Glycation of bovine serum albumin with monosaccharides inhibits heat-induced protein aggregation

Abstract

Glycation with different degree might result in different influence on heat-induced protein aggregation. Effects of glycation on heat-induced bovine serum albumin (BSA) aggregation was investigated, and the glycation degree was investigated. Glycation of BSA was carried out with xylose (Xyl) and galactose (Gal) via Maillard reaction at 60 °C and pH 9 for 1 and 3 days. The BSA band change was determined by SDS-PAGE, and their shape and size were then monitored by TEM and DLS. The molecule number of Xyl or Gal conjugated with BSA was determined by MALDI-TOF MS. Denaturation temperature was determined by DSC. Results show that high-molecular-weight substance and insoluble aggregates appeared when BSA was heated, while those changes were alleviated after glycation. Heat-induced changes in shape of BSA from spheroid particles to rod-like short chains, while the glycated BSA kept as spheroid particles. The size of BSA was increased by 115.7% and 204.3% after 1- and 3-day heating, respectively. The sizes of glycated BSAs were smaller than that of heated BSAs by about 37% for 1 day heating and by about 46% for 3-day heating, respectively. About 5 and 10 molecules of Xyl and about 2 and 4 molecules of Gal were conjugated with BSA after 1- and 3-day heating, respectively. The denaturation temperature of glycated BSA was higher than non-treated BSA and heated BSA. There was no obvious difference between Xyl and Gal on the inhibition effect of aggregation. In conclusion, glycation could hinder the heat-induced BSA aggregation. The higher glycation degree, the more hindrance.

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1. Introduction

Thermal processing is required for a variety of food products and remains a problem for food protein that undergo denaturation. This denaturation process is usually followed by aggregation.¹ As it is critical to control undesirable protein aggregation in food processing, regulation of aggregation has long been a subject of intensive investigation. Glycation with reducing sugars via Maillard reaction (MR) has been considered to be a green method to moderate protein aggregation,² as MR is a spontaneous and naturally-occurring reaction during food processing without adding extra additives. Recently studies showed glycation with glucose could stimulate aggregation of BSA as shown in Nile red and thioflavin T fluorescence photograph and TEM images,³ and in Congo red binding images.⁴ However, TEM images showed that glycation of human insulin with D-ribose strongly inhibits its ability to form large aggregates when stirring at 37 °C for 24 h.⁵ It is known that according to the glycation conditions, the target protein undergoes specific modifications that may either increase or suppress protein aggregation.⁶ However, a few attempts have been made to investigate how different degrees of protein glycation affect protein aggregation, so that this issue could be established.

BSA is a single polypeptide chain of 583 amino acid residues with known sequence.^7,8 The three-dimensional configuration of BSA is heart-like shape and composed of three homologous domains: domain I (residues 1–193), domain II (194–384) and III (385–583).⁹ BSA contains 35 cysteine, forming 17 pairs of disulphide bonds, leaving only cysteine 34 unpaired.⁷ These amino acid residues can form intermolecular disulfide bridges, resulting in protein aggregation during heating.¹⁰ BSA possess chaperone-like activity that be able to inhibit misfolding and aggregation of many client proteins (such as alcohol dehydrogenase, insulin and transthyretin).¹¹ Moreover, BSA is commonly used as model protein to investigate the effects of heat processing^12,13 and glycation,^4,14 because of its abundance, low-cost and ease of purification.¹⁵ Xylose (Xyl) and galactose (Gal) are common monosaccharides (MOS) with similar structure. Xyl and Gal are pentose and hexose, respectively. Xyl has been proved to have higher Maillard reactivity than fructose and glucose, according to the fluorescence of the advanced glycated end-products (AGEs) when incubated with BSA at 37 °C for 7 days.¹⁶ Gal was more reactive than glucose or lactose, as MALDI-TOF MS suggested more Gal (10.3) were added to BSA than glucose (3.3) and lactose (1.6) when incubated at 60 °C for 120 minutes.¹⁷ Our previous experiments, in which BSA was conjugated with different monosaccharides (xylose, glucose, and galactose) and disaccharides (lactose and maltose), showed that the glycation degree was in an order of BSA–Xyl > BSA–Gal > BSA–glucose, BSA–maltose, and BSA–lactose.¹⁸ Xyl and Gal were chosen due to their high glycation degree, and were used as representatives to investigate the effect of glycation on thermal stability of BSA.

