Characterization of heat-induced aggregates of globulin from common buckwheat (Fagopyrum esculentum Moench)

International Journal of Biological Macromolecules 39 (2006) 201–209

Characterization of heat-induced aggregates of globulin from common buckwheat (Fagopyrum esculentum Moench) Siu-Mei Choi a , Yoshinori Mine b , Ching-Yung Ma a,∗ a

Food Science Laboratory, Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China b Department of Food Science, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 13 February 2006; received in revised form 27 March 2006; accepted 27 March 2006 Available online 3 April 2006

Abstract Some physicochemical properties and the microstructure of heat-induced aggregates of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) formed at 100 ◦ C in 0.01 M phosphate buffer containing 1.0 M NaCl, pH 7.4 were studied. Differential scanning calorimetric (DSC) analysis shows a re-distribution of native and extensively denatured proteins in the heat-induced aggregates of BWG, particularly in the ISA fraction. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis suggests the occurrence of both dissociation and association of molecules and the involvement of intermolecular disulfide linkages during thermal aggregation. Transmission electron microscopy (TEM) reveals that native BWG appeared as uniform compact globules with diameters ranging between 11.7 and 12.5 nm. TEM examination of the buffer-soluble aggregates, fractionated by sucrose density gradient ultracentrifugation, demonstrates the formation of strand-like small aggregates and large compact globular soluble macroaggregates. © 2006 Elsevier B.V. All rights reserved. Keywords: Fagopyrum esculentum moench; Buckwheat globulin; Thermal aggregation; Transmission electron microscopy

1. Introduction Common buckwheat (Fagopyrum esculentum Moench), unlike most cereals, is a highly nutritious pseudocereal belonging to the Polygonaceae family. Buckwheat seeds are rich in starch (65–75%) and protein (10–12.5%) and contain many valuable compounds, such as anti-oxidative substances, minerals and dietary fibre [1–3]. The protein content in buckwheat flour is significantly higher than those in other cereals such as barley, rice, millet, and rye [4]. Buckwheat seed storage proteins are rich in globulin and albumin, but very low in glutelin and prolamin content [5]. According to net protein utilization (NPU), buckwheat proteins are classified close to animal proteins and considered to have excellent supplementary value to cereal grains [6]. They have a high biological value due to wellbalanced amino acid composition that is rich in lysine and arginine [7]. The salt-soluble protein, buckwheat globulin (BWG),

∗

Corresponding author. Tel.: +852 2299 0318; fax: +852 2858 3477. E-mail address: [email protected] (C.-Y. Ma).

0141-8130/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2006.03.025

represents the major Osborne fraction of buckwheat seed proteins and has been classified as a legumin-like storage protein. It has a typical hexameric structure of all legumin-like storage proteins, containing six non-identical disulfide-linked monomers. Each monomer is composed of one large acidic polypeptide (57.5 kDa) and one small basic polypeptide (23.5 kDa), and the six monomers are linked by noncovalent forces [5,8–12]. BWG has also been found to possess some favorable functional properties, such as good emulsion-forming and stabilizing capacity [13]. Hence, BWG could be a good candidate as an ingredient for fabricated foods, providing promising functional characteristics. Thermal aggregation and gelation are important functional properties of food proteins influencing their applications in various manufactured foods. Heat treatment is a commonly used processing during the food preparation. Upon heating, the thermal motion of globular proteins in water or solvent increases leading to the rupture of intramolecular and intermolecular bonds that stabilizes the native protein structure, resulting in a marked decrease in protein solubility, probably due to protein denaturation and subsequent aggregation [14–17]. Heat treatment may also cause certain

