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Effects of site-specific phosphorylation on the mechanical properties of ovalbumin-based hydrogels
International Journal of Biological Macromolecules 102 (2017) 1286–1296
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effects of site-specific phosphorylation on the mechanical properties of ovalbumin-based hydrogels Zhouyi Xiong, Meihu Ma ∗ , Guofeng Jin, Qi Xu National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
a r t i c l e
i n f o
Article history: Received 14 January 2017 Received in revised form 3 May 2017 Accepted 5 May 2017 Available online 8 May 2017 Keywords: Ovalbumin-based hydrogels Site-specific phosphorylation Rheological behavior
a b s t r a c t An efficient one-step grafting approach was developed to modify ovalbumin (OVA) by phosphorylation through selective reaction with the hydroxyl group of Ser and Thr residues present in OVA. The site-specific phosphorylated conjugates were characterized by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) and the results indicated that the Ser residue could be more readily phosphorylated, and the typical phosphopeptides 264 LTEWTSSNVMEER276 and 340 EVVGSAEAGVDAASVSEEFR359 demonstrated the formation of monophosphoester. Moreover, 13 C NMR analysis showed that the CH2 of Ser acted as a hydroxyl donor to react with sodium tripolyphosphate (STPP), and the conjugates with variable phosphorylation sites could improve the weak network and the resulting poor mechanical properties of ovalbumin-based hydrogels. Furthermore, small-amplitude oscillatory measurements, creep recovery tests and texture profile analysis of hardness and stickiness indicated that phosphorylation can strengthen the intermolecular cross-linking of protein molecules and produce significant influence on the rheological behavior and texture properties, suggesting that a suitable conjugation site is essential for the best gelation properties at a different pH. The integrated results indicate that phosphorylation change significantly modify the viscoelastic and mechanical properties of OVA-based hydrogels by changing molecular dynamics upon heating. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Ovalbumin is the most abundant protein in the egg white (54%–60%), with a relative molecular mass of 45 kDa and an isoelectric point of 4.5 [1]. It is a single peptide chain composed of 385 amino acids and contains six cysteine residues with a single disulfide bond between Cys121 and Cys74. This disulfide bond has an important effect on the morphology of the aggregates formed during the process of heating [2,3]. Moreover, the buried hydrophobic groups of ovalbumin was exposed upon heating, exhibiting a “reactive” conformational state thus improving protein intermolecular interaction, which contributes greatly to the hydration of ovalbumin in emulsibility, foamability and gelation [4–6]. At sufficiently high concentrations, thermally induced partial unfolding of OVA due to denaturation and aggregation can result in the formation of a spatial gel network. The formation of a three-dimensional network can contribute to food texture and hold
∗ Corresponding author. E-mail address: [email protected] (M. Ma). http://dx.doi.org/10.1016/j.ijbiomac.2017.05.028 0141-8130/© 2017 Elsevier B.V. All rights reserved.
moisture or functional components within the spatial gel network [7]. As OVA has a weak network structure, it is essential to improve its mechanical properties for a better application of hydrogels in food, pharmaceutical and biomaterial industries [8,9]. Chemical modification is a sound process that can be applied to improve the gelation of ovalbumin. Munialo et al. [10] have reported that the OVA gelation could be improved by conjugating some of its amino groups with fructooligosaccharide via Maillard reaction. However, the glycosylation of proteins by reaction of sugar with the -amino group of lysines reduces the nutritional value of proteins, and there is a limit for the chain length of saccharides because longer and thinner strands of network could increase the mesh size consequently reducing the gel strength. Since phosphorylation is an important chemical modification to improve the mechanical properties, functional properties and bioactivity of the biomaterials such as chitosan [11],cellulose [12], starch and protein [13,14]. Therefore, phosphorylation of OVA could be a desirable way to improve the mechanical properties of gel, in which phosphate could provide negatively charged domain for protein aggregation [15].
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Elucidation of protein-polymer conjugation sites in phosphorylation is important because of their significant effects on protein gelation. Matrix-Assisted Laser Desorption Ionization-Time-ofFlight Mass Spectrometry (MALDI-TOF/MS) has provided a major tool for structural characterization of protein modifications at the molecular level [16]. MALDI-TOF/MS is a very sensitive technique with the required amount of biological and chemical sample for analysis at sub-femtomole level, while it is insensitive to the buffer solutions which are essential for protein preparation [17]. Because covalent modification of polypeptides by phosphorylation can be measured by the increased molecular mass of protein, MALDITOF/MS is extensively used in identification of phosphopeptides [18].13 C nuclear magnetic resonance spectroscopy (13 C NMR) can provide direct information about the atomic coordinate, binding site and microenvironment around carbon atoms and has become a principal experimental technique in the investigation of the molecular structure and dynamics of proteins. Therefore, the phosphorylation sites of carbon atoms in the reaction can be identified by 13 C NMR [19,20]. Thus far, there have been few studies using the combination of MALDI-TOF/MS and 13 C NMR to explore the OVA and sodium tripolyphosphate (STPP) grafting reaction sites. A facile one-step grafting approach was developed to modify the OVA with STPP by phosphorylation, and the OVA-STPP conjugate was characterized by combined MALDI-TOF/MS and 13 C NMR to identify the site-specific phosphorylation. Moreover, the correlation of the molecular dynamics of OVA-based hydrogels with the mechanical properties was clarified by rheological characterization and texture analysis. The viscoelastic properties depend on frequency response and creep-recovery properties whereas hardness and stickiness depend on mechanical deformation and fracture stress, which were investigated to explore the network structure of hydrogels. The results of this investigation provide theoretical foundation and practical references for applications of OVA-based hydrogels as a green biological polymeric material.
