Probing Conformational Change of Bovine Serum Albumin–Dextran

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Probing Conformational Change of Bovine Serum Albumin−Dextran Conjugates under Controlled Dry Heating Shuqin Xia,†,‡ Yunqi Li,§ Qin Zhao,‡ Ji Li,‡ Qiuyang Xia,‡ Xiaoming Zhang,† and Qingrong Huang*,‡ †

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, Jiangsu 214122, People’s Republic of China ‡ Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, New Jersey 08901, United States § Key Laboratory of Synthetic Rubber and Laboratory of Advanced Power Sources, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, People’s Republic of China ABSTRACT: The time-dependent conformational change of bovine serum album (BSA) during Maillard reaction with dextran under controlled dry heating has been studied by small-angle X-ray scattering, fluorescence spectroscopy, dynamic light scattering, and circular dichroism analysis. Through the research on the radii of gyration (Rg), intrinsic fluorescence, and secondary structure, conjugates with dextran coating were found to inhibit BSA aggregation and preserve the secondary structure of native BSA against long-time heat treatment during Maillard reaction. The results suggested that the hydrophilic dextran was conjugated to the compact protein surface and enclosed it and more dextran chains were attached to BSA with the increase of the heating time. The study presented here will be beneficial to the understanding of the conformational evolution of BSA molecules during the dry-heating Maillard reaction and to the control of the protein−polysaccharide conjugate structure. KEYWORDS: BSA, dextran, dry heating, conformation, conjugation

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INTRODUCTION Protein is one of the major biopolymers involved in food. Heat treatments usually induce the structural changes and the denaturation and aggregation of food proteins, which are ubiquitous in food processing.1 Protein can also react with reducing sugars, such as oligosaccharides and polysaccharides, via Maillard reaction during heating. Maillard reaction has three stages, and the first is the initial glycation reaction.2,3 It relates to a condensation reaction of an unprotonated amino group in protein with a carbonyl group of reducing sugar.4 Maillard reaction can occur in both wet and dry conditions to synthesize glycoconjugates. Controlled dry heating has the advantages of mild reaction, easy control, and less outgrowth. It has received considerable attention to improve the solubility,5 emulsifying properties,6−10 gel properties,11,12 and thermal stability5,10,13 of various proteins. Ideally, a glycoconjugate is destined for improved functionality with minimal color or flavor development; thus, it is essential to control Maillard reaction carefully to avoid changes in the later stage.2 The reaction time is a crucial variable favoring the formation of glycoconjugates. The characteristic of the glycoconjugates correlates with the protein conformation and the molecular weight of the polysaccharide.4 Although many studies have been conducted to improve functionality of glycoconjugates, clear description of the correlation between the structural changes and the improvement of physical properties is still absent. It is challenging to address the complexity in the conformational change and the aggregation behavior of proteins during dry heating. Therefore, monitoring the conformational evolution of protein during the dry-heating period in the company of polysaccharide will be beneficinal to illuminate the influence of the reaction time and polysaccharide © XXXX American Chemical Society

on glycosylation and polymerization and the relationship between structure and functionality.14 For in-depth insight into the mechanism of protein conformational change during the dry-heating process in the presence of polysaccharide, bovine serum albumin (BSA) has been widely used as a model system to address critical problems in the food system. BSA is a globular protein, and 28 of 59 Lys residues are exposed to the solvent, which provide sufficient sites for Maillard reaction.15 It also contains 35 cysteines, which may participate in intermolecular conjugation upon heat treatment.16 The conformation of BSA in aqueous solutions was investigated by small-angle X-ray scattering (SAXS).17,18 In the presence of reducing sugars, including dextran19 and galactomannan,20 the structure and aggregation behavior of glycated BSA are sensitive to the protein concentration, solution pH, ionic strength, etc. To present a clear view on the conformational change of BSA and its conjugate during dry heating, we consider dextran with a single carbonyl group in the reducing end. During heating, protein might simultaneously experience glycosylation and aggregation. Studies via sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) and spectroscopic analysis have shown that glycation can prevent BSA aggregation in aqueous solution.21 To obtain further insight on the aggregation and glycosylation mechanism, the aggregation of BSA in the presence or absence of dextran as a function of the dry-heating time was investigated. SAXS, which Received: December 24, 2014 Revised: April 10, 2015 Accepted: April 14, 2015