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changes on the surface of protein molecule, stimulating specific aggregation, such as the formation of regular strands in soybean 11S globulin, glycinin. As temperature increases, certain regions of protein, e.g., hydrophobic or free sulfhydryl groups, become accessible and participate in intermolecular interactions, forming soluble aggregates through non-covalent and disulfide bonds [18]. Using differential scanning calorimetry (DSC) and Fouriertransform infrared (FTIR) spectroscopy, BWG has been shown to form aggregates at temperatures below its denaturation temperature of 100.4 ◦ C [19]. We have also studied thermal aggregation of BWG using size-exclusion chromatography combined with on-line multiangle laser light scattering and dynamic quasielastic light scattering (SEC-MALLS-QELS), in which molecular weight, hydrodynamic radius and conformational changes in molecules during thermal aggregation have been obtained. The results also demonstrated the involvement of both dissociation and association in aggregate formation [20]. In this investigation, the physicochemical properties and microstructure of heat-induced BWG aggregates will be evaluated by techniques including DSC, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sucrose gradient ultracentrifugation and transmission electron microscopy (TEM). The sulfhydryl and disulfide contents of the aggregate fractions will also be determined. The estimation of molecular size of aggregates and characterization of aggregate structure is of great importance to obtain the relevant information for network formation and gelation, which is critical in developing protein products for applications in the food industry. 2. Materials and methods 2.1. Preparation of buckwheat globulin Buckwheat flour with 84.6% carbohydrate, 10.7% protein, 2.9% lipid and 1.8% ash on dry weight basis [21], was obtained from Nikkoku Flour Milling Co. Ltd. (Japan) and was defatted by Soxhlet extraction with hexane. BWG was extracted from the defatted buckwheat flour using 0.5 M NaCl buffer in a ratio of 1:10 (w/v) according to the Osborne fractionation scheme [22]. The protein content of BWG was 93.9% (dry basis), as determined by the micro-Kjeldahl method [23] using a nitrogento-protein conversion factor of 5.53 [24]. 2.2. Heat treatments BWG solutions (1%, w/v) were prepared in 0.01 M phosphate buffer containing 1.0 M NaCl at pH 7.4 and stirred for 1.5 h at room temperature. The insoluble materials were removed by centrifugation at 10,000 × g for 20 min at 4 ◦ C. Aliquots (1 mL) of BWG solution were pipetted into resealable glass tubes and heated at 100 ◦ C in a boiling water bath for different time periods. The selection of a heating temperature at 100 ◦ C was based on the denaturation temperature (Td , 100.4 ◦ C) obtained in our previous DSC study [19]. After heating, tubes were immedi-

ately cooled in an ice bath for 5 min, and the solutions were centrifuged again at 10,000 × g for 20 min at 4 ◦ C to remove the insoluble aggregates. Protein contents of the supernatant and unheated globulin solution were determined according to the method of Lowry et al. [25]. 2.3. Preparation of heat-induced aggregates Buffer-soluble (SA) and buffer-insoluble (ISA) aggregates of BWG were prepared by centrifugation of the protein solution after heat treatment (100 ◦ C for 30 min). The supernatant containing SA was dialyzed exhaustively against distilled water at 4 ◦ C, while the precipitate containing the ISA was washed several times with distilled water. Both the SA and ISA were then recovered by freeze-drying and stored at −4 ◦ C. 2.4. Differential scanning calorimetry The thermal properties of the heated BWG and the SA and ISA fraction were examined by a TA 2920 Modulated DSC thermal analyzer (TA Instruments, New Castle, DE, USA) following the procedures as described by Choi and Ma [19]. Thermal transition characteristics including denaturation temperature (Td ), enthalpy (H), and the width at half-peak height (T1/2 ) were measured. All DSC measurements were performed in triplicates with standard deviations less than 0.85% for Td and 2.76% for H. 2.5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis The polypeptide compositions of the unheated BWG and the SA and ISA fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a Phast SystemTM (Pharmacia Biotech AB, Uppsala, Sweden) using 12.5% homogenous gels according to the method of Laemmli [26]. Protein samples (about 5 mg/mL) were treated with sample buffer containing 10 mM Tris–HCl, 1 mM EDTA, 2.5% SDS and 0.01% bromphenol blue. The samples were heated for 5 min in a boiling water bath, then cooled and centrifuged at 15,000 × g for 15 min. The amount of protein loaded was about 10–20 ␮g in 4 ␮L. The standard protein markers were from a Low Molecular Weight Electrophoresis calibration kit (Pharmacia Biotech, Piscataway, NJ, USA). The molecular weights of individual protein bands were determined using a Phoretix densitometric image analysis system (Phoretix International, Newcastle Upon Tyne, UK). 2.6. Analysis of sulfhydryl and disulfide contents The sulfhydryl (SH) and disulfide (SS) contents of the unheated BWG and the SA and ISA fractions were determined by the method of Beveridge et al. [27]. Protein samples (75 mg) were dissolved in 10 mL of Tris–Gly buffer (0.086 M Tris, 0.09 M glycine, 0.04 M EDTA, pH 8.0) containing 8 M urea. For SH content determination, 4 mL of the Tris–Gly buffer was added to 1 mL of protein solution. Then 0.05 mL of Ellman’s

