase as a consequence of the aggregation events amply discussed above. 4. Conclusions The use of front-face fluorescence to monitor conformational changes of soy proteins in emulsions made it possible to show that adsorption of the soy proteins changed the chemical environment of tryptophans in their structure. The data reported here confirm that the core structure of -conglycinin (that does not contain major fluorophores) adheres at the interface, and flexible moieties in its ␣ and ␣ subunits become even more exposed to the aqueous phase than they were in solution. Studies on the unadsorbed fraction of -conglycinin would suggest an exchange between proteins adsorbed at interface and those present in the continuous phase. A blue shift in the spectrum of -conglycinin present in the unadsorbed phase indicated aggregation of unadsorbed protein in solution. Glycinin showed remarkable changes in its structure when at the interface, whereas the fraction present in the continuous phase of the glycinin-stabilized emulsions did not show structural
rearrangements in the absence of a thermal treatment. The behavior of the unadsorbed fraction after heating at 95 ◦ C, was in line with what observed in the heated solutions, suggesting that the unadsorbed protein retained most of the structural features of the unheated protein in solution. This study also points out that heat treatments affected differently the structure of adsorbed and unadsorbed proteins. The adsorbed proteins were much less prone to temperature-induced structural rearrangements than those in solution. In the practically relevant case of SPI, the rearrangements in the system as a whole were less pronounced than for those expected from studies on the isolated protein components (i.e., glycinin and -conglycinin), most probably because of the presence of protein aggregates as a consequence of previous processing. This points out the importance of accurate characterization at the molecular level of materials that are of common use in the food industry and that are often characterized only in terms of composition and not in terms of their structure. References [1] A.H. Martin, M.B.J. Meinders, M.A. Bos, M.A. Cohen Stuart, T. van Vliet, Langmuir 19 (2003) 2922. [2] M. Keerati-u-rai, M. Corredig, Food Hydrocoll. 23 (2009) 2141. [3] M. Adachi, Y. Takenaka, A.B. Gidamis, B. Mikami, S. Utsumi, J. Mol. Biol. 305 (2001) 291. [4] C.M.M. Lakemond, H.H.J. de Jongh, M. Hessing, H. Gruppen, A.G.J. Voragen, J. Agric. Food Chem. 48 (2000) 1991. [5] J.M.S. Renkema, C.M.M. Lakemond, H.H.J. de Jongh, H. Gruppen, T. van Vliet, J. Biotechnol. 79 (2000) 223. [6] P.R. Shewry, Biol. Rev. 70 (1995) 375. [7] N. Maruyama, T. Katsube, Y. Wada, M.H. Oh, A.P. Barba de la Rosa, E. Okuda, S. Nakagawa, S. Utsumi, Eur J. Biochem. 258 (1998) 854. [8] N. Maruyama, M.R.M. Salleh, K. Akahashi, K. Yagasaki, H. Goto, N. Hontani, S. Nakagawa, S. Utsumi, J. Am. Oil Chem. Soc. 79 (2002) 139. [9] M. Keerati-u-rai, M. Corredig, J. Agric. Food Chem. 57 (2009) 3556. [10] F.E. Mitidieri, J.R. Wagner, Food Res. Int. 35 (2002) 547. [11] G.G. Palazolo, F.E. Mitidieri, J.R. Wagner, Food Sci. Technol. Int. 9 (2003) 409. [12] M. Keerati-u-rai, M. Corredig, J. Agric. Food Chem. 58 (2010) 9171. [13] G.G. Palazolo, D.A. Sorgentini, J.R. Wagner, J. Am. Oil Soc. 81 (2004) 625. [14] E. Molina, A. Papadopoulou, D.A. Ledward, Food Hydrocoll. 15 (2001) 263. [15] M. Miriani, M. Keerati-u-rai, M. Corredig, S. Iametti, F. Bonomi, Food Hydrocoll. 25 (2011) 620. [16] C. Castelain, C. Genot, Biochim. Biophys. Acta 1199 (1994) 59. [17] C. Granger, J.P. Da Costa, J. Toutain, M. Barey, M. Cansell, Int. Dairy J. 16 (2006) 489. [18] J. Christensen, L. Norgaard, R. Bro, S.B. Engelsen, Chem. Rev. 106 (2006) 1979. [19] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed., Springer, Singapore, 2006. [20] E. Dufour, M.F. Devaux, P. Fortier, H. Heymann, Int. Dairy J. 11 (2001) 465. [21] F. Bonomi, G. Mora, M.A. Pagani, S. Iametti, Anal. Biochem. 329 (2004) 104. [22] T. Nagano, M. Hirotsuka, H. Mori, K. Kohyama, K. Nishinari, J. Agric. Food Chem. 40 (1992) 941. [23] UniProtKB: Proteinknowledgebase [Internet]. [24] S. Cairoli, S. Iametti, F. Bonomi, J. Protein Chem. 13 (1994) 347. [25] K.W. Clara Sze, H.H. Ksnirsagar, M. Venkatachalam, S.K. Sathe, J. Agric. Food Chem. 55 (2007) 8745. [26] N. Catsimpoolas, J. Wang, T. Berg, Int. J. Protein Res. 2 (1970) 277. [27] N. Maruyama, R. Sato, Y. Wada, Y. Matsumura, H. Goto, E. Okuda, S. Nakagawa, S. Utsumi, J. Agric. Food Chem. 47 (1999) 5278. [28] L. Eynard, S. Iametti, P. Relkin, F. Bonomi, J. Agric. Food Chem. 40 (1992) 1731. [29] S. Iwabuchi, H. Watanabe, F. Yamauchi, J. Agric. Food Chem. 39 (1991) 34. [30] S. Iwabuchi, H. Watanabe, F. Yamauchi, J. Agric. Food Chem. 39 (1991) 27.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Structural changes of soy proteins at the oil–water interface studied by fluorescence spectroscopy Maneephan Keerati-u-rai a,∗ , Matteo Miriani a,b , Stefania Iametti b , Francesco Bonomi b , Milena Corredig a a b
Department of Food Science, University of Guelph, Ontario, N1G 2W1, Canada Università degli Studi di Milano, Department of Food Science, Milano, Via Celoria 2, Italy
a r t i c l e
i n f o
Article history: Received 14 September 2011 Received in revised form 30 November 2011 Accepted 2 December 2011 Available online 13 December 2011 Keywords: Soy proteins Soy protein isolate Glycinin -conglycinin Soy protein emulsion Front face fluorescence Heat treatment