A

DOI: 10.1021/jf506267r J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

ments, Walnut Creek, CA). A conjugate solution was prepared with 0.1 mg/mL BSA in deionized water. The quartz cuvette of a 1 cm path length was used for holding samples. The intrinsic fluorescence was detected at an excitation wavelength of 280 nm, and the emission wavelength ranged from 287 to 500 nm at 25 °C. The slit width of excitation and emission was 5 nm, respectively. Far-ultraviolet (UV) CD Spectroscopy. CD of BSA and glycoconjugates was investigated by an Aviv model 420SF CD spectrometer (Biomedical, Inc., Lakewood, NJ). The spectral bandwidth was set at 1 nm. The glycoconjugate was solubilized in deionized water with the BSA concentration of 0.1 mg/mL and then added to the quartz cell of 0.1 cm light path length. CD spectra was obtained from 190 to 250 nm at 25 °C. Molar ellipticity (θ) was analyzed to obtain the α-helix content via the CDPro2 software. DLS. The mean hydrodynamic diameters of protein and conjugates were determined with the methods described in our previous paper.25 A dynamic-light-scattering-based BIC 90 plus particle size analyzer was used, fitted with a BI-9000AT digital correlator (Brookhaven Instrument Corporation, Holtsville, NY). Measurements were carried out in triplicate at room temperature.

has permitted researchers to solve a lot of ineresting problems unaddressed using other structural methods alone, has created significant impacts on structural characterization of macromolecules during the past several decades.22 The combined studies via SAXS, circular dichroism (CD), fluorescence spectroscopy, and dynamic light scattering (DLS) have been conducted to investigate how the conformation and structure of BSA change as a function of the reaction time coexisting with dextran.

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MATERIALS AND METHODS

Materials. BSA (fraction V) with a molecular weight of 66 kDa was obtained from Sigma Chemical Co. (St. Louis, MO). Technical-grade dextran (DT10) with an average molecular weight of 10 kDa was provided by Pharmacosmos (Danmark). The other chemicals, such as sodium chloride (NaCl), potassium chloride (KCl), Na2HPO4·7H2O, and NaH2PO4·H2O, were of analytical reagent grade and purchased from Sigma Chemical Co. (St. Louis, MO). Preparation of BSA−Dextran Conjugates. The conjugates were synthesized through Maillard reaction by dry heating based on the method introduced by Kato et al.,9 with minor modification. The BSA−dextran mixture in a 1:12 molar ratio was solubilized in phosphate buffer solution (PBS, pH 7.0, 0.1 M NaCl), followed by freezing dry. The freeze-dried mixtures were then put into a desiccator containing saturated KCl solution and incubated at 60 °C for different times (8, 19, and 26 h). The samples with BSA alone were also treated in the same way for comparison. After incubation, the samples were solubilized in deionized water (0.25%, w/v) and then pre-filtered via a 0.45 μm pore membrane filter (Millipore Corp., Bedford, MA). A 20 mL Microsep advance centrifugal device (Pall Corporation, Port Washington, NY) was used for ultrafiltration with the molecular weight cut-off of 100 000. The samples were centrifuged at 1000 revolutions/min for 30 min to 1 /8 of the initial volume to maintain. The deionized water was added to complement the original volume. This procedure was repeated in triplicate to remove excess dextran and BSA. Afterward, the retentates (conjugates) were freeze-dried. It has been proven that the native or dry-heated BSA and 10 kDa dextran could penetrate through the cartridges.4 The control samples containing pure protein or dextran were also treated accordingly. SDS−PAGE. Electrophoresis analysis was conducted on the basis of the method provided by Jung et al.,19 with minor modification. A 10% acrylamide separating gel and a 5% stacking gel containing 0.1% SDS were used. Samples were dissolved in phosphate buffer solution (pH 7.4, 10 mM). Afterward, samples were incubated for 3 min at 100 °C. Electrophoresis was conducted at about 10 mA until the dye front arrived at the bottom of the separating gel. The Coomassie Brilliant Blue R solution was used for gel staining to visualize protein. Later, it was discolored with a solution composed of 10% methanol and 10% acetic acid. Binding Ratio of DT10 to BSA. Colorimetric methods for determing protein and polysaccharide concentrations were used to calculate the binding ratio of DT10 to BSA. The BSA content was assayed using the Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories, Hercules, CA).23 The dextran content was quantified by the phenol− sulfuric acid method.24 SAXS. BSA and BSA−dextran conjugate were dissolved in deionized water with 2 mg/mL BSA. The content of BSA in solustions was adjusted using the Bio-Rad Protein Assay Kit II for SAXS measurements. SAXS experiments were carried out at the BioCAT, 18-ID beamline of the Advanced Photon Source (Argonne National Laboratory) according to the way that we used previously.25,26 A sample−detector distance of 3.5 m was taken to lay over a combined Q range from 0.006 to 0.37 Å−1. The scattering data were obtained by a single exposure of 1 s, and 15 replicas were measured and averaged. Fluorescence Spectroscopy. Fluorescence spectra were recorded with a Cary Eclipse fluorescence spectrophotometer (Varian Instru-