(1) Sample preparation:The P-OVA bands were excised from a Coomassie Brilliant Blue-stained SDS-PAGE gel and typically ingel digested according to the standard protocol as described by Shevchenko et al. [24]. (2)MS detection:The P-OVA detection was performed on a TripleTOF 5600 System (AB SCIEX, Foster City, CA) coupled with a splitless Ultra 1D Plus (Eksigent, Dublin, CA) system. The desalted peptides of N-OVA and P-OVA were dissolved in 0.1% formic acid containing 98% H2 O and 2% acetonitrile, and loaded into the C18 trap column (5 × 0.3 mm, 5 m, Agilent Technologies, Inc.) at a flow rate of 5 L/min, followed by elution from the trap column over a C18 analytic column (0.075 × 150 mm, 3 m particle size, 100 Å pore size, Eksigent) at a flow rate of 300 L/min in a 100 min gradient. The mobile phase consisted of two components: component A was 3% DMSO/97% acetonitrile with 0.1% formic acid, and component B was 3% DMSO/97% H2 O with 0.1% formic acid. The IDA (information dependent acquisition) mode was used to acquire MS/MS. The precursor ion range was set from m/z 35 to m/z 1500, and the product ion range was set from m/z 100 to m/z 1500. Survey scans were acquired in 250 ms and 20 product ion scans were collected in 100 ms/per scan. (3) Database searching: Raw data from MS were searched with ProteinPilot software against the Uniprot Gallus gallus (Chicken) database using following parameters: Digestion (Trypsin), Sample Type (identification), Special factors (phosphorylation emphasis), Cys Alkylation (Iodoacetamide), and Search Effort (Rapid) [25].
2. Materials and methods
2.4.
2.1. Materials
Natural-abundance 13 C NMR measurements were carried out with a Bruker AV400 spectrometer (Bruker AG, Karlsruhe, Germany) operating at 600 MHz and 25 ◦ C. 40 mg of N-OVA and P-OVA were dispersed in 0.6 mL of D2 O (66.7%, w/v) containing 0.1 mol/L of DSS as an internal chemical shift standard. The number of scans was 4096, the number of dummy scans was 4, the dimension of accumulation loop was 4 and pulprog was zgpg30 [26].
Sodium tripolyphosphate (STPP) was purchased from Aladdin Industrial Co. Ltd. (Shanghai, China). Q Sepharose Fast Flow was supplied by RuiDaHengHui Science & Technology Development Co. Ltd. (Beijing, China). The D2 Oused (D, 99.9%) was obtained from Sigma Chemical Co. (St. Louis, MO), Sodium 2,2-dimethyl2-silapentane-5-sulfonate (DSS) was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai China). Polyethylene glycol 8000 (PEG-8000) was made by Merck Chemicals Co., Ltd. (Shanghai China). All other chemicals used in the experiment were produced by Sinopharm ChemicalReagent Co., Ltd. (Shanghai, China), and were of analytical grade. 2.2. Preparation of ovalbumin-based hydrogels The OVA-STPP conjugates were prepared as previously reported with modifications [21]. First, ovalbumin (OVA) was extracted and purified by following Geng et al. [22]. Second, OVA was dissolved at 20 g/L in 0.1 M sodium tripolyphosphate at pH values of 5.0, 7.0, and 9.0 by adjusting the pH with 1 N HCl or NaOH, followed by lyophilization. Third, the lyophilized samples were incubated at 45 ◦ C for 12 h. Finally, the dry-heated samples were dissolved and dialyzed against deionized water for 2 days to remove free pyrophosphate and then lyophilized. The phosphorylation degrees of OVAs phosphorylated by dry-heating at three different pH values (5.0, 7.0 and 9.0) were determined to be 3.373, 3.715 and 2.491 mg/g and named as P-OVA5, P-OVA7, and P-OVA9, respectively.