a b s t r a c t Fluorescence spectroscopy was used to acquire information on the structural changes of proteins at the oil/water interface in emulsions prepared by using soy protein isolate, glycinin, and -conglycinin rich fractions. Spectral changes occurring from differences in the exposure of tryptophan residues to the solvent were evaluated with respect to spectra of native, urea-denatured, and heat treated proteins. The fluorescence emission maxima of the emulsions showed a red shift with respect to those of native proteins, indicating that the tryptophan residues moved toward a more hydrophilic environment after adsorption at the interface. The heat-induced irreversible transitions were investigated using microcalorimetry. Fluorescence spectroscopy studies indicated that while the protein in solution underwent irreversible structural changes with heating at 75 and 95 ◦ C for 15 min, the interface-adsorbed proteins showed very little temperature-induced rearrangements. The smallest structural changes were observed in soy protein isolate, probably because of the higher extent of protein–protein interactions in this material, as compared to the -conglycinin and to the glycinin fractions. This work brings new evidence of structural changes of soy proteins upon adsorption at the oil water interface, and provides some insights on the possible protein exchange events that may occur between adsorbed and unadsorbed proteins in the presence of oil droplets. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Soy proteins are widely employed as ingredients in foods because of their nutritional and processing functionality. These proteins have the ability to adsorb at the oil–water or air–water interfaces [1,2], reducing interfacial tension and forming a thick layer which provides stability to the oil droplets. Soy protein isolate (SPI) is mostly constituted of glycinin and -conglycinin. Glycinin is a hexamer, composed of two trimers stacking on top of one another [3]. Its molecular structure consists of an acidic and a basic polypeptide linked by a disulfide bond, which gives structural rigidity to the protein. Soy glycinin is a very heterogeneous protein, with various subunit combinations and a molecular mass in the 300–380 kDa range. The structure is held together by hydrogen bonds and electrostatic interactions, and it can be dissociated using thermal treatment, pH, or chaotropic agents such as urea [4–6]. The second most abundant protein in SPI is -conglycinin, a glycoprotein present as a trimer with a molecular mass in the 150–200 kDa
∗ Corresponding author. Tel.: +1 519 824 4120x56101; fax: +1 519 824 6631. E-mail address: [email protected] (M. Keerati-u-rai). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.12.002
range. This protein is composed of three different subunits; ␣, ␣ and  [7]. Studies using recombinant proteins have demonstrated that the three polypeptides have a similar core structure, and ␣, ␣ have acidic glycosilated extension regions [7,8]. Unlike glycinin, -conglycinin does not have disulfide bridges. Both these proteins are present in an aggregated form in solution [9]. It has been previously demonstrated that both major soy proteins adsorb at the oil–water interface [10,11]. When oil-in-water emulsions are prepared with SPI, the oil droplets show a negatively charged layer with a -potential of about −15 mV at neutral pH [12]. Furthermore, the proteins adsorb as a monolayer with a thickness corresponding to that of the average size of the protein oligomers, around 30–40 nm [12]. Although a number of studies have been published on the emulsifying properties of soy proteins and on the stability of the emulsions to various environmental and processing conditions [11,13,14], only a few of them addressed fundamental questions related to structural rearrangements at the interface. A recent study using fluorescence spectroscopy allowed deriving some hypotheses on the changes occurring during thermal treatment on glycinin and -conglycinin in solution, or after adsorption at interface [15]. In the present work, the changes occurring in SPI, glycinin, and -conglycinin proteins after adsorption
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at oil-in-water interfaces, as well as after thermal treatment of the solutions or emulsions, were followed using fluorescence spectroscopy. Fluorescence spectroscopy is a well established technique to observe protein conformational changes as well as to study the changes in protein-based matrices during processing or storage [16–19]. Front face fluorescence spectroscopy has been introduced to overcome some of the limitations related to the analysis of solid, turbid, viscous, and concentrated samples [20,21]. In proteins, the dominant fluorophore is the indole group of tryptophan, and its emission is very sensitive to solvent polarity. Hence, the fluorescence spectrum of a protein is determined by the chemical environment of the fluorescent amino acids, and changes in the tryptophan (Trp) spectrum can be used to identify conformational changes of proteins, occurring, for example, upon adsorption onto an oil–water interface. When Trp residues move from a more hydrophobic environment to a hydrophilic one, the emission maximum shifts to longer wavelengths (red shift), whereas the orientation toward a more hydrophobic location causes a blue shift. The intensity of these shifts is a function of both the extent of exposure, and of the number of involved residues [19]. Results obtained using micro-DSC suggests that small structural changes occur to soy proteins after adsorption at the oil/water interface [2,12]. In addition, only limited conformational changes have been reported for glycinin protein adsorbed at the air–water interface using infrared reflection–absorption spectroscopy [1]. The aim of this work was to study the structural changes of soy proteins when present at the oil–water interface in emulsions. The nature and extent of these changes was compared to changes induced on the soluble protein by physical (heat) or chemical (chaotropes) denaturation. Furthermore, to discriminate between structural changes occurring at the interface from those affecting the non-adsorbed proteins, a structural analysis was performed on both the cream and serum fractions, separated with centrifugation. Finally, structural characterizations of proteins in heated emulsions and of emulsions prepared from heated proteins allowed discriminating the structural changes of native, heat-denatured, and surface-denatured proteins.