Heat could induce BSA to aggregate under a broad range of incubation conditions, in most of the cases, either in food processing progress with elevated temperatures (60–90 °C) and short time (4 h to 6 d),^13,19 or at accelerated storage condition with moderate temperature (usually 37 °C) and long term (up to 7 weeks).^16,20 In the past, the effects of glycation with MOS on heat-induced BSA aggregation were intensively studied at accelerated storage condition,^16,20 but their result were not consist with each other. However, rarely studies were conducted in the food processing condition. Besides, thermal processing is usually required to ensure microbiological safety and quality in food processing.¹ Moreover, the previous studies on heat-induced BSA aggregation were mainly focusing on the condition of acidic pH^13,21,22, and less on neutral pH^23,24 but rarely on alkaline pH¹⁹. However, almost all the studies of the effect of glycation on the BSA aggregation were carried out at neutral pH.^16,20,25 Since rare attempts have been made to control the aggregation process at alkaline condition, such that a more detailed description of the impact of the attachment of MOS to protein stability could be established. Thus, the current work is concerned with determining the effect of glycation with MOS on BSA aggregation at elevated temperature (60 °C) and alkaline condition (pH 9) in the short term (1 to 3 days).

The focus of current research is toward an extended investigation about the effect of glycation on heat-induced BSA aggregation, and the glycation degree was investigated. The BSA band change was determined by SDS-PAGE, and their shape and size were then monitored by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The molecule number of Xyl or Gal conjugated with BSA was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Denaturation temperature was determined by differential scanning calorimetry (DSC).

2. Materials and methods

2.1. Materials

BSA (fraction V, purity ≥ 98%, A-0332) with a molecular weight of 66.43 kDa was purchased from Amresco Inc. (Solon, OH, USA). D(+)-xylose (Xyl) and D(+)-galactose (Gal) were of biological reagent grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sinapic acid (purity ≥ 99.0%) was from Sigma Chemical Co. (St. Louis, MO, USA). Dialysis bags (MWCO 8–14 kDa) were purchased from Biodee Biotechnology Co., Ltd. (Beijing, China). Carbon-coated copper grids were obtained from Beijing Zhongjingkeyi Technology Co., Ltd. (Beijing, China). Low-molecular-weight protein markers kit (14.4 to 97.4 kDa) was provided by Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences (Shanghai, China). All other chemicals were of analytical grade.

2.2. Methods

2.2.1. Preparation of glycated BSA via Maillard reaction. Phosphate-buffered saline (PBS), containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 2.0 mM KH₂PO₄, was adjusted to the pH 9.0 with 1 M NaOH. Stock solutions of BSA (100 mg mL⁻¹) and Xyl or Gal (0.2 M) were mixed in PBS in a molar ratio of 1 : 666.7 (equal to mass ratio 1 : 1.5 for BSA/Xyl, and 1 : 1.8 for BSA/Gal), at the final concentrations of BSA 0.15 mM (10 mg mL⁻¹) and Xyl or Gal 0.1 M (Xyl 15 mg mL⁻¹, Gal 18 mg mL⁻¹). The BSA and Xyl or Gal mixture solutions were heated in tightly-capped plastic tubes (to prevent evaporation) at 60 °C water bath for 1 or 3 days, and then were extensively dialyzed against PBS (pH 7.4) or deionized water (dH₂O). The samples dialyzed in PBS were used for SDS-PAGE, DLS, TEM; dialyzed in dH₂O for MALDI-TOF MS, which were also further lyophilized for DSC assessment. The dialyzed samples were named as BSA–Xyl or BSA–Gal. All samples were separated into aliquots and stored at −20 °C before further analysis. Three independent replications were conducted for each sample preparation.