The hydrogels were prepared by dissolving N- and P-OVA in deionized water at concentrations of 8% and 10% (w/v). Then the protein solutions were heated in a water bath at 90 ◦ C for 30 min to form heat-induced gels. The gels were immediately cooled to room temperature by ice-water bath for 15 min, and allowed to stand at room temperature for 12 h [23]. 2.3. MALDI-TOF mass spectroscopy
13 C
nuclear magnetic resonance spectroscopy (13 C NMR)
2.5. Texture profile analysis (TPA) The gel samples were equilibrated for 12 h at room temperature (25 ◦ C) before texture analysis. The dimension of cylindrical samples was 2.5 cm in diameter and 2.5 cm in height. The texture analysis was performed using a Model TA-XT. PLUS Texture Analyzer (Stable Micro System, Surrey, UK), with the measurement parameters as follows: pretest speed of 2 mm/s, test speed of 1 mm/s, post-test speed of 1 mm/s, target distance of 4 mm. The maximum force was recorded when the penetration distance reached 4 mm. The probe used was a P/0.5 cylindrical probe [27]. The hardness (force at the peak of the first curve) and stickiness (area of the negative force curve) were analyzed from the test curves by Texture Loader software [28]. 2.6. Rheological measurements Dynamic rheological measurements and creep-recovery tests were carried out by using an AR2000ex stress controlled rheometer
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Fig. 1. MALDI spectra of typical phosphorylated peptides 264 LTEWTSSNVMEER276 at P-OVA5 (a–c), P-OVA7 (d–e) and P-OVA9 (f–g).
(TA Instruments Ltd., Crawley, UK) with a parallel-plate geometry(diameter 40 mm; gap 1 mm).The gels were formed in the rheometer and covered with a thin layer of silicone oil to prevent evaporation. The temperature of the plate was programmed
to increase from 25 to 90 ◦ C at a rate of 2.0 ◦ C/min, and was equilibrated for 10 min at 90 ◦ C, then the plate was cooled to 25 ◦ C at a rate of 5 ◦ C/min [29].
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Small-amplitude oscillatory measurements were carried out over the range of 0.1–100 Hz. The storage modulus (G ), loss modulus (G ) and loss factor (tan ␦) were all determined as functions of frequency with a constant strain of 1%, which was within the linear viscoelastic range determined by strain sweep (at constant shear frequency of 1 Hz) [9]. Creep recovery tests were conducted at 25 ◦ C after formation of gels in the rheometer. The samples were subjected to a constant stress of 0.7958 Pa within the linear viscoelastic regime for a period of 120 s (creep time), then the applied stress was suddenly removed and sample recovery was recorded for a period of 120 s (recovery time).The recovery measurements were started immediately after the creep process [30].
3. Results and discussion 3.1. MALDI-TOF/MS analysis The possible phosphorylation sites, number of phosphate groups and chemical bond connections in tryptic peptides of P-OVA were identified by MALDI-TOF/MS. The typical phosphorylation forms of the peptides 264 LTEWTSSNVMEER276 and 340 EVVGSAEAGVDAASVSEEFR359 of P-OVA5, P-OVA7 and P-OVA9 are shown in Fig. 1 and Fig. 2. From Fig. 1a, the ion peak with m/z 846.39+ [y7-P(S)]+ and 944.36+ (y7)+ in the gure matched well with the fragmentation of peptide 264 LTEWTSSNVMEER276 of P-OVA5, confirming that the phosphate group was linked to Ser270. Theoretically, if a peptide is mono-phosphorylated, the corresponding m/z peaks with 1, 2 or 3 charges will display a mass increase of ∼98.0 Da, with m/z change of ∼98.0, ∼49.0 and ∼32.7, respectively. For dualphosphorylated peptides, the mass increase will be ∼196.0 Da. The ion peak with m/z 846.39+ [y7-P(S)]+ was found to have a mass shift of 97.97 Da from the phosphorylation site (Ser270) of the peptide 264 LTEWTSSNVMEER276 with m/z 944.36+ (y7)+ , and the M of P(S) was calculated to be ∼98 Da [(y7)+ -(y7-P(S))+ = 944.36846.39 ≈ 98], equivalent to the neutral loss of H3 PO4 . Similarly, in Fig. 1b-c, the ion peak with m/z 933.41+ [y8-P(S)]+ , 1031.37+ [y8]+ , 132.45+ [y9]+ and 1034.47+ [y9-P(T)]+ in the gure matched well with the fragmentation of peptide 264 LTEWTSSNVMEER276 of P-OVA5, confirming that the phosphate group was linked to Ser269 and Thr268. Thus, the M of P(S) or P(T) was calculated to be ∼98 Da [(y8)+ -(y8-P(S))+ ≈98 or (y9)+ -(y9-P(T))+ ≈98], equivalent to the neutral loss of H3 PO4 , respectively. From Fig. 1a-c, it can be seen that there are three possible phosphorylation sites (Ser270, Ser269 and Thr268) on this peptide 264 LTEWTSSNVMEER276 of P-OVA5. Furthermore, from Fig. 1d-g, it can be observed that there are two possible phosphorylation sites (Ser270 and Ser269) on this peptide 264 LTEWTSSNVMEER276 of P-OVA7 and P-OVA9, respectively [31]. In Fig. 2a,b, a single phosphorylation