2. Materials and methods 2.1. Protein preparation Soy protein isolate (SPI) was prepared by dispersing (1:10 ratio, w/v) defatted soy flakes (a gift from the Solae Company, St. Louis, MO, USA dispersion index 90) in 100 mM Tris–HCl buffer, pH 8.0. After stirring at room temperature for 1 h, the soluble fraction was separated by centrifugation at 12,000 × g for 30 min at 10 ◦ C (Beckman Coulter, Model J2-21, Fullerton, CA, USA). The centrifuged dispersion was then adjusted to pH 4.8 with 2 M HCl, and refrigerated at 4 ◦ C for 2 h. The precipitated protein was recovered by centrifugation as described above, washed with 8 volumes of 10 mM sodium acetate, pH 4.8, solubilized in ultrapure water (Barnstead International, E-pureTM D4641, Ames, IA, USA), and adjusted to pH 7.5 with 2 M NaOH. After overnight dialysis against water at 4 ◦ C, the material was freeze dried and stored at −20 ◦ C. Fractions rich in glycinin and -conglycinin were isolated as previously described [22] with minor modifications. Defatted soy flakes (The Solae Company) were suspended in ultrapure water in 1:15 ratio (w/v) and the pH adjusted to 8.0 with 2 M NaOH. After stirring the suspensions for 2 h at room temperature, the insoluble fractions were separated by centrifugation at 9000 × g for 30 min at 20 ◦ C. Sodium bisulfite (0.98 g/l) was added to the soluble fraction after adjusting the pH to 6.4 with 1 M HCl. After overnight
standing at 4 ◦ C, the protein suspension was centrifuged at 7000 × g for 20 min at 4 ◦ C. A glycinin-rich fraction was recovered in the precipitate, solubilized in ultrapure water, and adjusted to pH 7.5 with 2 M NaOH. Sodium chloride (0.25 M final concentration) was added to the remaining supernatant, which was adjusted to pH 5.0 with 1 M HCl and stirred for 1 h in an ice bath. After centrifugation at 9000 × g for 30 min at 4 ◦ C the supernatant was diluted with cold ultrapure water in 2:1 ratio (v/v) and adjusted to pH 4.8 with 2 M HCl. A -conglycinin rich fraction was recovered by centrifugation at 7000 × g for 20 min at 4 ◦ C. The precipitate was resolubilized with ultrapure water and adjusted to pH 7.5 with NaOH. Both isolated fractions (-conglycinin and glycinin rich fractions) were dialyzed overnight at 4 ◦ C against ultrapure water, and freeze-dried. The freeze dried proteins were stored at −20 ◦ C. Protein content was determined using the combustion method for nitrogen (Leco FP-528, Mississauga, ON, Canada), and using N × 6.25 for calculation (approved method 46-30 AACC, 2000). The purity of polypeptides was assessed by using SDS-PAGE. The glycinin-rich fraction was 90% pure, and -conglycinin about 86%. 2.2. Emulsion preparation Previous work demonstrated that 1% (w/v) SPI and conglycinin were necessary to prepare a stable emulsion of 10% soybean oil with a monomodal distribution of droplet size [2,12], whereas 2% protein (w/v) was needed to prepare an emulsion with glycinin [12]. Protein suspensions prepared in 50 mM sodium phosphate buffer, pH 7.4, were left overnight at 4 ◦ C to fully hydrate, and then were equilibrated at room temperature prior to homogenization. A pre-homogenization step with 10% soybean oil (w/w) (Sigma–Aldrich, St. Louis, MO, USA) was performed by using a shear dispersing unit (PowerGen 125, Fisher Scientific, Ottawa, ON, Canada) for approximately 1 min, and followed immediately by homogenization in a microfluidizer (110S model, Newton, MA, USA) for five passes with an input pressure of 300 kPa, corresponding to an overall pressure of 40 MPa. Emulsions were stored at 4 ◦ C, and analyzed the next day. 2.3. Heat treatment Thermal treatment of solutions and emulsions was carried out in a circulating water bath (Thermo Haake DC10, Sigma–Aldrich, St. Louis, MO, USA) at two different temperatures (75 ◦ C and 95 ◦ C) for 15 min. These temperatures were chosen based on the denaturation temperatures of -conglycinin and glycinin, as measured by microDSC [2]. Heating at 75 ◦ C for 15 min was performed for emulsions stabilized with a -conglycinin-rich fraction, while 95 ◦ C for 15 min was used for emulsions stabilized with a glycinin-rich fraction. Emulsions prepared with SPI were treated at either 75 or 95 ◦ C for 15 min. In addition to heating the emulsions after homogenization, emulsions were also prepared with heated protein dispersions. In this case, after cooling the heated protein solution in an ice bath, the protein solutions were immediately homogenized with 10% soy oil as reported above. 2.4. Microcalorimetry Protein samples were diluted to 5 mg/ml with 50 mM sodium phosphate buffer, pH 7.4, and analyzed by micro-calorimetry (VPDSC, MicroCal Inc., North Hampton, MA, USA) to verify the presence of thermal transitions in the original protein samples or to verify complete denaturation in heated samples. Both emulsions and cream samples were analyzed. The cream layer separated from the serum phase by centrifugation at 12,000 × g for 15 min (Brinkmann Instruments, Westbury, NY, USA) was carefully removed and dried on filter paper (Whatman # 1, Fisher Scientific, ON, Canada). The