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reagent (5,5 -dithio-bis-2-nitrobenzoic acid in Tris–Gly buffer, 4 mg/mL) was added, and absorbance was measured at 412 nm after 5 min. For total SH content [SH + reduced SS] analysis, 0.05 mL of ␤-ME and 4 mL of Tris–Gly buffer were added to 1 mL of the protein solution. The mixture was incubated for 1 h at room temperature. After additional 1 h incubation with 10 mL of 12% TCA, the mixtures were centrifuged at 5000 × g for 10 min. The precipitate was twice re-suspended in 5 mL of 12% TCA and centrifuged to remove ␤-ME. The precipitate was dissolved in 10 mL of Tris–Gly buffer. Then 0.04 mL of Ellman’s reagent was added to 4 mL of this protein solution, and the absorbance was measured at 412 nm after 5 min. Sample and reagent blanks were prepared for each determination. The calculation was as followed: μMSH/g = 73.53 × A412 × D/C where A412 is the absorbance at 412 nm, C is the sample concentration (mg/mL), D is the dilution factor: 5 and 10 are used for SH and total SH (SH + reduced SS) content analysis, respectively, 73.53 is derived from 106 /(1.36 × 104 ); 1.36 × 104 is the molar absorptivity (Ellman, 1959) and 106 is for the conversion from molar basis to ␮M/mL basis and from mg solids to g solids. Half of the value after subtracting the SH value from the total SH value was defined as the SS content.

Fig. 1. Differential scanning calorimetric thermograms of (a) buckwheat globulin (unheated control), (b) buffer-soluble aggregates and (c) buffer-insoluble aggregates formed after heating at 100 ◦ C for 30 min.

3. Results and discussion 2.7. Sucrose density gradient ultracentrifugation and transmission electron microscopy The buffer-soluble fractions prepared under different heat treatments were separated by sucrose density gradient ultracentrifugation. A sucrose gradient (15–40%, mass/volume) was prepared in a 5.2 mL ultracentrifuge tube by a gradient maker. An aliquot (0.5 mL) of heated protein sample (supernatant fraction) was applied on top of the sucrose gradient, and centrifuged at 250,000 × g and at 4 ◦ C for either 1 h or 17 h with a Beckman XL-90 Ultracentrifuge, using a SW 55 Ti rotor (Beckman Co., Sunnyvalle, CA, USA). After ultracentrifugation, an aliquot (0.5 mL) of the sample was collected using a fraction collector, and absorbance was monitored at 280 nm. Fractions from the top, middle and bottom peaks were examined by transmission electron microscopy (TEM). A droplet of protein sample was deposited onto a 200 mesh copper grid coated in FormVar (polyvinyl formal) as a support film for electron microscopy and covered with a very thin amorphous carbon film (about 20 nm). After several minutes, the excess was removed using a piece of filter paper. Then a small drop of 2% potassium phosphotungstic acid stain solution (pH 7.0) was added for 2 min. The grid was put into a petri dish and the stain was allowed to dry in an oven at 35 ◦ C for 1 min. The sample was then loaded onto a sample holder for examination by TEM. A Philips EM 208S transmission electron microscope (Philips Optics, Eindhoven, Netherlands) was used, and micrographs were taken by a BioScan Model 792 Camera (JEOL Active Co., Ltd, Akishima, Tokyo, Japan). Samples were examined typically at a magnification of × 52,000 at 25 kV.