2.2.2. SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted according to the method described by Jing and Kitts (2004)²⁶ with minor modification. The sample was diluted with PBS (pH 7.4) to a protein concentration of 2 mg mL⁻¹, and then was mixed (1 : 1) with loading buffer containing 5% (v/v) β-mercaptoethanol and heated at 100 °C for 3 min. 8 μL of the sample was loaded onto the gel. A 5% (w/v) acrylamide stacking gel and a 12% (w/v) running gel containing 0.1% (w/v) SDS were used for electrophoresis at constant voltage (110 V), and a set of low-molecular-weight protein markers (14.4 to 97.4 kDa) was used to estimate the molecular weight of samples. Subsequently, the gels were stained with 0.05% (w/v) Coomassie brilliant blue R250 (in methanol/acetic acid/dH₂O, 50/10/40, v/v/v), and distained in a solution of methanol/acetic acid/dH₂O (20/10/70, v/v/v).

2.2.3. Transmission electron microscopy (TEM). An aliquot (10 μL) of dH₂O diluted sample (at 0.2 mg mL⁻¹ protein) was placed onto a carbon-coated copper grid (300 mesh), which was pre-treated with plasma cleaner (Solarus 950 Advanced plasma system, Gatan Inc., CA, USA). Then (after 1 min) the residual solution was gently blotted away by filter paper. 5 μL phosphotungstic acid solution (0.2 M, pH 6.5) was added onto the grid and stayed for 1 min in dark. The residual solution was gently blotted away again and the grid was allowed to air-dry. TEM images were obtained using an FEI Tecnai 20 microscope (FEI, OR, USA) at an acceleration voltage of 120 kV.

2.2.4. Dynamic light scattering (DLS). The samples were diluted with PBS (pH 7.4) to a protein concentration of 2 mg mL⁻¹. z-Average hydrodynamic diameter (d, nm) and polydispersity index (PdI) were collected by automatic runs at least five measurements and calculated based on cumulant analysis using Zetasizer software on Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK). Samples were tested using disposable cuvettes at 25 °C and a fixed scattering angle of 90° was used. ζ-Potential of the particles was measured in disposable folded capillary cells, and calculated model was set to Smoluchowski.

2.2.5. MALDI-TOF MS. The samples were diluted with dH₂O to a protein concentration of 1 mg mL⁻¹. An aliquot (0.5 μL) of diluted sample and 0.5 μL of matrix (saturated sinapic acid in 50% acetonitrile and 0.1% trifluoroacetic acid) were successively placed onto a MALDI-TOF target steel plate, mixed with an Eppendorf pipette, and air-dried. The mass spectra were recorded in the reflector mode with an acceleration voltage of 20 kV and an effective flight path of 200 cm on Autoflex II TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA). External calibration was performed using protein calibration standard II kit (Part No. 207234, Bruker Daltonics, MA, USA) containing BSA and trypsinogen. Mass-to-charge ratio (m/z) (equaling to molecular weight, MW) of heated BSA and glycated BSA was obtained. The number (N) of Xyl or Gal conjugated with BSA molecule was calculated as follows:

N = (MW_{BSA–Xyl/Gal} − MW_{BSA heated})/MW_Xyl/Gal

2.2.6. Differential scanning calorimetry (DSC). The thermal properties of samples were investigated according to the process of Chen, et al. (2015)²⁷ with slight modification. 2.0 mg of lyophilized samples were weighed into aluminum pans, and 10 μL of dH₂O was added. The pans were hermetically sealed and heated from 30 to 100 °C at a rate of 2 °C min⁻¹. A sealed empty pan was used as a reference. Onset temperature (T_onset), peak denaturation temperature (T_d) and enthalpy of denaturation (ΔH) were determined using TA60 software on the different scanning calorimeter (DSC-60, Shimadzu, Tokyo, Japan).

2.2.7. Statistical analysis. Each experiment was performed in triplicate. Results were presented as the mean ± standard deviations (SD). Data were analyzed by one-way ANOVA, followed by Tukey's multiple comparison, using Minitab software (Minitab Inc., State College, PA, USA). The level of statistical significance was set at p < 0.05.