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Tryptophan fluorescence emission spectra (ex , 280 nm; em , 300–450) were measured in a solid sample holder fitted to a spectrofluorometer (Shimadzu, RF5301, Mandel Scientific co. Inc., Mississauga, ON, Canada). Excitation and emission slits were set at 5 nm. Prior to analysis, emulsions were diluted 1:10 with 50 mM sodium phosphate buffer, pH 7.4. In the case of cream analysis, the cream layer blotted on filter paper, as described above, was washed with buffer to remove the excess protein, and re-suspended in buffer to its initial volume fraction (10% oil). The serum was obtained from centrifugation of emulsions at 10,000 × g for 20 min, removed using a needle and syringe, and filtered through a 0.22 m HPLC filter. Both cream and serum were diluted 1:1 with buffer immediately before fluorescence measurements. 2.6. Statistical analysis All experiments were conducted in triplicate (three separate emulsion preparations) and data are reported as averages and standard deviations. 3. Results and discussion 3.1. Spectroscopic features of urea-denatured, heat-denatured, and surface adsorbed soy proteins in whole emulsions Emission fluorescence spectra of proteins in solution and in the corresponding emulsions are shown in Fig. 1 along with those of the urea-denatured proteins. In the conditions used here, Trp residues are sole responsible of the observed signal, and contributions from other fluorescent amino acid residues may be considered negligible. Trp are found in both the acidic and basic polypeptides of glycinin. In general, the acidic subunit has more Trp than the basic subunit, which has one Trp, except for the B3 variant, which has two. There are more Trp residues (three to five) in each glycinin monomer than in -conglycinin [23]. In fact, only one Trp residue is found in the ␣ subunit, two in ␣ , and none in the  subunit of -conglycinin. The spectra of native proteins in Fig. 1 and the relative abundance of Trp residues discussed above highlight that structural information may only be derived by an analysis of the position of the fluorescence emission maximum, and not by the intensity of the emission, as pointed out by almost all studies in which fluorescence has been used to monitor loss or acquisition of protein structure [19]. Shifts of fluorescence emission to longer wavelengths (red shift) are indicating exposure of the fluorophores to a hydrophilic environment [15,19,24]. Previous reports have shown that the structure of soy proteins is lost upon treatment with chaotropes like urea or guanidine hydrochloride [15,25,26]. Treatment of the various protein solutions with urea results in a marked red shift of fluorescence
Fluorescence Intensity (A.U.)
A
300 250 200 150 100 50 900
Fluorescence Intensity (A.U.)
2.5. Fluorescence measurements
350
Fluorescence Intensity (A.U.)
cream was then resuspended in 50 mM sodium phosphate buffer, pH 7.4, to its initial volume fraction (10%). Samples were held at 20 ◦ C for 15 min prior to heating from 20 to 115 ◦ C at a 1 ◦ C/min scan rate, and then cooled to 20 ◦ C. Reference samples were either 50 mM sodium phosphate buffer, pH 7.4, or 10% soybean oil in emulsion prepared with 2% Tween 80 (Sigma–Aldrich, ON, Canada). Enthalpy changes (H) and denaturation temperature midpoint (Td ) were calculated using the OriginTM software version 7.0 (OriginLab, Northampton, MA, USA), as the area under the peak and the temperature at peak maximum, respectively.
43
B
800 700 600 500 400 300 200 100 500
C
400 300 200 100 0
320
340
360
380
400
Wavelength (nm) Fig. 1. Emission fluorescence spectra of (A) -conglycinin (10 mg/ml), (B) glycinin (20 mg/ml) and (C) soy protein isolate (10 mg/ml). Emission spectra (ex : 280 nm) were recorded on protein solutions in buffer (solid lines), in buffer containing 8 M urea (dotted lines), or on 10% soy oil emulsions (dashed lines). All samples were diluted ten-fold in respective buffer prior to measurements. Note the difference in the Y axis. Spectra are representative, for statistical analysis of maximum peak (see Table 1).