3.1. Thermal transition characteristics of heat-aggregated fractions The DSC thermograms and thermal transition characteristics of BWG and its SA and ISA fractions are shown in Figs. 1 and 2, respectively. BWG has a relatively high thermal stability (Td at 100.4 ◦ C) and a large content of ordered structure (H of 17.6 (J/g)). The SA fraction exhibited a slight increase in Td and decrease in T1/2 with no marked change in H value when compared to the unheated control (Fig. 2). Upon further heating (100 ◦ C for 120 ), the SA had similar higher Td and lower T1/2 and H values (data not shown) when compared with the control. The denaturation temperature (Td ) can be used as a measure of thermal stability of proteins while the enthalpy value (H) is correlated with the content of ordered secondary structure of a protein [28], indicated by the proportion of undenatured protein [29]. In comparison with the unheated control, the slight increase in thermal stability as indicated by higher Td in the SA fraction may be due to rearrangement of the protein to assume a more compact conformation or the association of the protein molecules to a complex network structure of aggregates resulting in higher thermal stability. In addition, unfolding of native protein may lead to the exposure of buried apolar groups, enhancing protein–protein interactions by hydrophobic association. Subsequent heating of the heat-associated (SA) BWG would therefore require the rupture of more hydrophobic groups than in the native protein, resulting in higher Td . Similar high molecular weight associated molecules have also been found in heated oat globulin solution with a compact, ordered structure which would

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and the exothermic reactions of aggregation and breakup of hydrophobic interactions were more prominent. On the other hand, the ISA fraction only showed little DSC endothermic response (Fig. 1) suggesting a large proportion of protein components in the ISA fraction were extensively unfolded and denatured. In comparison with the unheated control, there was a pronounced decrease in both Td and H and a dramatic increase in T1/2 value in the ISA fraction (Fig. 2). The almost complete loss of endothermic response in the ISA fraction (Fig. 1) suggests extensive protein denaturation, although the formation of insoluble aggregates, an exothermic process, could also lead to a net loss in enthalphy value [32]. The broadening of the peak (Fig. 1) and marked increase in T1/2 value (Fig. 2) in the ISA fraction indicate the loss of cooperativity in the denaturation process. As shown in Fig. 2, the pronounced decrease in both Td and H in the ISA fraction after heating implies that the proportion of undenatured protein or ordered structure found in ISA were decreased upon heating, probably due to thermal denaturation since a partially unfolded protein would require less heat energy (lower H) to denature completely than a native protein [33], and lower thermal stability was resulted due to the presence of highly denatured protein. The present DSC data suggest a re-distribution of native and denatured protein in the aggregates of BWG, particularly in the ISA fraction as more pronounced changes in thermal characteristics were observed. Similar to other oligomeric proteins with complex quaternary structures, heating may cause association/dissociation of BWG molecules and disruption of the quaternary structure resulting in aggregation [34–38].

Fig. 2. Thermal characteristics of buckwheat globulin (BWG) (unheated control) and the buffer-soluble (SA) and buffer-insoluble (ISA) aggregate fractions formed after heating at 100 ◦ C for 30 min. Error bars represent standard deviations of triplicate determinations.