site can be observed. The ion peak with m/z 623.26+ [b6]+ , 525.26+ [b6-P(S)]+ , 995.482+ {[M]-P(S)}2+ and 1044.482+ {([M]2+ )-([M]-P(S))}2+ in the gure matched well with the fragmentation of peptide 340 EVVGSAEAGVDAASVSEEFR359 of P-OVA5, confirming that the phosphate group was linked to Ser344 and Ser 353. Meanwhile, in Fig. 2c,d, there are potential double phosphorylation sites. The ion peak with m/z 525.26+ [b6-P(S)]+ , 623.26+ [b6]+ , 654.312+ [b15-2P(S)]2+ ,752.292+ [b15]2+ ,525.26+ [b6P(S)]+ ,623.26+ [b6]+ , 795.312+ [b16]2+ and 746.352+ [b16-P(S)]2+ in the gure matched well with the fragmentation of peptide 340 EVVGSAEAGVDAASVSEEFR359 of P-OVA5, confirming that the phosphate group was linked to Ser344, Ser353 and Ser344, Ser355. Wang et al. [31] reported that in natural ovalbumin, phosphorylation has been found on two of the serine residues (Ser68 and Ser344), indicating that Ser353 and Ser355 on the 340 EVVGSAEAGVDAASVSEEFR359 peptide could be considered as
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potential novel phosphorylation sites. According to the previous study, the naturally phosphorylated peptides of OVA were considered hard to be further phosphorylated due to stronger repulsive force interaction between the natural and grafted phosphate groups, however, it was successfully phosphorylated in this study [32]. Furthermore, the effect of phosphorylation on the conformation, reaction site, and three-dimensional structure of OVA were explored.From Fig. 3a, it can be observed that the Ser269 and Ser270 were located at the random coil region of 264 LTEWTSSNVMEER276 , which were prone to a grafting reaction and resulted in phosphorylation reaction in POVA to form a phosphate ester (Fig. 1) [33]. Additionally, analysis found that phosphorylation occurred in Ser353 located on the loose structure of random coil in 340 EVVGSAEAGVDAASVSEEFR359 of P-OVA5, P-OVA7 and P-OVA9 (Fig. 3b). However, in natural Ser344, the phosphorylation site is located in the orderly ␣-helix region, and combines two glutamic acid residues of the C-terminus to form part of the recognition site for a protein kinase [1] [34]. According to the above analysis, we could exactly describe the loss process of the neutral molecules H3 PO4 of phosphorylated peptides. For example, according to the structure of the object peptides 264 LTEWTSSNVMEER276 and 340 EVVGSAEAGVDAASVSEEFR359 , a neutral loss of H3 PO4 was probably formed by loss of H in ␣-C and H2 PO4 in -C on Ser270 of 264 LTEWTSSNVMEER276 and Ser353 of 340 EVVGSAEAGVDAASVSEEFR359 .
3.2.
13 C
NMR analysis
Representative 13 C NMR spectra, the peak assignment, chemical shifts and carbon content of N- and P-OVA are shown in Fig. 4 and Table 1. A significant difference could be observed in the carbon content of OVA after the phosphorylation reaction (Table 1). Carbon contents of CH2 (Ala), ␣CH (Phe), and ␥CH2 (Lys) were increased after phosphorylation. Meanwhile, it was found that carbon contents of CH2 (Ser) and C(>C-O, >C-N,or >CH-O) were obviously decreased compared with N-OVA, which suggested the comformational change of OVA amino acid residue induced by phosphorylation reaction [12,35]. From Fig. 4, it can also be seen that the four peaks at ∼17.64, ∼21.73, ∼57.00 and ∼72.23 ppm were assigned to the chemical shift of CH2 (Ala), ␣CH (Phe),␥ CH2 (Lys) and C (>C-O, > C-N,or >CH-O) in N OVA, respectively. Similarly, the four peaks at ∼17.65, ∼21.74, ∼57.01 and ∼72.25 ppm were assigned to the chemical shift of CH2 (Ala), ␣CH (Phe), ␥ CH2 (Lys) and C (>C-O, >C-N, or >CH-O) in P-OVA, respectively, indicating that the chemical shift of the four peaks was not obviously changed by phosphorylation. Moreover, in the 13 C NMR spectrum of N-OVA and P-OVA, a peak at ∼63.00 ppm could be attributed to CH2 (Ser) group in N-OVA and P-OVA. When compared to N-OVA (63.91 ppm), the spectrum showed a chemical shift to a higher binding energy by ∼0.88 ppm in the P-OVA(63.03 ppm), indicating that the chemical environment of C atoms of CH2 (Ser) was changed after phosphorylation, further confirming the occurrence of phosphorylation reaction between the OVA and STPP, which could also support the conclusion that the major phosphate sites in OVA are CH2 group of Serine molecule [14]. The integrated results from MALDI-TOF/MS and 13 C NMR analyses indicated that the phosphate groups are introduced into OVA by phosphorylation in the presence of sodium tripolyphosphate, and the majority of phosphate sites of the ovalbumin are CH2 of serine residue side. The possible reactions between STPP and OVA are listed as follows:
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Fig. 2. MALDI spectra of typical phosphorylated peptide 340 EVVGSAEAGVDAASVSEEFR359 in P-OVA5 (a–d), P-OVA7 (e–f) and P-OVA9 (g–h).