3. Results and discussion

3.1. SDS-PAGE

SDS-PAGE of BSA heated without or with Xyl/Gal was shown in Fig. 1. A major band around 66 kDa with high density was observed for BSA (B₀). The band density was low (B₁) and much low (B₃) after heating 1- and 3-day. The band densities of heated BSAs (B₁ and B₃) were also lower than glycated BSAs (BX and BG). While similar band density was observed between BSA–Xyl and BSA–Gal (BX₁ and BG₁; BX₃ and BG₃).

Fig. 1 SDS-PAGE of BSA heated and/or glycated with Xyl/Gal. BSA (B, 10 mg mL⁻¹) was heated without or with Xyl (X, 0.1 M) or Gal (G, 0.1 M), and then dialyzed against PBS (pH 7.4). The subscripts of 0, 1, 3 indicate unheated, heated (60 °C) for 1 and 3 days, respectively.

A new band of BSA appeared in the high-molecular-weight (HMW) area after heating, and the band density was lower for 3-day than for 1-day heating. The band density was higher for glycated BSA than for heated BSA. It seems that the band density was higher for BSA–Xyl than for BSA–Gal. Insoluble protein bands at the bottom of the sample wells were observed for heated BSAs (B₁ and B₃), but not for glycated BSAs. The band smearings (66–97 kDa; 150–180 kDa) were observed for glycated BSAs, but not for heated BSAs. The band densities seem higher for BSA–Xyl than for BSA–Gal.

When BSA was heated, its band density (66 kDa) had been changed from high to low, a new band of BSA appeared in the HMW area, and the insoluble protein bands were also observed at the bottom of the sample wells. All those changes were alleviated when BSA was glycated with Xyl or Gal. While there was no obvious difference in alleviating effect between BSA–Xyl and BSA–Gal.

It has been reported that the new band in HMW area appeared in native PAGE when BSA was heated at 58 °C for 10–250 min.²⁵ In presence of glucose and under 37 °C and 6 days, BSA new band in HMW area also appeared in SDS-PAGE.³ The density of HMW species was much less for glucose–glycated BSA than non-glycated BSA when heated at 58 °C for 30–120 min, and a correlation of the higher concentrations of glucose and the lower density of HMW species was observed.²⁵ Heat-treated protein could become partly unfolded and result in the exposure of hydrophobic core because of the disruption of the hydrogen bonds.^28,29 The unfolded proteins tend to aggregates through the forces of hydrophobic interactions, hydrogen bonds,^30,31 ionic bonding,⁶ and disulfide bonds.¹⁰ Heat could induce β-sheet increase,^13,19,32 and through hydrogen bond between the NH or CO group these β-sheets could inter-/over-lap and lead to proteins aggregates.³³ As some of these forces are interrupted by SDS and β-mercaptoethanol during sample treatment for SDS-PAGE,^34,35 hydrophobic interactions and hydrogen bonds linked β-sheet network could be predominant force for protein aggregation (formation of HMW species).^30,36–38

3.2. TEM

BSA exhibited as a spheroid particle with a diameter of 5–10 nm, and became rod-like short chains with length of 50–120 nm and width of ∼20 nm after heating. The glycated BSA was also a spheroid particle with a diameter of 10–20 nm. The sizes of BSA–Xyl and BSA–Gal were similar (Fig. 2). In summary, when BSA was heated, its size and shape had been changed from spheroid particle to short chain. Both BSA–Xyl and BSA–Gal were similar in size and kept in the shape of spheroid particle. Since the unconjugated Xyl or Gal were removed by dialysis, impedance of BSA aggregation could be attributed to glycation.

Fig. 2 TEM images of BSA heated and/or glycated with Xyl/Gal. BSA (10 mg mL⁻¹) was heated at 60 °C for 3 days without or with Xyl/Gal (0.1 M) in PBS (pH 9), and then were dialyzed against dH₂O.