have higher thermal stability than the un-associated protein [30]. A slight decrease in T1/2 value in SA, suggesting protein denaturation and aggregation occurred in a highly cooperative manner. No marked change in enthalpy value in SA formed after 30 min heating, suggests the possible occurrence of both exothermic and endothermic processes simultaneously. Thermal treatments could disrupt the chemical forces that maintain the structural integrity of protein molecules, such as hydrophobic and ionic interactions, hydrogen bonds and disulfide bonds, resulting in protein denaturation [31]. Rupture of hydrogen bonds is considered an endothermic reaction which could increase the net endothermic contribution whereas breakup of hydrophobic interactions and aggregation are exothermic reactions which could lower the net endothermic contribution causing a decrease in H [32]. The possible occurrence of both endothermic and exothermic reactions may cause no net gain or loss in the value of H, making this enthalpy value remains unchanged in the SA fraction. However, SA formed after longer heating time (120 min) caused the decrease in H value, implying that more protein molecules became unfolded

3.2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis To elucidate the nature of the chemical forces involved in aggregated formation, SDS-PAGE analysis was performed. The SDS-PAGE patterns of unheated BWG, SA and ISA fractions are shown in Fig. 3. Under non-reducing condition, the unheated control (Fig. 3, lane 2) showed a large number of bands with molecular weight (Mw ) ranging from about 17 to 65 kDa. The major protein bands were in the 50–65 and 34–36 kDa regions. In comparison with the unheated control, the SA fraction (Fig. 3, lane 3) also exhibited a large number of protein bands, but there were decreases in the intensity of the protein bands at 34–36 kDa, and the disappearance of a component at 21–22 kDa. This may suggest that some polypeptides with higher Mw of 34–36 and 21–22 kDa have been broken down or dissociated into smaller polypeptides with lower Mw in range of 18–14.4 kDa during the formation of SA. In contrast, under non-reducing condition, the SDS-PAGE profile of ISA was markedly different from the control, showing that the majority of protein components in ISA had high Mw and some of which could not enter the separating gel (Fig. 3, lane 4). In addition, some minor protein bands were also found in Mw of 45–66 kDa region and similar protein patterns were also observed in the control and SA fraction. When compared to the control or SA fraction, however, the intensities of such protein bands were reduced in the ISA fraction. The difference in

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Fig. 3. SDS-PAGE patterns of soluble and insoluble aggregates of buckwheat globulin heated at 100 ◦ C for 30 min. Lanes 2, 3, 4 without ␤-mercaptoethanol. Lanes 5, 6, 7 with ␤-mercaptoethanol. Lanes 1 and 8, low molecular weight marker. Lanes 2 and 5, unheated buckwheat globulin (control). Lanes 3 and 6, soluble aggregates. Lanes 4 and 7, insoluble aggregates.

the electrophoretic pattern observed may suggest the dissociation/association and re-distribution of protein molecules during the thermal aggregation of BWG. Under reducing conditions (Fig. 3), similar SDS-PAGE patterns showing the disappearance of the high Mw components (50–65 kDa) and the appearance of additional small Mw bands were observed in both the unheated control (lane 5) and the soluble and insoluble aggregates (lane 6 and 7). A major band at 23 kDa and several minor bands between 31 and 47 kDa, corresponding to the basic and acidic polypeptides, respectively, were observed after reduction by ␤-mercaptoethanol (␤-ME) (Fig. 3, lane 5–7). With the addition of ␤-ME, the electrophoretic pattern of SA (Fig. 3, lane 6) resembled closely to that of the unheated control (Fig. 3, lane 5) whereas ISA (Fig. 3, lane 7) showed a similar pattern but with three major bands at Mw of 22–24, 34–36 and 44–48 kDa regions. The present data show that heating of BWG led to both the disappearance of protein bands and the appearance of new bands, suggesting the occurrence of both dissociation and association of polypeptides during heat treatment. Under reducing condition, disappearance or dissociation of higher Mw protein bands to smaller components in the control (Fig. 3, lane 5) suggests that these high Mw components (50–65 kDa) may correspond to the BWG monomers and each monomer is made up of an acidic and a basic polypeptide linked by disulfide bonds. The estimated Mw of BWG hexamers falls in the range of 300–390 kDa, which is in agreement with data deduced from genomic clones (342,258 Da) and MALLS (342,000 Da) studies [20,39]. The presence of high Mw bands near the origin of the ISA lane (Fig. 3, lane 4) and their dissociation by ␤-ME (Fig. 3, lane 7) suggests the involvement of disulfide linkages for the formation of large size aggregates. Since BWG is structurally similar to soy glycinin and oat globulin, it would be interesting to compare the thermal aggregation mechanisms of these proteins. Thermal changes in soy