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Fig. 3. The cartoon diagram of the phosphorylated peptides of ovalbumin (PDB IJTI), 264 LTEWTSSNVMEER276 (a) and 340 EVVGSAEAGVDAASVSEEFR359 (b).
Table 1 Peak assignment, chemical shifts and carbon content of N -and P-OVA (P-OVA5, P-OVA7 and P-OVA9) by 13 C NMR. Sample
Peak assignment
Chemical shift(ppm)
Carbon content (%)
N OVA
CH2 (Ala) ␣CH (Phe) ␥ CH2 (Lys) -CH2 (Ser) C(>C-O,>C-N, or >CH-O) CH2 (Ala) ␣CH (Phe)
17.64 21.73 57.00 63.91 72.23 17.65 21.74
4.2 4.78 4.13 5.14 81.75 6.32 8.94
P-OVA5
␥ CH2 (Lys) -CH2 (Ser) C(>C-O,>C-N, or >CH-O) CH2 (Ala) ␣CH (Phe)
57.01 63.03 72.25 17.65 21.74
7.10 2.73 76.34 7.42 8.82
P-OVA7
␥ CH2 (Lys) CH2 (Ser) C(>C-O,>C-N, or >CH-O) CH2 (Ala) ␣CH (Phe)
57.01 63.03 72.25 17.65 21.74
7.07 0.95 75.74 6.90 7.38
P-OVA9
␥ CH2 (Lys) -CH2 (Ser) C( >C-O, >C-N, or >CH-O)
57.01 63.02 72.25
6.24 1.01 78.48
3.3. Mechanical properties of OVA-based hydrogels The texture properties of N- and P-OVA gels at concentrations of 8% and 10% are shown in Fig. 5. It can be found that hardness was positively correlated with stickiness (with the correlation coefficient of 0.994** and 0.918 at concentrations of 8% and 10%, respectively), and both hardness and stickiness were increased
with increasing concentration. As shown in Fig. 5a, P-OVA7 had the highest value of gel strength, but the P-OVA5 and P-OVA9 showed a decrease in gel strength compared with N-OVA. It can also observed that the stickiness, which attribute to the combined effect of adhesive, cohesive forces, viscosity and viscoelasticity, was improved by phosphorylation at pH 7.0 (Fig. 5b) [36,37]. The distinctive crosslinking density depended on the polymerization
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Fig. 4.
13
C NMR spectra of N-OVA (a), P-OVA5 (b), P-OVA7(c), and P-OVA9 (d).
Fig. 5. Hardness (a) and stickiness (b) of N- and P-OVA hydrogels (pH 5.0, 7.0 and 9.0) at concentrations of 8% and 10%.
mechanism at different phosphorylation pH values and resulted in the different arrangement and association of OVA molecules in the gel matrix, thereby directly contributed to variations in texture properties [38]. The storage modulus (G ) represents the stored energy of a viscoelastic material resulting from the elastic deformation, while the loss modulus (G ) represents the energy loss caused by viscous flow. Fig. 6a–d shows the G and G as a function of frequency for N- and P-OVA. It can be found that OVA-based hydrogels showed a typical gel-like behavior, with higher G than G over the entire frequency range without crossover, indicating a more solid-like behavior of network structure, and both G and G increased with an increase of frequency. The N-OVA, P-OVA5 and P-OVA9 exhib-
ited a great frequency-dependence corresponding to higher slopes of the frequency sweep curve, but P-OVA7 showed a very weak dependency with smaller slopes (which were almost parallel). This indicated that P-OVA7 gel had a strong gel network structure with more rigidity than N-OVA gel system [39]. Moreover, the viscoelastic moduli of P-OVA7 gel (G = 3952 Pa, G = 1472 Pa at 1 Hz) increased significantly relative to that of N-OVA(G = 315 Pa, G = 44 Pa at 1 Hz) at protein concentration of 8%, demonstrating that OVA phosphorylated at pH 7.0 enhanced the viscoelastic behavior of OVA hydrogel by strengthening intermolecular interaction [40,41]. Furthermore, the increasing concentration of N- and P-OVA led to an obvious increase in both G and G , thus improving viscoelastic properties of each sample significantly. The G of
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Fig. 6. Changes of G (filled symbols) and G (open symbols) in frequency sweep of N OVA,P-OVA5,P-OVA7 and P-OVA9 hydrogels (a-d) at concentrations of 8% and 10%; dynamic viscosity measurements of N- and P-OVA (pH 5.0, 7.0 and 9.0) hydrogels at concentrations of 8% (e) and 10% (f) from frequency 0.1 to 10 Hz.