It has been reported that the size of BSA in solution is approximated 3.4 nm × 8.4 nm × 8.4 nm.³⁹ When BSA was heated at 70 °C for 96 h, curly chain structure (>100 nm) with a diameter around 10 nm was formed.²⁴ The chain structure with length of ∼350 nm appeared when BSA was heated at 62 °C for 240 min.¹⁹ The length of chain structure was increased (400 → 900 → 1300 nm) as heating time was extended (6 → 40 → 145 h) at 90 °C.¹³ The long chains (up to 900 nm) of β-lactoglobulin became short (50–200 nm in length) when heated in the presence of glucose.³¹ While sheet-like structure was observed for BSA when it was heated with D-glucose-6-phosphate disodium at 37 °C and for 23 weeks.⁴⁰ Heat-induce BSA aggregation and formation of short-chain could be attributed to hydrophobicity interaction, ionic bonding, and hydrogen bonds linked β-sheet network, and disulfide bonds, especially β-sheet network.^6,30,36–38 Glycation could hinder β-sheet network by inhibiting α-helix to β-sheet conversion,^20,25 and the repulsive steric interactions.¹

3.3. Particle size, PdI and ζ-potential

The size of BSA (14.0 nm) was increased by 115.7% and 204.3% after 1- and 3-day heating, respectively. The sizes of glycated BSAs were smaller than that of heated BSAs by about 37% for 1-day heating and by about 46% for 3-day heating, respectively. Although inhibition effect in size enlargement was stronger for glycated BSAs of 3-day heating than that of 1-day heating, there was no significant difference in size observed between BSA–Xyl and BSA–Gal (Table 1). In summary, the size of BSA became large after heating. The inhibition in size enlargement was observed for glycated BSAs, with higher glycation degree and greater inhibition in size enlargement.

Table 1 Particle size, PdI and ζ-potential of BSA heated and/or glycated with Xyl/Gal^a

Sample	Heating time (days)	Size (d, nm)	PdI	ζ-Potential (mV)
a BSA (10 mg mL^−1) was heated at 60 °C without or with Xyl/Gal (0.1 M) in PBS (pH 9), and then were dialyzed against PBS (pH 7.4). Different superscript letters in the same column indicate significant (p < 0.05) differences.
BSA	0	14.0 ± 1.0^a	0.492 ± 0.022^a	−12.4 ± 0.7^a
BSA	1	30.2 ± 1.3^b	0.243 ± 0.020^b	−15.2 ± 0.4^b
BSA	3	42.6 ± 1.0^c	0.293 ± 0.029^b	−14.5 ± 0.8^bc
BSA–Xyl	1	19.0 ± 0.9^d	0.305 ± 0.039^b	−15.0 ± 0.4^b
BSA–Xyl	3	23.0 ± 0.9^e	0.399 ± 0.008^c	−13.5 ± 0.5^ac
BSA–Gal	1	18.7 ± 1.0^d	0.276 ± 0.052^b	−15.2 ± 0.1^b
BSA–Gal	3	23.6 ± 0.3^e	0.410 ± 0.006^c	−15.1 ± 0.6^b

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The PdI of BSA (0.492) was decreased by 50.6% and 40.5% after 1- and 3-day heating, respectively. The PdI of glycated BSA was larger than that of heated BSA by about 20% for 1-day heating and by about 37% for 3-day heating, respectively. Although inhibiting effect in PdI change was stronger for glycated BSAs of 3-day heating than that of 1-day heating, there was no significant difference in PdI observed between BSA–Xyl and BSA–Gal (Table 1). In summary, the PdI of BSA was decreased after heating, which was inhibited by glycation of BSA, with higher glycation degree and more inhibition.

ζ-Potential of BSA was decreased from −12.4 mV to −15.2 to −14.5 mV after 1- and 3-day heating, respectively. ζ-Potential of glycated BSAs was not significantly different from heated BSAs (Table 1). In summary, ζ-potential of BSA was decreased after heating, and was similar for both glycated BSAs and heated BSAs.

It has been reported that the size was enlarged from 9 nm to 21 nm when BSA was heated at 58 °C for 210 min, but it was from 9 nm to 7 nm when BSA was heated in presence of glucose at 37 °C for 7 weeks.²⁰ The ζ-potential of whey proteins isolate (WPI) decreased from −16 mV to −20 mV, when WPI was heated in presence of glucose at 80 °C for 2 h.¹ Generally, a high (>30 mV) or a low magnitude (<−30 mV) of ζ-potential imparts a good particle dispersion stability.⁴¹ After heating and/or glycation, the ζ-potential of BSA became lower and was toward to stable status.