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RESULTS AND DISCUSSION Binding of BSA with DT10. SDS−PAGE patterns of BSA−DT10 conjugates with different Maillard reaction times were used to prove the covalent binding between DT10 and BSA (Figure 1). It was found that the high-molecular-weight

Figure 1. SDS−PAGE pattern of the BSA−DT10 mixture after Maillard reaction.

bands were broad using protein staining. There were bands (∼205 kDa) at the head of the separating gel, implying the existence of high-molecular-weight product. Under the conditions of dry heating in this study, oligomers might first arise based on monomeric BSA; subsequently, Maillard reaction proceeded.19 Therefore, larger oligomeric BSA−DT10 conjugates might exist in the product. In the electropherogram of the reactant (8 h), there was almost no band from the unreacted BSA. We also measured the composition of the conjugates with three reaction times. It was displayed that about 1.68 M DT10 was attached to 1 M BSA. With the reaction time prolonging to 19 and 26 h, about 2.50 and 3.12 M DT10 were bound to 1 M BSA, respectively. SAXS Profiles. Heating of proteins may lead to conformational changes.6 The scattering intensity profiles of pure BSA, dextran (DT10), and BSA−DT10 conjugate solutions were presented in Figure 2. B

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Figure 2. (a) Scattering intensity profiles of BSA and dextran (DT10) during the controlled dry heating period. (b) Spherical shell-fitting plots for scattering intensity profiles of BSA during the dry-heating period in the presence of dextran. Scaling exponents were obtained through the best fit in a log−log plot. The solid line is the fit to a Schultz polydisperse core spherical shell.

intermolecular β-sheet (i.e., β-amyloid).30 The dry state may prefer β-amyloid because of the lower conformational relaxation freedom; this is also indicated from the CD results. For BSA dry heated from 8 to 19 h in the presence of dextran (Figure 2b), the slopes also changed when the heating time prolonged in the small q range. It was noted that the slopes did not increase significantly, provided that the heating time prolonged to 26 h. This suggested that the conjugation reaction reached a steady state with the prolonging of the reaction time. The phenomenon was associated with the steric hindrance existing near ε-lysyl amino groups in BSA and branched structure in DT10.19 Furthermore, the steric effect deriving from the attached dextran might bring an obstacle for further binding. Guinier Analysis. The quantity radius of gyration (Rg) was commonly used to reflect the volume of solution occupied by the extended micromolecule.20 Guinier analysis of the scattering curve gave an estimation of Rg. The Rg of particles in solutions was determined using the Guinier relationship.