emission [15]. The observed red shift with denaturation by 8 M urea was highest for glycinin (18 nm, from 329 to 347), followed by conglycinin (10 nm, from 334 to 344) and by SPI (10 nm, from 332 to 342) (Fig. 1; Table 1). The emission maximum wavelengths for all the spectra collected in this work are summarized in Table 1, for ease of comparison. In the case of -conglycinin, the Trp residues are part of the solvent-exposed N-terminal extended domains of the ␣ and ␣ subunits (Trp63 and Trp63, Trp100, respectively). Hence, the smaller red shift recorded for -conglycinin in the presence of Urea, compared to glycinin. In the case of SPI, the shift in the spectrum resembled that of the -conglycinin in solution. This behavior may be caused by the relative ratio of the two proteins in the SPI, by the presence of aggregates of the various proteins, and/or by differences in processing history, which may result in a lower
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Table 1 Position of the emission maximum in fluorescence spectra. Values are the average of three separate experiments. The only sample showing a blue shift with respect to native proteins is highlighted in bold.a Sample
Fluorescence emission maximum (nm) -Conglycinin Unheated
Native protein Urea-treated protein Heated protein Unheated emulsions Cream from unheated emulsions Serum of unheated emulsions Cream from heated emulsions Serum from heated emulsions Cream from emulsions prepared with heated protein Serum from emulsions prepared with heated protein
◦
75 C
Unheated
334.2 ± 0.8 344.0 ± 0.0
Soy protein isolate ◦
95 C
Unheated
329.1 ± 0.2 347.2 ± 0.1 335.6 ± 0.0
338.0 ± 0.3 341.2 ± 0.3 327.3 ± 3.0 343.1 332.0 343.4 332.2
± ± ± ±
333.8 ± 0.2
0.3 0.7 0.3 0.6
75 ◦ C
95 ◦ C
333.7 ± 0.2
335.6 ± 0.4
332.4 ± 0.2 342.1 ± 0.2
335.8 ± 0.6 340.9 ± 0.2 329.7 ± 0.2
334.2 ± 0.1 340.1 ± 0.6 331.8 ± 0.4 341.2 334.9 339.4 336.1
± ± ± ±
0.4 0.2 0.2 0.2
343.3 333.3 340.2 332.2
± ± ± ±
0.2 0.4 0.6 0.2
341.8 337.6 339.6 353.2
± ± ± ±
0.2 0.4 0.6 1.7
1.8e-3
Heat Capacity (cal/oC)
A 1.2e-3
6.0e-4
0.0
Heat Capacity (cal/oC)
4.8e-3
B
3.6e-3
2.4e-3
1.2e-3
Fluorescence Intensity (A.U.)
Value are the average of emission spectra ± standard deviation of three replicate experiments.
Fluorescence Intensity (A.U.)
a
Glycinin
Heat Capacity (cal/oC)
1.8e-3
C
1.2e-3
6.0e-4
0.0
Fluorescence Intensity (A.U.)
0.0
350
250 200 150 100 50 900 0
40
60
80
o
100
120
Temperature ( C) Fig. 2. Thermal transitions measured using microcalorimetry for 10% oil-in-water emulsions (solid line) and corresponding solutions (dotted lines) of -conglycinin 10 mg/ml (A), glycinin 20 mg/ml (B) and soy protein isolate 10 mg/ml (C).
B
800 700 600 500 400 300 200 100 500
C
450 400 350 300 250 200 150 100 50 0
20
A
300
320
340
360
380
400
Wavelength (nm) Fig. 3. Intrinsic fluorescence spectra of (A) -conglycinin (10 mg/ml), (B) glycinin (20 mg/ml) and (C) soy protein isolate (10 mg/ml). Emission spectra were recorded for the whole emulsions (solid line), the adsorbed proteins in the cream phase (broken line), and the unadsorbed protein in the continuous phase (centrifugal serum) (dotted line). The maximum wavelength values are summarized in Table 1.
A
1.5e-3
Heat Capacity (Cal/oC)
Fluorescence Intensity (A.U.)
M. Keerati-u-rai et al. / Colloids and Surfaces B: Biointerfaces 93 (2012) 41–48
1.0e-3
5.0e-4
0.0
Fluorescence Intensity (A.U.)
Heat Capacity (Cal/oC)
4.0e-3 B
3.0e-3 2.0e-3 1.0e-3 0.0
Fluorescence Intensity (A.U.)
Heat Capacity (Cal/oC)
2.0e-3 C
1.5e-3 1.0e-3 5.0e-4 0.0 0
20
40
60
80
100
120
o
Temperature ( C) Fig. 4. Thermal transitions measured using microcalorimetry for (A) -conglycinin, (B) glycinin and (C) soy protein isolate solutions in 50 mM sodium phosphate, pH 7.4. Unheated (solid line), heated at 75 ◦ C (dashed lines) and at 95 ◦ C (dotted lines) for 15 min.
susceptibility of the glycinin aggregates in the SPI to denaturation by chaotropes. Fig. 1 also shows the fluorescence spectra of the various proteins when present in the emulsions (both in solution and adsorbed at the interface). Compared to their corresponding solutions, the spectra also showed a red shift in all cases, although to a smaller extent than in the presence of urea, suggesting an increased exposure to the aqueous phase of the Trp residues after adsorption of the proteins at the oil–water interface, in agreement with previous reports [15]. If Trp residues were present in the portion of the proteins interacting with the oil interface, a blue shift would be expected. However, the fluorescence spectrum of -conglycinin in emulsion showed a red shift of 4 nm (from 334 to 338) compared to that of the protein in solution, whereas glycinin showed a red shift of 7 nm (from 329 to 336). It has been previously proposed [15] that the Trp residues located in the extended region of the -conglycinin subunits protrude in the aqueous phase, whereas the Trp-free core hydrophobic region of -conglycinin may act as
45
350
A 300 250 200 150 100 50 900
B
800 700 600 500 400 300 200 100 0 450
C
400 350 300 250 200 150 100 50 0
320
340
360
380
400
Wavelength (nm) Fig. 5. Emission fluorescence spectra of (A) -conglycinin (10 mg/ml), (B) glycinin (20 mg/ml) and (C) soy protein isolate (10 mg/ml). Emission spectra (ex : 280 nm) were recorded for unheated protein solutions (solid line), heated protein at 75 ◦ C for 15 min (dotted line), and heated protein solution at 95 ◦ C for 15 min (dashed line). All samples were diluted ten-fold in buffer prior to the measurement.