3.4. MALDI-TOF MS

The m/z of heated BSA was larger (+124.50) for 1-day heating and even larger (+272.94) for 3-day heating than non-treated BSA. By subtracting the masses of corresponding heated BSAs, 714.45 and 1546.42 more masses for BSA–Xyl and 393.62 and 709.60 more masses for BSA–Gal were obtained after 1- and 3-day heating, respectively (Fig. 3). Based on the molecular weights 150.13 and 180.15 for Xyl and Gal, respectively, it could be calculated that about 5 and 10 molecules of Xyl and about 2 and 4 molecules of Gal were conjugated with BSA after 1- and 3-day heating, respectively. By calculation based on subtracting the mass of non-treated BSA, it could be about 6 and 12 molecules of Xyl and about 3 and 6 molecules of Gal were conjugated with BSA after 1- and 3-day heating, respectively. As heating time increased from 1 to 3 days, the degree of glycation (the number of Xyl/Gal conjugated with BSA) was also increased.

Fig. 3 MALDI-TOF MS spectra of BSA heated and/or glycated with Xyl/Gal. BSA (10 mg mL⁻¹) was heated at 60 °C without or with Xyl/Gal (0.1 M) in PBS (pH 9), and then were dialyzed against dH₂O.

As the charge number z is always 1, the mass-to-charge ratio (m/z) value is often considered to represent molecular mass. It has been reported that the molecules conjugated to BSA were 10 for galactose and 2 for lactose under the reaction conditions of 60 °C and 120 min.¹⁷ The molecules conjugated to whey protein were 10 for glucose and 8 for lactose under the conditions of 80 °C and 2 h.¹ The conjugated molecules of fructose were increased from 2 to 6 after heating (60 °C) time increasing from 1 h to 5 h.²⁹ Unexpectedly, an increase of MW for BSA upon thermal treatment has been observed. It might be due to the reaction of BSA with matrix small molecular substances during heating. Further study is needed to explore the possible mechanism. As mass detection range was up to 130 kDa by using Autoflex II MALDI-TOF/TOF mass spectrometer, the mass larger than 130 kDa, such as HMW species (>180 kDa) appeared on SDS-PAGE, could not be detected by this instrument.

3.5. DSC

The determined onset temperature (T_onset), peak denaturation temperature (T_d) and enthalpy of denaturation (ΔH) were obtained by DSC for BSA heated and/or glycated with Xyl/Gal (Table 2). Lower values of T_onset (52.77 °C), T_d (53.38 °C) and ΔH (4.06 kJ mol⁻¹) were observed for the heated BSA, and higher values of T_onset (∼55.66 °C), T_d (∼56.40 °C) and ΔH (∼11.31 kJ mol⁻¹) for the glycated BSA, than that non-treated BSA (T_onset 54.58 °C; T_d 55.48 °C; ΔH 12.95 kJ mol⁻¹). There was no significant difference in T_onset, T_d and ΔH observed between BSA–Xyl and BSA–Gal. In summary, T_onset, T_d and ΔH were increased after glycation of BSA.

Table 2 DSC denaturation parameters of BSA heated and/or glycated with Xyl/Gal^a

Sample	Denaturation parameters
	T onset (°C)	T d (°C)	ΔH (kJ mol^−1)
a BSA (10 mg mL^−1) was heated at 60 °C for 3 days without or with Xyl/Gal (0.1 M) in PBS (pH 9), and then were dialyzed against dH2O and further lyophilized. Different superscript letters in the same column indicate significant (p < 0.05) differences.
BSA	54.58 ± 0.19^a	55.48 ± 0.03^a	12.95 ± 0.58^a
BSA, heated	52.77 ± 0.03^b	53.38 ± 0.04^b	4.06 ± 0.88^b
BSA–Xyl	55.65 ± 0.06^c	56.65 ± 0.07^c	10.10 ± 0.25^c
BSA–Gal	55.67 ± 0.08^c	56.20 ± 0.12 ^cd	12.52 ± 2.14^ac