For the dry-heated BSA alone, the slopes in the large q range transformed little (Figure 2a). At small q range, the slopes increased sharply with the heating time prolonged. It changed from −0.23 to −1.19 in dilute solutions when the dry heating time prolonged up to 19 h, suggesting that large aggregates formed by interchain correlation of BSA. Such an obvious transformation also suggested that the aggregation of BSA during dry heating might undergo a rod-like shape (slope of −1).26 However, at a longer heating time (26 h), the slope changed little, revealing that protein did not aggregate further. The 17 disulfide bridges inside BSA make the tertiary structure stable at room temperature, while they do not prevent changes of shape and size as a function of the temperature. Furthermore, a free thiol group (Cys34) is present in the BSA structure, which has been verified to be involved in heatinduced aggregation pathways of BSA. According to the literature, heat-induced aggregation of BSA takes place via two ways in solution. One is the formation of intermolecular disulfide bonds,27−29 and the other is the formation of C

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Journal of Agricultural and Food Chemistry ln[I(Q )/(Q = 0)] = −(QR g)2 /3

has already been found that free cysteine plays a notable role on the aggregate growth of BSA in the aqueous system via intermolecular disulfide bridges and S−S exchange.31 The participation of the unique free cysteine in the aggregation pathway can be affected by the formation of glycosylated albumin. In fact, Maillard reaction of proteins generally involved amino groups (lysine and arginine residues) and thiol groups of the cysteine residue, owing to its powerful nucleophilic property.32,33 Therefore, the adduction of dextran on the unique sulphydryl group could occur, which might prevent the cysteine residue from taking part in the formation of intermolecular bonds.30 The pure dextran was incubated in the same dry-heating condition too. The SAXS results indicated that the structure of DT10 was unchanged (Figure 2a). The dry heating has no significant impact on the conformation of dextran. The gyration radius is slightly changed from 3.6 to 3.8 nm with 26 h of heating. Spherical Core−Shell Fitting. Conferred from the spherical core−shell fitting (Figure 2b), the core (BSA) has a radius of around 3.6−4.2 nm. When reaching a steady state, the conjugate has a shell with a thickness of around 6.8 nm. BSA conjugates with dextran are slightly smaller in shell thickness (∼5.8 nm) for 8 h of dry heating compared to 19 h (∼7.0 nm) and 26 h (∼6.6 nm). The core radii (BSA) increased slightly with the increase of the dry-heating time. Hence, hydrophilic dextran was bound to the protein surface, followed by enclosing the protein, and the dextran reacted more extensively as the heating time increased. It might also be deduced that the conjugation between DT10 and BSA had no obvious influence on the BSA peptide backbone. In addition, because the steric effect depended upon the polymer chain density of the colloidal particle surface, the shell, owing to the attached dextran with a longer reaction time, agreed with the better stability against heat-induced aggregation of protein. DLS Measurements. It was also found that the prolonging of the heating time was accompanied by the formation of larger molecules (Figure 4). This tendency could relate to the

(1)

The data range for Guinier analysis was in accordance with QRg < 1.57, which is appropriate to Guinier fitting.26 Figure 3

Figure 3. Guinier plots for scattering intensity profiles of BSA during the dry-heating period in the (a) absence and (b) presence of dextran (DT10, 10 kDa). Scaling exponents were obtained through the best fit in a log−log plot.

displayed the fitting line and the influence of heating time on Rg. With the prolonging of the reaction time, I(Q) increased in a small Q range, which might be attributed to the aggregation of protein.26 The reaction time and dextran presence dependence of Rg are listed in Table 1. The Rg values of pure BSA increased from 3.8 nm (native) to 5.3 nm (8 h), 7.6 nm (19 h), and 8.9 nm (26 h) as the reaction time increased. However, the Rg values of conjugates just increased from 3.8 nm (native BSA) to 5.6 nm (8 h), 6.8 nm (19 h), and 6.8 nm (26 h). The Rg difference corresponding to the same reaction time may come from the competition between glycosylation and aggregation of BSA. It

Figure 4. Average diameter of BSA for dry-heated [60 °C and 79% relative humidity (RH)] time periods in the absence and presence of dextran (DT10, 10 kDa).