an anchor at the interface [15,27]. It is also established that the ␣ and ␣ subunits show better emulsifying properties than the  subunit of -conglycinin because of the extension regions [27]. In the case of the emulsions prepared with SPI, a very modest red shift was shown (2 nm, from 332 to 334 nm, see Table 1), again suggesting the presence of extensive intermolecular interactions in SPI, in agreement with recent microcalorimetry studies, where small thermal transitions were observed for soy proteins once adsorbed at the interface compared to the original proteins in solution [2]. In the frame of these studies, it must be noted that it was previously demonstrated that the microfluidization of the proteins in the absence of oil did not affect their emission fluorescence spectra [15].
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Fluorescence Intensity (A.U.)
Fluorescence Intensity (A.U.)
46
600
A
B
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Fig. 6. Emission fluorescence spectra of (A) -conglycinin heated at 75 ◦ C for 15 min, (B) glycinin heated at 95 ◦ C for 15 min and (C and D) soy protein isolate heated at 75 ◦ C (C) and 95 ◦ C (D) for 15 min. Spectra were collected for cream from unheated emulsion (solid line); cream from heated emulsion (dashed line); cream from emulsion prepared with heated solution (dotted line). The maximum wavelength values are summarized in Table 1.
3.2. Thermal transitions of the adsorbed proteins in emulsions Microcalorimetry has been employed to study the thermal transitions of soy proteins emulsion, this approach allows to appreciate thermodynamically relevant transitions associated to changes in the structural features of adsorbed proteins [9]. Homogenization of -conglycinin and glycinin in the absence of oil did not alter the DSC profile of either protein, confirming once again that the treatment “per se” did not promote major structural changes [9,15]. Two main thermal transition peaks were observed in both solutions and emulsions, at 69 ◦ C and 84 ◦ C (Fig. 2), that essentially correspond to the reported thermal transitions for -conglycinin and glycinin, respectively. In the -conglycinin containing emulsion, a second peak at 95 ◦ C was evident, that was absent in the DSC tracings obtained for the protein in solution. The appearance of a transition at high temperature may be consequent to rearrangements of the supramolecular structure of this protein at the interface, which could have led to a fraction of the protein transitioning at a higher temperature [12]. In the case of emulsions prepared with SPI, the transitions were in full agreement to those of the emulsions prepared with individual fractions, suggesting the absence of intermolecular interactions involving both proteins. 3.3. Structural characterization of the proteins present in individual phases of emulsions It is important to note that results shown in Figs. 1 and 2 were obtained with emulsion samples containing both absorbed and unadsorbed proteins. To distinguish between the structural changes occurring at the interface and those involving the protein in solution, fluorescence measurements were carried out on
emulsions after separation of the cream and serum phases. As shown by the fluorescence emission spectra in Fig. 3 and by the data in Table 1, the shift in the maximum emission wavelength in emulsions may be ascribed to contributions from both the protein adsorbed at the interface in the cream phase and from the unadsorbed protein present in the serum phase. The observed shifts were larger for the proteins at the interface than for the unadsorbed protein in the serum (Fig. 3); although, in all cases, the extent of the spectral changes was much lower than those observed for proteins treated with 8 M urea (Fig. 1; Table 1). As reported in Table 1, the fluorescence spectrum of -conglycinin on the oil droplet was red shifted 7 nm with respect to that of the native protein, compared to a 7 nm blue-shift of the protein in the serum phase versus the protein in buffer. In the case of glycinin, red shifts were 12 nm (cream versus original solution) and 1 nm (serum versus original solution). SPI samples showed a red shift of 8 nm (cream versus solution), and no shifts were noticeable in the proteins remaining in the serum phase. This latter observation once again suggested that the glycinin present in SPI contributes less to the observed changes than the glycinin in the glycinin-rich fraction. The 7 nm blue shift (from 334 to 327 nm) recorded for the conglycinin present in the serum, may come from an exchange between the adsorbed and unadsorbed proteins occurring at the interface, followed by aggregation of the protein released from the droplet surface. The aggregation itself may shift tryptophan residues to a more hydrophobic environment, as reported for other food proteins [19,24,28]. It is important to note that any interpretation of these spectroscopic shifts to assess the amounts of individual protein forms in the system is improper, as the data cannot provide relative figures for the various protein forms (i.e.,
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900 Fluorescene Intensity (A.U)
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Fig. 7. Emission fluorescence spectra of (A) -conglycinin heated at 75 ◦ C for 15 min, (B) glycinin heated at 95 ◦ C for 15 min and (C and D) soy protein isolate heated at 75 ◦ C (C) and 95 ◦ C (D) for 15 min. Spectra of centrifugal serum from unheated emulsions (solid line), heated protein solutions (dotted line), serum from heated emulsion (dashed line) and serum from emulsions prepared with heated solutions (grey line). The maximum wavelength values are summarized in Table 1.