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It has been reported that T_d value of BSA was 55.9 °C.⁴² The heat-induced T_d decrease of BSA might be contributed by the unstable aggregates forming by non-covalent linkages. Glycation of BSA was toward to an enhanced resistance against thermal denaturation. A significant increase of T_d from 75 °C to 81 °C was observed when β-lactoglobulin heated in presence of glucose at 60 °C for 5 h.²⁹ An increase of T_d for trypsin (56.8 → 60.8 °C) and chymotrypsin (54.3 → 55.8 °C) was observed when heated in presence of glucose at 85 °C for 40 h.⁴³ The heat stability after glycation was improved because the integrity of the secondary, tertiary and quaternary structures of the unmodified protein was retained.²⁹

4. Conclusions

When BSA was heated, high-molecular-weight substance and insoluble aggregates appeared. Its shape had been changed from spheroid particle to short chain. The size of BSA became large and the PdI was decreased. All those changes were alleviated after glycation. The higher glycation degree, the more hindrance in size enlargement and PdI decrease. After heating and/or glycation, the BSA particles were toward to a good dispersion stability. The degree of glycation was increased with heating time. The resistance against thermal denaturation was enhanced after glycation. Glycation with Xyl or Gal could hinder the heat-induced BSA aggregation. The higher glycation degree, the more hindrance. Similar inhibition effects were observed for Xyl and Gal in BSA aggregation. Glycation could be considered as a control strategy in protein aggregation during thermal process.

Acknowledgements

This work was supported by the National Scientific Foundation of China (No. 31171676).