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Table 2 The power-law parameters fitted by frequency sweep of N- and P-OVA hydrogels. Parameter
G0
n
R2a
G0
n
R2a
NOVA (8%) NOVA (10%) POVA5 (8%) POVA5 (10%) POVA9 (8%) POVA9 (10%)
274.74 1396.5 136.82 635.1 214.23 1881.7
0.077 0.0593 0.097 0.0642 0.0963 0.0795
0.9915 0.9878 1.00 0.994 0.9999 0.9993
39.109 306.14 23.443 133.34 36.416 421.88
0.0793 0.0468 0.1099 0.0556 0.1141 0.0707
0.9516 0.9372 0.986 0.9635 0.9809 0.9752
a
Coefficient of determiatnion.
Fig. 7. The compliance of creep phase (a) and (b), creep-recovery curves(c) and (d) of N- and P-OVA (pH 5.0,7.0 and 9.0) hydrogels at concentrations of 8% and 10% from frequency 0.1 to 10 Hz.
P-OVA7 increased from 3952 Pa to 55352 Pa as the protein concentration increased from 8% to 10%, indicating its large concentration dependence. This result demonstrated that more protein molecules were involved in the gel matrices with the increase of protein concentration and formation of stronger and firmer hydrogels [42]. According to the polymer dynamics theory, as N-OVA, P-OVA5 and P-OVA9 showed a linear frequency dependence of G and G in log—log plot, the strong power-law relation between G , G and can be fitted using the following equations: G = G0 × n
(1)
G = G0 × n
(2)
where intercepts G0 and G0 are related to the strength of intermolecular interactions of network and the slopes n and n represent the stability of the protein matrix [43]. The fitted parameter values are shown in Table 2. It can be observed that the both n and n were decreased with the protein concentration increased from 8% to 10%, indicating the decrease of frequency dependency with
the reinforcement of network structures [39,40]. At the concentration of 8%, N-OVA showed better viscoelasticity than P-OVA5 and P-OVA9 with higher G0 and G0 , but P-OVA9 had higher viscoelastic modulus than N-OVA at the concentration of 10%. Fig. 6e and f shows the curves of dynamic viscosity vs. frequency for N- and P-OVA gels at concentrations of 8% and 10%. The viscosity values of OVA hydrogels decreased with increasing frequency, indicating the N-OVA and P-OVA hydrogels were non-Newtonian fluids with shear thinning behavior [44]. The slope of log for P-OVA5, POVA7 and P-OVA9 was −0.873, −0.885 and −0.887, respectively, higher than that of N-OVA (-0.834) in the concentration of 8%. This result revealed that the STPP grafted into the OVA backbone structure could be transformed to cross-linked chain, and participate in the formation of a cross-linked polymer, thus strengthening the intermolecular entanglements and showing increased resistance to frequency initially, leading to a more obviously shear-thinning behavior [45].
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The curves of compliance (J(t)) represents the deformation per unit stress for creep process are presented in Fig. 7a, b. As shown by the J(t) values, the softness of the material was reduced with elevated concentration, suggesting the formation of stronger hydrogel structure. From Fig. 7a, b, it can also be found that phosphorylation causes an decrease in compliance, indicative of the increased rigidity of network [46]. The creep-recovery curves of the N- and P-OVA gels are shown in Fig. 7c, d. The strain as a function of time was decreased with the increase of concentration, indicating the strong concentration dependence of hydrogel resistance against mechanical deformation. This suggested the entanglement of protein molecule was enhanced by the increase of OVA concentration, resulting in a harder three-dimensional network structure. The response of phosphorylated OVA showed the instantaneous reduction of elastic strain with the application of constant stress, and the maximum deformation (JMAX ) at 120 s was decreased from 7.49% for N-OVA to 4.74, 2.16 and 3.70% for P-OVA5, P-OVA7 and POVA9 at concentration 8%, and from 1.78% for N-OVA to 1.45, 0.57 and 1.32% for P-OVA5, P-OVA7 and P-OVA9 at concentration 10%, respectively, which means phosphorylation reinforced the elastic bonds of hydrogels, leading to stronger resistance to mechanical deformation [47]. Moreover, the residual deformation (J∞) caused by irreversible viscosity flow strain was reduced by phosphorylation compared with N-OVA, indicating that the network is more stable with a characteristic elastic behavior and the mechanical deformation of P-OVA hydrogels is mostly reversible. These results demonstrate that by grafting electronegative phosphate groups, the cross-linking of gels network could be enhanced via non-covalent intermolecular interaction, thus improving their mechanical properties by promoting the formation of hydrogen bond and intermolecular bridge bond between grafted phosphate groups and water molecules [45,48].