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Fig. 4. Sulfhydryl (SH) and disulfide (SS) contents of buckwheat globulin (BWG) (unheated control) and the buffer-soluble (SA) and buffer-insoluble (ISA) aggregate fractions formed after heating at 100 ◦ C for 30 min. The error bars represent standard deviations of the means.

glycinin was proposed to include the formation of soluble aggregates, dissociation of the soluble aggregates to acidic and basic polypeptides, as well as the formation of insoluble aggregates from dissociated basic polypeptides, whereas acidic polypeptides remain soluble [35]. Similar dissociation of soluble aggre-

Fig. 5. Fractionation of heated buckwheat globulin by sucrose density gradient (15–40%) ultracentrifugation for 1 h (A) or 17 h (B). Sedimentation was from left to right. Samples from peak 1 (Fraction 1), peak 2 (Fraction 2) and peak 3 (Fraction 3) were examined by transmission electron microscopy.

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gates was also observed in oat globulin [30], particularly at higher temperatures, but the predominant product was monomer, with little acidic and basic polypeptides. Since monomers were the major dissociated molecules in oat globulin, no significant redistribution of acidic and basic polypeptides into the soluble and insoluble aggregates was observed. This suggests that insoluble aggregates form directly from dissociated monomers in oat globulin, unlike those formed from dissociated polypeptide chains in soy glycinin [35]. It was also suggested that SH-SS interchange is limited in oat globulin, and heating only disrupts noncovalent bonds linking the monomers into soluble aggregates [30]. In the present study, dissociation of BWG and association to high Mw aggregated molecules were demonstrated, which have also been reported by laser light scattering study [20]. The results also imply the involvement of both non-covalent interactions including hydrogen bonding and hydrophobic interactions and the covalent forces such as disulfide bonding during the thermal aggregation of BWG. Similar to oat globulin, redistribution of acidic and basic polypeptides into soluble and insoluble aggregates was not indicated. 3.3. Sulfhydryl and disulfide contents of heat-aggregated BWG The SH and SS contents of BWG (unheated control) and the SA and ISA fractions formed after 30 min heating at 100 ◦ C were

measured (Fig. 4). BWG has high SS content (36.4 ␮M/g protein) and low SH content (3.2 ␮M/g protein). SA has a slightly lower SS content and a much lower SH content when compared to the unheated control. In contrast, the ISA fraction has a markedly higher SH content and a lower SS content than the control (Fig. 4). The decrease in SH content in SA may imply the formation of disulfide bonds. Heat treatment could lead to cleavage of existing disulfide bonds or activation of buried SH groups through protein unfolding. These newly formed or activated SH groups may form new intermolecular disulfide bonds which are essential for aggregate formation. It is generally accepted that disulfide linkage is involved in the cross-linking of denatured protein molecules to form aggregates [40,41]. It has been suggested that the ability of some proteins to form intermolecular disulfide bonds during thermal treatment is a pre-requisite for their coagulation and gelation [36]. The findings are consistent with the SDS-PAGE data which showed the involvement of disulfide bonds in SA formation (Fig. 3). The decrease in free SH groups in the SA fraction may cause by the processes such as ␤-elimination or oxidation which are directly responsible for loss of SH groups [42]. There was no marked change in the SS content in SA. This could be due to a balance of a decrease in SS content from the cleavage of existing disulfide bonds from native BWG and an increase in SS content caused by the newly formed disulfide-linked aggregates, resulting in a relatively constant SS content.