Table 1. Variation of Radii of Gyration (Rg) during the Dry-Heating Period in the Absence and Presence of Dextran (DT10) time (h)

0

8

19

26

Rg (Å) of BSA Rg (Å) of BSA−DT10

38.0 ± 0.3

52.8 ± 0.1 56.1 ± 0.3

75.7 ± 0.5 67.7 ± 0.4

89.3 ± 0.4 68.4 ± 0.2

D

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Figure 5. Intrinsic fluorescence spectra excited at 280 nm of BSA for dry-heated (60 °C and 79% RH) time periods in the (a) absence and (b) presence of dextran (DT10, 10 kDa). Fluorescence spectra of BSA samples were measured at 0.1 mg/mL.

Figure 6. CD spectra of BSA for dry-heated (60 °C and 79% RH) time periods in the (a) absence and (b) presence of dextran (DT10, 10 kDa).

Once dry heating alone, the alternation of fluorescence intensity of BSA was significant, which came with an obvious shift of the emission maximum to 340.9 (19 h) and 343.9 (26 h), respectively. It is common knowledge that the protein aggregation produces a decrease of the fluorescence intensity, which is accompanied by a blue shift of the maximum emission intensity wavelength. Considering the results, it was implied that the protein underwent conformation change, resulting in the Trp residue exposure to a nonpolarity microenvironment when dry heating alone. In contrast, there was little change in the emission maximum wavelength of glycoconjugates (λmax = 347.81 nm at 19 and 26 h), suggesting that the conformation around the Trp microenvironment of glycoconjugates resembled with that of native BSA and BSA/DT10 mixture. Whereas in comparison to the controlled BSA by dry heating alone and BSA/DT10 mixture without the heating process, the fluorescence intensity in each glycoconjugate was lowered. Therefore, it could be postulated that the attachment of dextran to BSA took place on the surface of the protein molecule, which corresponded to the core−shell microstructure deduced from SAXS results. It might also be assumed that the binding between BSA and dextran had no significant influence on the conformation of BSA. A previous study of BSA−dextran conjugates with Maillard reaction time reaching 48 and 72 h showed little change in the fluorescence intensity when compared to the native protein.4 On the contrary, the fluorescence intensity in native BSA alone increased upon dry heating at the same time period.

oligomerization of BSA.19 Thermally induced denaturation and aggregation of BSA have been widely studied. The thiol groups and nonpolar amino acid residues are exposed because of denaturation, which results in the formation of irreversible aggregates via intermolecular disulfide bonds and hydrophobic attraction. This was the reason that the BSA formed aggregates when it was dry-heated alone. Whereas the aggregation and glysolation may occur simultaneously provided that there is dry heating in the presence of dextran, the shell by glycosylation may form a barrier to prevent BSA from aggregation further. This difference is in virtue of the introduction level of hydrophilic dextran to protein. The DLS results are in agreement with the SAXS experiments, in which the aggregation of BSA was concomitant with the reaction time and the extra aggregation was inhibited by the presence of dextran upon the increase of the dry-heating time. Fluorescence Measurements. There are two tryptophan (Trp) residues possessing intrinsic fluorescence in BSA. Trp212 lies in a hydrophobic pocket of BSA, and Trp134 lies in a hydrophilic region of the molecule surface.16 Moreover, tryptophan displays an emission maximum near 348 nm in water.20 The intrinsic fluorescence spectra were used to elucidate the conformation change around the Trp microenvironment. As shown in Figure 5, the fluorescence emission spectra of native BSA and BSA/DT10 mixture were compared to BSA dry heated alone and glycoconjugate. It was observed that native BSA presented fluorescence emission maxima at 350.92 nm. E