4. Conclusions A facile one-step grafting approach was developed to modify the OVA by phosphorylation using STPP. The site-specific phosphorylation was investigated by 13 C NMR and MALDI-TOF/MS, and it was found that the specific derivatization of OVA could be directed to Ser and Thr residues, and Ser residue could be more readily phosphorylated owing to the higher steric hindrance of methyl group in -C of Thr. As a result, CH2 of Ser acted as a hydroxyl donor to react with STPP and form phosphoester bonds, and the P-OVA5 with flexible protein loops had more potential phosphorylation sites. The phosphorylation showed a significant effect on molecular dynamics of OVA-STPP conjugates, specially the mechanical properties of hydrogels. The P-OVA7 formed a quasisolid viscoelastic hydrogel (G and G exceeding 104 Pa) with the highest value of hardness and stickiness (corresponding to highest viscosity), which represents a weak frequency-dependence. This result suggests that grafting electronegative phosphate groups at an appropriate level could enhance the cross-linking of hydrogels via non-covalent intermolecular interaction, thus forming a strong and firm three-dimensional network structure. However, the P-OVA5 showed lower hardness and stickiness than N-OVA with weaker viscoelastic behavior (102 < G and G < 103 ), and the increased slopes of dynamic rheological curves demonstrated a strong frequency-dependence, indicating the formation of more flexible hydrogels. Consistent with our previous study [21], the P-OVA5 had high surface hydrophobicity, resulting in a strong hydrophobic interaction between protein molecules, thus hindering their entanglement with gel matrix and causing the formation of more heterogeneous gels. Although the distinctively crosslinking density at a different phosphorylation pH could display the form
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of homogeneous or heterogeneous network, however, dynamic viscosity measurements demonstrated that all the phosphorylated OVAs had a more obviously shear-thinning behavior than N-OVA, which confirmed that phosphorylation strengthens the inter-molecular cross-linking. It can also be concluded from creeprecovery test results that phosphorylation caused a decrease in compliance and mechanical deformation to enhance the resistance against constant mechanical stress. This study demonstrates that the mechanical properties of polymeric hydrogels could be improved by phosphorylation as a natural protein-based soft material which offering a promising practical route to applicate in the food and biomedical field.
Acknowledgment This work was funded by the Modern Agro-industry Technology Research System (Project code no. nycytx-41-g22) and the National Natural Science Foundation of China (grant no. 31571784).
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effects of site-specific phosphorylation on the mechanical properties of ovalbumin-based hydrogels Zhouyi Xiong, Meihu Ma ∗ , Guofeng Jin, Qi Xu National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
a r t i c l e
i n f o
Article history: Received 14 January 2017 Received in revised form 3 May 2017 Accepted 5 May 2017 Available online 8 May 2017 Keywords: Ovalbumin-based hydrogels Site-specific phosphorylation Rheological behavior
a b s t r a c t An efficient one-step grafting approach was developed to modify ovalbumin (OVA) by phosphorylation through selective reaction with the hydroxyl group of Ser and Thr residues present in OVA. The site-specific phosphorylated conjugates were characterized by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) and the results indicated that the Ser residue could be more readily phosphorylated, and the typical phosphopeptides 264 LTEWTSSNVMEER276 and 340 EVVGSAEAGVDAASVSEEFR359 demonstrated the formation of monophosphoester. Moreover, 13 C NMR analysis showed that the CH2 of Ser acted as a hydroxyl donor to react with sodium tripolyphosphate (STPP), and the conjugates with variable phosphorylation sites could improve the weak network and the resulting poor mechanical properties of ovalbumin-based hydrogels. Furthermore, small-amplitude oscillatory measurements, creep recovery tests and texture profile analysis of hardness and stickiness indicated that phosphorylation can strengthen the intermolecular cross-linking of protein molecules and produce significant influence on the rheological behavior and texture properties, suggesting that a suitable conjugation site is essential for the best gelation properties at a different pH. The integrated results indicate that phosphorylation change significantly modify the viscoelastic and mechanical properties of OVA-based hydrogels by changing molecular dynamics upon heating. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Ovalbumin is the most abundant protein in the egg white (54%–60%), with a relative molecular mass of 45 kDa and an isoelectric point of 4.5 [1]. It is a single peptide chain composed of 385 amino acids and contains six cysteine residues with a single disulfide bond between Cys121 and Cys74. This disulfide bond has an important effect on the morphology of the aggregates formed during the process of heating [2,3]. Moreover, the buried hydrophobic groups of ovalbumin was exposed upon heating, exhibiting a “reactive” conformational state thus improving protein intermolecular interaction, which contributes greatly to the hydration of ovalbumin in emulsibility, foamability and gelation [4–6]. At sufficiently high concentrations, thermally induced partial unfolding of OVA due to denaturation and aggregation can result in the formation of a spatial gel network. The formation of a three-dimensional network can contribute to food texture and hold
∗ Corresponding author. E-mail address: [email protected] (M. Ma). http://dx.doi.org/10.1016/j.ijbiomac.2017.05.028 0141-8130/© 2017 Elsevier B.V. All rights reserved.
moisture or functional components within the spatial gel network [7]. As OVA has a weak network structure, it is essential to improve its mechanical properties for a better application of hydrogels in food, pharmaceutical and biomaterial industries [8,9]. Chemical modification is a sound process that can be applied to improve the gelation of ovalbumin. Munialo et al. [10] have reported that the OVA gelation could be improved by conjugating some of its amino groups with fructooligosaccharide via Maillard reaction. However, the glycosylation of proteins by reaction of sugar with the -amino group of lysines reduces the nutritional value of proteins, and there is a limit for the chain length of saccharides because longer and thinner strands of network could increase the mesh size consequently reducing the gel strength. Since phosphorylation is an important chemical modification to improve the mechanical properties, functional properties and bioactivity of the biomaterials such as chitosan [11],cellulose [12], starch and protein [13,14]. Therefore, phosphorylation of OVA could be a desirable way to improve the mechanical properties of gel, in which phosphate could provide negatively charged domain for protein aggregation [15].