References

1. G. Liu and Q. Zhong, Food Hydrocolloids, 2013, 32, 87–96.
2. F. Taghavi, M. Habibi-Rezaei, M. Amani, A. A. Saboury and A. A. Moosavi-Movahedi, Int. J. Biol. Macromol., 2016 DOI:10.1016/j.ijbiomac.2015.12.085.
3. T. K. Girish and R. U. Prasada, J. Sci. Food Agric., 2016, 96, 4973–4983.
4. S. Awasthi and N. T. Saraswathi, Int. J. Biol. Macromol., 2016, 87, 1–6.
5. C. Iannuzzi, M. Borriello, V. Carafa, L. Altucci, M. Vitiello, M. L. Balestrieri, G. Ricci, G. Irace and I. Sirangelo, Biochim. Biophys. Acta, Mol. Basis Dis., 2016, 1862, 93–104.
6. C. Iannuzzi, G. Irace and I. Sirangelo, Front. Mol. Biosci., 2014, 1, 9.
7. D. C. Carter and J. X. Ho, Adv. Protein Chem., 1994, 45, 153–203.
8. T. Peters, All about Albumin: Biochemistry, Genetics, and Medical Applications, Academic Press, San Diego, 1996.
9. A. Bujacz, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2012, 68, 1278–1289.
10. I. Rombouts, B. Lagrain, K. A. Scherf, P. Koehler and J. A. Delcour, Sci. Rep., 2015, 5, 12210.
11. T. E. Finn, A. C. Nunez, M. Sunde and S. B. Easterbrook-Smith, J. Biol. Chem., 2012, 287, 21530–21540.
12. R. Lu, W. Li, A. Katzir, Y. Raichlin, H. Yu and B. Mizaikoff, Analyst, 2015, 140, 765–770.
13. I. Usov, J. Adamcik and R. Mezzenga, ACS Nano, 2013, 7, 10465–10474.
14. S. Xia, Y. Li, Q. Zhao, J. Li, Q. Xia, X. Zhang and Q. Huang, J. Agric. Food Chem., 2015, 63, 4080–4086.
15. A. O. Elzoghby, W. M. Samy and N. A. Elgindy, J. Controlled Release, 2012, 157, 168–182.
16. Y. Wei, L. Chen, J. Chen, L. Ge and R. Q. He, BMC Cell Biol., 2009, 10, 10.
17. A. I. Ledesma-Osuna, G. Ramos-Clamont and L. Vazquez-Moreno, Acta Biochim. Pol., 2008, 55, 491–497.
18. J. L. Liu, Q. Li, X. J. Xing and H. Jing, Shipin Anquan Zhiliang Jiance Xuebao, 2015, 6, 1819–1827.
19. V. Vetri, M. D. Amico, V. Foderà, M. Leone, A. Ponzoni, G. Sberveglieri and V. Militello, Arch. Biochem. Biophys., 2011, 508, 13–24.
20. P. Rondeau, G. Navarra, F. Cacciabaudo, M. Leone, E. Bourdon and V. Militello, Biochim. Biophys. Acta, Proteins Proteomics, 2010, 1804, 789–798.
21. C. Veerman, L. Sagis, J. Heck and E. van der Linden, Int. J. Biol. Macromol., 2003, 31, 139–146.
22. V. Vetri, F. Librizzi, M. Leone and V. Militello, Eur. Biophys. J., 2007, 36, 717–725.
23. R. Su, W. Qi, Z. He, Y. Zhang and F. Jin, Food Hydrocolloids, 2008, 22, 995–1005.
24. N. K. Holm, S. K. Jespersen, L. V. Thomassen, T. Y. Wolff, P. Sehgal, L. A. Thomsen, G. Christiansen, C. B. Andersen, A. D. Knudsen and D. E. Otzen, Biochim. Biophys. Acta, Proteins Proteomics, 2007, 1774, 1128–1138.
25. P. Rondeau, S. Armenta, H. Caillens, S. Chesne and E. Bourdon, Arch. Biochem. Biophys., 2007, 460, 141–150.
26. H. Jing and D. D. Kitts, Food Chem. Toxicol., 2004, 42, 1833–1844.
27. F. Chen, B. Li and C. Tang, J. Agric. Food Chem., 2015, 63, 3559–3569.
28. C. Schmitt, C. Sanchez, S. Desobry-Banon and J. Hardy, Crit. Rev. Food Sci. Nutr., 1998, 38, 689–753.
29. K. Broersen, A. G. Voragen, R. J. Hamer and H. H. De Jongh, Biotechnol. Bioeng., 2004, 86, 78–87.
30. C. Ren, L. Tang, M. Zhang and S. Guo, J. Agric. Food Chem., 2009, 57, 1921–1926.
31. M. S. Pinto, J. Léonil, G. Henry, C. Cauty, A. F. Carvalho and S. Bouhallab, Food Res. Int., 2014, 55, 70–76.
32. M. Bhattacharya, N. Jain and S. Mukhopadhyay, J. Phys. Chem. B, 2011, 115, 4195–4205.
33. M. Sunde and C. Blake, Adv. Protein Chem., 1997, 50, 123–159.
34. H. Schägger, Nat. Protoc., 2006, 1, 16–22.
35. N. J. Turro, X. G. Lei, K. P. Ananthapadmanabhan and M. Aronson, Langmuir, 1995, 11, 2525–2533.
36. A. Okuno, M. Kato and Y. Taniguchi, Biochim. Biophys. Acta, Proteins Proteomics, 2007, 1774, 652–660.
37. E. Saguer, P. A. Alvarez, J. Sedman and A. A. Ismail, Food Hydrocolloids, 2013, 33, 402–414.
38. C. M. Dobson, Nature, 2003, 426, 884–890.
39. F. Zhang, M. W. A. Skoda, R. M. J. Jacobs, R. A. Martin, C. M. Martin and F. Schreiber, J. Phys. Chem. B, 2007, 111, 251–259.
40. B. Bouma, L. M. J. Kroon-Batenburg, Y. P. Wu, B. Brunjes, G. Posthuma, O. Kranenburg, P. G. de Groot, E. E. Voest and M. F. B. G. Gebbink, J. Biol. Chem., 2003, 278, 41810–41819.
41. C. Freitas and R. H. Müller, Int. J. Pharm., 1998, 168, 221–229.
42. A. Michnik, K. Michalik, A. Kluczewska and Z. Drzazga, J. Therm. Anal. Calorim., 2006, 84, 113–117.
43. V. T. Pham, E. Ewing, H. Kaplan, C. Choma and M. A. Hefford, Biotechnol. Bioeng., 2008, 101, 452–459.

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