in solution, at the interface, denatured and aggregated, denatured and non-aggregated). 3.4. Thermal treatment of proteins and emulsions, and properties of emulsion stabilized by heat-treated proteins The structural changes occurring to the proteins after heating solutions and emulsions were also studied, also in consideration of the practical relevance of thermal processes. The various samples were heated either at 75 or 95 ◦ C for 15 min, in the attempt to selectively denature individual protein fractions (see Fig. 2). Changes in the thermal transitions of the protein solutions before and after these thermal treatments are shown in Fig. 4, and reflect the differences in denaturation temperature between -conglycinin and glycinin. In particular, -conglycinin was fully denatured (no residual thermal transition) after having been heated at 75 ◦ C for 15 min, whereas glycinin lost its thermal transition after having been heated at 95 ◦ C for 15 min. This selective denaturation also occurred in the SPI samples, since the denaturation peak of conglycinin disappeared after heating at 75 ◦ C and that of glycinin was no longer present after heating at 95 ◦ C. Intrinsic fluorescence spectra for the various protein solutions before and after heating are shown Fig. 5. There were no differences (see also Table 1) in the maximum emission wavelength for -conglycinin after heating at 75 ◦ C for 15 min (Fig. 5A) compared to the unheated protein, in spite of the full loss of the thermal transition peak after this temperature (Fig. 4A). On the other hand, it has been reported that heating -conglycinin at 75 ◦ C increases the overall surface hydrophobicity of the protein and causes the formation of soluble aggregates upon cooling via hydrophobic interactions [15,29]. Also, it has been shown that heat
dissociated -conglycinin could partially rearrange into non-native trimers after cooling [15,30]. It is important to note that the spectra of -conglycinin reflect changes in the extended region of the protein, and changes in the core structure of the protein cannot be observed by fluorescence spectroscopy, due to the lack of fluorophores in the protein structural core domain. There was a 5 nm red shift after heating glycinin at 95 ◦ C for 15 min (Fig. 5B; Table 1), in agreement with earlier studies [15]. The red shift may indicate either a dissociation of the native quaternary structure (or of some undescribed supramacromolecular aggregates) or the formation of a more open and solvent-accessible tertiary structure. In the case of SPI solutions, there was a small shift (2 nm) in the fluorescence spectrum after heating at 75 ◦ C for 15 min and a 4 nm red shift after heating at 95 ◦ C for 15 min (Fig. 5C; Table 1). The higher shift at the higher temperature is in line with the behavior of glycinin, as discussed above. Fluorescence measurements were also performed on the cream and serum phases of emulsions subjected to heating, either at 75 ◦ C or 95 ◦ C for 15 min, and on emulsions prepared from heated proteins. The fluorescence spectra of the protein present at the interface (cream) and in the unadsorbed phase (serum) are shown in Figs. 6 and 7, respectively, whereas figures for maximum emission wavelengths are summarized in Table 1. Fig. 6 compares the differences in the cream phases in heated and unheated emulsions prepared with the various proteins before and after heating. In the case of -conglycinin (Fig. 6A), spectra of the proteins in the various cream sample were almost overlapping. There were no statistically significant changes also in the emission maximum of glycinin when adsorbed at the interface before or after heating at 95 ◦ C (Fig. 6B; Table 1), suggesting that adsorption prevented further structural rearrangements of glycinin at
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interface. The fluorescence spectra of proteins in SPI-stabilized cream showed a red shift of 3 nm after heating at 75 ◦ C (Fig. 6C), but no further changes after heating at 95 ◦ C (Fig. 6D). It could be hypothesized that in the case of SPI, the red shift is attributable to the structural change occurring to -conglycinin, as there is no further change at the higher temperature. In the case of SPI-based emulsions prepared with heated protein solutions, no significant change was observed for the emission maximum (Fig. 6C and D; Table 1) after heating at either 75 or 95 ◦ C. Fig. 7 compares the fluorescence emission spectra of the serum phases for the various emulsions. In the case of -conglycinin, the original, unheated protein solution showed a peak at 334 nm, with very little change after heating (Fig. 6A) or when a heated solution was used to prepare the emulsion. It has been previously demonstrated that under these conditions, a higher amount of protein adsorbs at the interface [12]. The unadsorbed fraction in heated glycinin-stabilized emulsions showed a red shift at 335 nm (heated after homogenization) or 336 nm (heated prior to homogenization, Fig. 7B). These figures are significantly higher than those for the native protein (329 nm) or for the unadsorbed protein in the unheated emulsion (330 nm, Fig. 6 and Table 1), and close to what observed when the protein was heated in solution. In the case of SPI-stabilized emulsions, while very little change was noted in the unadsorbed fractions after heating the emulsions at 75 ◦ C (Fig. 7C), or when emulsions were prepared from proteins heated at 75 ◦ C, in agreement with results obtained with -conglycinin (Fig. 7A). After heating the emulsion at 95 ◦ C for 15 min, the unadsorbed proteins showed a red shift similar to that described for the glycinin-based system (Fig. 7D). The largest fluorescence shift (17 nm, from 336 to 353 nm, Table 1) was observed when heated SPI were used. However, the fluorescence intensity in these samples was very low, because of the low residual protein present in the serum phase as a consequence of the aggregation events amply discussed above. 