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The reason for the discrepancy may be correlated with the long reaction time. As in our preliminary experiments, it was found that the solubility of the conjugates was very low and floccules formed in the aqueous dispersion when the reaction time reached 36 h or even higher. Jung et al. also characterized the structure of BSA−dextran (69 000 Da) conjugates after controlled dry heating for 7 days by fluorescence spectroscopy and found the similar fluorescence shielding effect by the dextran molecule bound to BSA.19 Far-UV CD Measurements. CD spectroscopy was used to investigate the influence of dextran on the characteristics of BSA at the secondary structure level during controlled dry heating. As shown in Figure 6, the helix presented two negative absorption bands at around 209 and 222 nm. Native BSA was reported to have a fraction of about 55% α-helix and 19% βsheet at pH 7.34 Herein, the α-helix content of native BSA was about 49% in deionized water, and it almost stayed constant in dry-heating BSA samples containing dextran (Table 2). In view

BSA

*Telephone: 848-932-5514. Fax: 732-932-6776. E-mail: [email protected]. Funding

This work was supported by projects of the National Natural Science Foundation of China (31471624), the National 125 Program of China (2012BAD33B04), and 111 Project (B07029). This research used resources of the Advanced Photon Source by Argonne National Laboratory under Contract DE-AC02-06CH11357. This project was also supported by Grant 9 P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. Notes

The authors declare no competing financial interest.

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8h

19 h

26 h

8h

19 h

26 h

α-helix (%)

48.9

46.5

38.2

34.9

50.8

50.5

49.9

REFERENCES

(1) Da Silva Pinto, M.; Bouhallab, S. D.; De Carvalho, A. F.; Henry, G.; Putaux, J.-L.; Leonil, J. Glucose slows down the heat-induced aggregation of β-lactoglobulin at neutral pH. J. Agric. Food Chem. 2012, 60, 214−219. (2) Oliver, C. M.; Melton, L. D.; Stanley, R. A. Creating proteins with novel functionality via the Maillard reaction: A review. Crit. Rev. Food Sci. Nutr. 2006, 46, 337−350. (3) Liu, J.; Ru, Q.; Ding, Y. Glycation a promising method for food protein modification: Physicochemical properties and structure, a review. Food Res. Int. 2012, 49, 170−183. (4) Jiménez-Castaño, L.; Villamiel, M.; López-Fandiño, R. Glycosylation of individual whey proteins by Maillard reaction using dextran of different molecular mass. Food Hydrocolloids 2007, 21, 433−443. (5) Wang, Q.; Ismail, B. Effect of Maillard-induced glycosylation on the nutritional quality, solubility, thermal stability and molecular configuration of whey protein. Int. Dairy J. 2012, 25, 112−122. (6) Einhorn-Stoll, U.; Ulbrich, M.; Sever, S.; Kunzek, H. Formation of milk protein−pectin conjugates with improved emulsifying properties by controlled dry heating. Food Hydrocolloids 2005, 19, 329−340. (7) Yadav, M. P.; Parris, N.; Johnston, D. B.; Onwulata, C. I.; Hicks, K. B. Corn fiber gum and milk protein conjugates with improved emulsion stability. Carbohydr. Polym. 2010, 81, 476−483. (8) O’Regan, J.; Mulvihill, D. M. Heat stability and freeze−thaw stability of oil-in-water emulsions stabilised by sodium caseinate− maltodextrin conjugates. Food Chem. 2010, 119, 182−190. (9) Kato, A.; Minaki, K.; Kobayashi, K. Improvement of emulsifying properties of egg white proteins by the attachment of polysaccharide through Maillard reaction in a dry state. J. Agric. Food Chem. 1993, 41, 540−543. (10) Á lvarez, C.; García, V.; Rendueles, M.; Díaz, M. Functional properties of isolated porcine blood proteins modified by Maillard’s reaction. Food Hydrocolloids 2012, 28, 267−274. (11) Sun, W.-W.; Yu, S.-J.; Yang, X.-Q.; Wang, J.-M.; Zhang, J.-B.; Zhang, Y.; Zheng, E.-L. Study on the rheological properties of heatinduced whey protein isolate−dextran conjugate gel. Food Res. Int. 2011, 44, 3259−3263. (12) Matsudomi, N.; Nakano, K.; Soma, A.; Ochi, A. Improvement of gel properties of dried egg white by modification with galactomannan through the Maillard reaction. J. Agric. Food Chem. 2002, 50, 4113− 4118. (13) Xu, C.-h.; Yang, X.-q.; Yu, S.-j.; Qi, J.-r.; Guo, R.; Sun, W.-W.; Yao, Y.-J.; Zhao, M.-M. The effect of glycosylation with dextran chains of differing lengths on the thermal aggregation of β-conglycinin and glycinin. Food Res. Int. 2010, 43, 2270−2276. (14) Enomoto, H.; Li, C.-P.; Morizane, K.; Ibrahim, H. R.; Sugimoto, Y.; Ohki, S.; Ohtomo, H.; Aoki, T. Glycation and phosphorylation of