Fig. 6. Transmission electron micrographs of buckwheat globulin aggregates formed by heating at 100 ◦ C for 30 min and fractionated by sucrose density gradient ultracentrifugation for 17 h. (a) Unheated buckwheat globulin (control). (b) Fraction 1. (c) Fraction 2. (d) Fraction 3. Magnification: 52,000× (The bars in the micrographs represent 200 nm in length).

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Heating may disrupt both covalent and non-covalent interactions leading to the exposure of previously buried SH groups and hydrophobic groups to the surface of protein molecules. Upon extended heating of BWG, the formation of larger aggregates via covalent intermolecular disulfide bonds or via noncovalent interactions between exposed hydrophobic groups of unfolded BWG and aggregates may occur simultaneously. The marked increase in free SH content in the ISA fraction can be attributed to both the breaking of disulfide bonds and activation of buried SH groups during heating. Only some exposed SH groups may be involved in intermolecular disulfide bonds formation and extensive exposure of the buried hydrophobic groups may form molecules with highly disordered structures, resulting in a net increase in SH content and a net decrease in SS content in ISA. The data suggest that ISA contained denatured protein molecules with highly disordered structures which are consistent with the DSC data (Fig. 2). The formation of larger aggregates via hydrophobic interactions between exposed hydrophobic groups of smaller aggregates may reduce the accessibility of the intermolecular disulfide bonds of such smaller aggregates to be broken down by heating. The involvement of both covalent intermolecular disulfide bonds and non-covalent hydrophobic interactions for ISA formation was also demonstrated by SDS-PAGE (Fig. 3).

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3.4. Sucrose density gradient ultracentrifugation and transmission electron microscopy BWG solutions heated at 100 ◦ C for 30 and 60 min were fractionated by sucrose density gradient ultracentrifugation. As shown in Fig. 5A, heating for 30 min and centrifugation for 1 h resulted in the formation of slowly sedimenting fraction only (Peak 1) whereas heating for 60 min (and centrifuged for the same period) led to the formation of three peaks representing, respectively, slowly sedimenting fraction (Peak 1), faster sedimenting fraction containing smaller molecular size SA (Peak 2), and fastest sedimenting fraction containing large macroaggregates (Peak 3). In order to obtain better separation and to determine the presence of various components in these fractions, the heated protein solutions were centrifuged for a longer time (17 h). The SA fraction (Peak 2) became detectable after 30 or 60 min of heating (Fig. 5B). Heating for 60 min decreased the amount of SA (Fig. 5B, Peak 2) with concomitant increase in the proportion of soluble macroaggregates (Fig. 5B, Peak 3). When examined by TEM, native BWG was visualized as a dispersion of globules containing uniformly compact protein molecules with estimated diameters from 11.7 to 12.5 nm (Fig. 6a). The value is comparable with laser light scattering data which showed that BWG exist as a hexamer with a diameter of 12.8 nm [20]. The compact globular conformation of BWG

Fig. 7. Transmission electron micrographs of buckwheat globulin aggregates formed by heating at 100 ◦ C for 60 min and fractionated by sucrose density gradient ultracentrifugation for 1 h. (a) Unheated buckwheat globulin (control). (b) Fraction 1. (c) Fraction 2. (d) Fraction 3. Magnification: 52,000× (The bars in the micrographs represent 200 nm in length).

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molecules observed by TEM has also been confirmed by the log–log plot of hydrodynamic radii (rh ) versus Mw , giving a slope of 0.38 which is close to that expected for a spherical molecule [20]. The TEM micrographs of SA formed after heating for 30 min and centrifuged for 1 h show that some native BWG molecules were still present in the SA (not shown). Poor resolutions were found in these micrographs, probably due to insufficient centrifugation time to sediment the aggregates. The images of SA from different fractions show different appearances, and particles with aggregated structures were distinguishable from the native protein globules. In the top fraction (Peak 1), a combination of random strand-like structures with estimated length of 200–400 nm and some native BWG was observed. The formation of strands may be attributed to unfolding of the native BWG or association of partially denatured molecules. Aggregation of protein changed the initial uniform distribution of the native protein. Similar microstructures were observed in both the middle (Peak 2) and the bottom (Peak 3) fractions, but with larger aggregates. After 17 h centrifugation (Fig. 6b–d), separation of aggregates with different sizes and globular molecules with increased size were clearly observed due to better resolution. Large soluble macroaggregates were well separated from each other, together with some strand-like structures in Peak 3 (Fig. 6d). The results suggest a change of conformation from an extended strand struc-