4. Conclusions The use of front-face fluorescence to monitor conformational changes of soy proteins in emulsions made it possible to show that adsorption of the soy proteins changed the chemical environment of tryptophans in their structure. The data reported here confirm that the core structure of -conglycinin (that does not contain major fluorophores) adheres at the interface, and flexible moieties in its ␣ and ␣ subunits become even more exposed to the aqueous phase than they were in solution. Studies on the unadsorbed fraction of -conglycinin would suggest an exchange between proteins adsorbed at interface and those present in the continuous phase. A blue shift in the spectrum of -conglycinin present in the unadsorbed phase indicated aggregation of unadsorbed protein in solution. Glycinin showed remarkable changes in its structure when at the interface, whereas the fraction present in the continuous phase of the glycinin-stabilized emulsions did not show structural
rearrangements in the absence of a thermal treatment. The behavior of the unadsorbed fraction after heating at 95 ◦ C, was in line with what observed in the heated solutions, suggesting that the unadsorbed protein retained most of the structural features of the unheated protein in solution. This study also points out that heat treatments affected differently the structure of adsorbed and unadsorbed proteins. The adsorbed proteins were much less prone to temperature-induced structural rearrangements than those in solution. In the practically relevant case of SPI, the rearrangements in the system as a whole were less pronounced than for those expected from studies on the isolated protein components (i.e., glycinin and -conglycinin), most probably because of the presence of protein aggregates as a consequence of previous processing. This points out the importance of accurate characterization at the molecular level of materials that are of common use in the food industry and that are often characterized only in terms of composition and not in terms of their structure. References [1] A.H. Martin, M.B.J. Meinders, M.A. Bos, M.A. Cohen Stuart, T. van Vliet, Langmuir 19 (2003) 2922. [2] M. Keerati-u-rai, M. Corredig, Food Hydrocoll. 23 (2009) 2141. [3] M. Adachi, Y. Takenaka, A.B. Gidamis, B. Mikami, S. Utsumi, J. Mol. Biol. 305 (2001) 291. [4] C.M.M. Lakemond, H.H.J. de Jongh, M. Hessing, H. Gruppen, A.G.J. Voragen, J. Agric. Food Chem. 48 (2000) 1991. [5] J.M.S. Renkema, C.M.M. Lakemond, H.H.J. de Jongh, H. Gruppen, T. van Vliet, J. Biotechnol. 79 (2000) 223. [6] P.R. Shewry, Biol. Rev. 70 (1995) 375. [7] N. Maruyama, T. Katsube, Y. Wada, M.H. Oh, A.P. Barba de la Rosa, E. Okuda, S. Nakagawa, S. Utsumi, Eur J. Biochem. 258 (1998) 854. [8] N. Maruyama, M.R.M. Salleh, K. Akahashi, K. Yagasaki, H. Goto, N. Hontani, S. Nakagawa, S. Utsumi, J. Am. Oil Chem. Soc. 79 (2002) 139. [9] M. Keerati-u-rai, M. Corredig, J. Agric. Food Chem. 57 (2009) 3556. [10] F.E. Mitidieri, J.R. Wagner, Food Res. Int. 35 (2002) 547. [11] G.G. Palazolo, F.E. Mitidieri, J.R. Wagner, Food Sci. Technol. Int. 9 (2003) 409. [12] M. Keerati-u-rai, M. Corredig, J. Agric. Food Chem. 58 (2010) 9171. [13] G.G. Palazolo, D.A. Sorgentini, J.R. Wagner, J. Am. Oil Soc. 81 (2004) 625. [14] E. Molina, A. Papadopoulou, D.A. Ledward, Food Hydrocoll. 15 (2001) 263. [15] M. Miriani, M. Keerati-u-rai, M. Corredig, S. Iametti, F. Bonomi, Food Hydrocoll. 25 (2011) 620. [16] C. Castelain, C. Genot, Biochim. Biophys. Acta 1199 (1994) 59. [17] C. Granger, J.P. Da Costa, J. Toutain, M. Barey, M. Cansell, Int. Dairy J. 16 (2006) 489. [18] J. Christensen, L. Norgaard, R. Bro, S.B. Engelsen, Chem. Rev. 106 (2006) 1979. [19] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed., Springer, Singapore, 2006. [20] E. Dufour, M.F. Devaux, P. Fortier, H. Heymann, Int. Dairy J. 11 (2001) 465. [21] F. Bonomi, G. Mora, M.A. Pagani, S. Iametti, Anal. Biochem. 329 (2004) 104. [22] T. Nagano, M. Hirotsuka, H. Mori, K. Kohyama, K. Nishinari, J. Agric. Food Chem. 40 (1992) 941. [23] UniProtKB: Proteinknowledgebase [Internet]. [24] S. Cairoli, S. Iametti, F. Bonomi, J. Protein Chem. 13 (1994) 347. [25] K.W. Clara Sze, H.H. Ksnirsagar, M. Venkatachalam, S.K. Sathe, J. Agric. Food Chem. 55 (2007) 8745. [26] N. Catsimpoolas, J. Wang, T. Berg, Int. J. Protein Res. 2 (1970) 277. [27] N. Maruyama, R. Sato, Y. Wada, Y. Matsumura, H. Goto, E. Okuda, S. Nakagawa, S. Utsumi, J. Agric. Food Chem. 47 (1999) 5278. [28] L. Eynard, S. Iametti, P. Relkin, F. Bonomi, J. Agric. Food Chem. 40 (1992) 1731. [29] S. Iwabuchi, H. Watanabe, F. Yamauchi, J. Agric. Food Chem. 39 (1991) 34. [30] S. Iwabuchi, H. Watanabe, F. Yamauchi, J. Agric. Food Chem. 39 (1991) 27.