BSA−DT10

native BSA

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Table 2. α-Helix Content of BSA by Dry Heating Alone and in the Presence of Dextran dry-heating time

Article

of the minor difference in the CD spectra, the changes in the secondary structure of BSA upon the formation of the glycoconjugate were small. In contrast, the CD spectra changed significantly when the protein alone was dry-heated for 19 and 26 h (Figure 6a), and the α-helix content was found to decrease significantly (Table 2). This suggested that BSA underwent secondary structure changes because of thermal treatment and the presence of dextran might serve to maintain the structural integrity of BSA during dry-heat treatment. This behavior is consistent with the SAXS experimental finding, in which minor loss of secondary structure is accompanied with the prolonged reaction time upon glycosylation with dextran. Spectroscopic analysis by Kim et al. also found that the surface of glycoconjugate was covered with galactomannan and the hydrophobic interior conformation as well as the secondary structure were unchanged obviously.20 In addition, alteration of the albumin tertiary structure with the glycation process by glucose in the aqueous system had no significant impact on the secondary structure of protein, which was probed after glycation for 7 weeks.21,35 In summary, SAXS, fluorescence, CD, and DLS were employed to investigate the structural change of BSA and BSA−dextran conjugate as a function of dry-heating time. Dextran was found to be able to protect BSA from denaturation and aggregation during dry heating. During incubation in controlled dry heating, BSA endures an obvious change in secondary structure, tertiary structure, and aggregation. Because of the protection from the conjugated dextran layer, the secondary structure of native BSA is well-preserved. The conjugate thickness after reaching a steady state is around 7 nm, with a core of 4 nm. It is necessary to study further to have good knowledge of the relationship between the glycoconjugate structure and functionality. F