ture to a more compact spherical form. The formation of strands may be caused by the breakdown of hydrogen bonds and unfolding of protein molecules upon heating, probably during the early stage of aggregation. The observation of random strandlike structures is in agreement with previous analysis carried out using SEC-MALLS technique, in which the conformation slope of BWG hexamers from the log–log plot of rh versus Mw increased from 0.38 (spherical) to 0.52 (random-coiled) after 30 min of heating, implying unfolding of BWG hexamers and the formation of random-coiled molecules. At the high Mw portion of the plot, corresponding to large soluble macroaggregates, a decline in the slope was observed, suggesting the formation of compact spherical structure from an initially more extended conformation [20]. The microstructures of SA formed after heating at 100 ◦ C for 60 min and centrifuged for 1 and 17 h are shown in Figs. 7 and 8, respectively. After centrifugation for 1 h, highly branched aggregates were cross-linked to form a network-like structure as shown in Peak 1 and 2 (Fig. 7b and c). The evolution of these network-like strand structures became more obvious upon prolonged heating, since more extensive unfolding of the protein molecules may lead to increased exposure of hydrophobic groups, resulting in a closer alignment of polypeptide chains and the formation of much stronger hydrogen bonds. Clearer separation of the large soluble macroaggregates was visualized after 17 h ultracentrifugation (Fig. 8b–d).

Fig. 8. Transmission electron micrographs of buckwheat globulin aggregates formed by heating at 100 ◦ C for 60 min and fractionated by sucrose density gradient ultracentrifugation for 17 h. (a) Unheated buckwheat globulin (control). (b) Fraction 1. (c) Fraction 2. (d) Fraction 3. Magnification: 52,000× (The bars in the micrographs represent 200 nm in length).

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Polydisperse aggregates of BWG with different size and shape were observed in this study, suggesting the spontaneous interactions arising from partial unfolding of the molecules, releasing previously committed hydrogen-bonded protein groups. The oligomeric structures of legumin-like proteins are stabilized by disulfide bonds between the acidic and basic polypeptides. Dissociation of the oligomers into monomers may promote the formation of aggregates. BWG monomers, similar to other legume 11S globulins, are linked by non-covalent forces [43]. Heating may disrupt these forces and dissociate the hexamers, thereby facilitating the association of BWG molecules, leading to the formation of strand-like aggregates and large compact macroaggregates. 4. Conclusions The present data show that BWG is a heat-coagulable protein, which could form aggregates at temperatures below its Td . Thermal aggregation of BWG was controlled by both non-covalent chemical forces such as hydrogen bonding, hydrophobic, and electrostatic interactions and covalent interactions including disulfide bonding and SH–SS interchange. DSC data suggest a re-distribution of the native and extensively denatured proteins in the heat-induced aggregates of BWG, particularly in the ISA fraction. Based on both SDS-PAGE and TEM observations, both dissociation and association of BWG molecules were involved in the formation of heat-induced aggregates. A morphological examination of BWG aggregates by TEM, in combination with our previous MALLS measurements, can lead to better understanding of the relationship between heat treatments and the size and shape of heat-induced aggregates. This would provide a knowledge base for controlling aggregation, and may lead to the development of protein products that meet the properties required for specific food applications. Acknowledgements The research project was supported by a Hong Kong University Conference and Research grant and a Hong Kong University Seed Fund. The authors thank M.K. Rout for technical assistance in the project. References [1] H. Taira, in: A.M. Johnson, M.J. Peterson (Eds.), Encyclopedia of Food Technology, Avi Publ. Co., Westport, CT, 1974, p. 139.

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