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Journal of Agricultural and Food Chemistry β-lactoglobulin by dry-heating: Effect on protein structure and some properties. J. Agric. Food Chem. 2007, 55, 2392−2398. (15) Li, Y.; Lee, J.; Lal, J.; An, L.; Huang, Q. Effects of pH on the Interactions and Conformation of Bovine Serum Albumin: Comparison between Chemical Force Microscopy and Small-Angle Neutron Scattering. J. Phys. Chem. B 2008, 112, 3797−3806. (16) Bourassa, P.; Kanakis, C. D.; Tarantilis, P.; Pollissiou, M. G.; Tajmir-Riahi, H. A. Resveratrol, genistein, and curcumin bind bovine serum albumin. J. Phys. Chem. B 2010, 114, 3348−3354. (17) Zhang, F.; Skoda, M. W. A.; Jacobs, R. M. J.; Martin, R. A.; Martin, C. M.; Schreiber, F. Protein interactions studied by SAXS: Effect of ionic strength and protein concentration for BSA in aqueous solutions. J. Phys. Chem. B 2007, 111, 251−259. (18) Gospodarczyk, W.; Szutkowski, K.; Kozak, M. Interaction of Bovine Serum Albumin (BSA) with Novel Gemini Surfactants Studied by Synchrotron Radiation Scattering (SR-SAXS), Circular Dichroism (CD), and Nuclear Magnetic Resonance (NMR). J. Phys. Chem. B 2014, 118, 8652−8661. (19) Jung, S. H.; Choi, S. J.; Kim, H. J.; Moon, T. W. Molecular characteristics of bovine serum albumin-dextran conjugates. Biosci., Biotechnol., Biochem. 2006, 70, 2064−2070. (20) Kim, H. J.; Choi, S. J.; Shin, W.-S.; Moon, T. W. Emulsifying Properties of Bovine Serum Albumin−Galactomannan Conjugates. J. Agric. Food Chem. 2003, 51, 1049−1056. (21) Rondeau, P.; Navarra, G.; Cacciabaudo, F.; Leone, M.; Bourdon, E.; Militello, V. Thermal aggregation of glycated bovine serum albumin. Biochim. Biophys. Acta, Proteins Proteomics 2010, 1804, 789− 798. (22) Graewert, M. A.; Svergun, D. I. Impact and progress in small and wide angle X-ray scattering (SAXS and WAXS). Curr. Opin. Struct. Biol. 2013, 23, 748−754. (23) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (24) Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956, 28, 350−356. (25) Huang, Y.; Yu, H.; Guo, L.; Huang, Q. Structure and selfassembly properties of a new chitosan-based amphiphile. J. Phys. Chem. B 2010, 114, 7719−7726. (26) Li, Y.; Xia, Q.; Shi, K.; Huang, Q. Scaling behaviors of α-zein in acetic acid solutions. J. Phys. Chem. B 2011, 115, 9695−9702. (27) Rondeau, P.; Armenta, S.; Caillens, H.; Chesne, S.; Bourdon, E. Assessment of temperature effects on β-aggregation of native and glycated albumin by FTIR spectroscopy and PAGE: Relations between structural changes and antioxidant properties. Arch. Biochem. Biophys. 2007, 460, 141−150. (28) Rombouts, I.; Lagrain, B.; Delcour, J. A. Heat-Induced CrossLinking and Degradation of Wheat Gluten, Serum Albumin, and Mixtures Thereof. J. Agric. Food Chem. 2012, 60, 10133−10140. (29) Luthuli, S. D.; Chili, M. M.; Revaprasadu, N.; Shonhai, A. Cysteine-capped gold nanoparticles suppress aggregation of proteins exposed to heat stress. IUBMB Life 2013, 65, 454−461. (30) Saguer, E.; Alvarez, P. A.; Sedman, J.; Ismail, A. A. Study of the denaturation/aggregation behaviour of whole porcine plasma and its protein fractions during heating under acidic pH by variabletemperature FTIR spectroscopy. Food Hydrocolloids 2013, 33, 402− 414. (31) Militello, V.; Vetri, V.; Leone, M. Conformational changes involved in thermal aggregation processes of bovine serum albumin. Biophys. Chem. 2003, 105, 133−141. (32) Zeng, J.; Davies, M. J. Evidence for the Formation of sdducts and S-(Carboxymethyl)cysteine on reaction of α-dicarbonyl compounds with thiol groups on amino acids, peptides, and proteins. Chem. Res. Toxicol. 2005, 18, 1232−1241. (33) Thorpe, S. R.; Baynes, J. W. Maillard reaction products in tissue proteins: New products and new perspectives. Amino Acids 2003, 25, 275−281.

(34) Quiming, N. S.; Vergel, R. B.; Nicolas, M. G.; Villanueva, J. A. Interaction of bovine serum albumin and metallothionein. J. Health Sci. 2005, 51, 8−15. (35) Mendez, D. L.; Jensen, R. A.; McElroy, L. A.; Pena, J. M.; Esquerra, R. M. The effect of non-enzymatic glycation on the unfolding of human serum albumin. Arch. Biochem. Biophys. 2005, 444, 92−99.

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DOI: 10.1021/jf506267r J. Agric. Food Chem. XXXX, XXX, XXX−XXX