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Elucidation of protein-polymer conjugation sites in phosphorylation is important because of their significant effects on protein gelation. Matrix-Assisted Laser Desorption Ionization-Time-ofFlight Mass Spectrometry (MALDI-TOF/MS) has provided a major tool for structural characterization of protein modifications at the molecular level [16]. MALDI-TOF/MS is a very sensitive technique with the required amount of biological and chemical sample for analysis at sub-femtomole level, while it is insensitive to the buffer solutions which are essential for protein preparation [17]. Because covalent modification of polypeptides by phosphorylation can be measured by the increased molecular mass of protein, MALDITOF/MS is extensively used in identification of phosphopeptides [18].13 C nuclear magnetic resonance spectroscopy (13 C NMR) can provide direct information about the atomic coordinate, binding site and microenvironment around carbon atoms and has become a principal experimental technique in the investigation of the molecular structure and dynamics of proteins. Therefore, the phosphorylation sites of carbon atoms in the reaction can be identified by 13 C NMR [19,20]. Thus far, there have been few studies using the combination of MALDI-TOF/MS and 13 C NMR to explore the OVA and sodium tripolyphosphate (STPP) grafting reaction sites. A facile one-step grafting approach was developed to modify the OVA with STPP by phosphorylation, and the OVA-STPP conjugate was characterized by combined MALDI-TOF/MS and 13 C NMR to identify the site-specific phosphorylation. Moreover, the correlation of the molecular dynamics of OVA-based hydrogels with the mechanical properties was clarified by rheological characterization and texture analysis. The viscoelastic properties depend on frequency response and creep-recovery properties whereas hardness and stickiness depend on mechanical deformation and fracture stress, which were investigated to explore the network structure of hydrogels. The results of this investigation provide theoretical foundation and practical references for applications of OVA-based hydrogels as a green biological polymeric material.
(1) Sample preparation:The P-OVA bands were excised from a Coomassie Brilliant Blue-stained SDS-PAGE gel and typically ingel digested according to the standard protocol as described by Shevchenko et al. [24]. (2)MS detection:The P-OVA detection was performed on a TripleTOF 5600 System (AB SCIEX, Foster City, CA) coupled with a splitless Ultra 1D Plus (Eksigent, Dublin, CA) system. The desalted peptides of N-OVA and P-OVA were dissolved in 0.1% formic acid containing 98% H2 O and 2% acetonitrile, and loaded into the C18 trap column (5 × 0.3 mm, 5 m, Agilent Technologies, Inc.) at a flow rate of 5 L/min, followed by elution from the trap column over a C18 analytic column (0.075 × 150 mm, 3 m particle size, 100 Å pore size, Eksigent) at a flow rate of 300 L/min in a 100 min gradient. The mobile phase consisted of two components: component A was 3% DMSO/97% acetonitrile with 0.1% formic acid, and component B was 3% DMSO/97% H2 O with 0.1% formic acid. The IDA (information dependent acquisition) mode was used to acquire MS/MS. The precursor ion range was set from m/z 35 to m/z 1500, and the product ion range was set from m/z 100 to m/z 1500. Survey scans were acquired in 250 ms and 20 product ion scans were collected in 100 ms/per scan. (3) Database searching: Raw data from MS were searched with ProteinPilot software against the Uniprot Gallus gallus (Chicken) database using following parameters: Digestion (Trypsin), Sample Type (identification), Special factors (phosphorylation emphasis), Cys Alkylation (Iodoacetamide), and Search Effort (Rapid) [25].
2. Materials and methods
2.4.
2.1. Materials
Natural-abundance 13 C NMR measurements were carried out with a Bruker AV400 spectrometer (Bruker AG, Karlsruhe, Germany) operating at 600 MHz and 25 ◦ C. 40 mg of N-OVA and P-OVA were dispersed in 0.6 mL of D2 O (66.7%, w/v) containing 0.1 mol/L of DSS as an internal chemical shift standard. The number of scans was 4096, the number of dummy scans was 4, the dimension of accumulation loop was 4 and pulprog was zgpg30 [26].
Sodium tripolyphosphate (STPP) was purchased from Aladdin Industrial Co. Ltd. (Shanghai, China). Q Sepharose Fast Flow was supplied by RuiDaHengHui Science & Technology Development Co. Ltd. (Beijing, China). The D2 Oused (D, 99.9%) was obtained from Sigma Chemical Co. (St. Louis, MO), Sodium 2,2-dimethyl2-silapentane-5-sulfonate (DSS) was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai China). Polyethylene glycol 8000 (PEG-8000) was made by Merck Chemicals Co., Ltd. (Shanghai China). All other chemicals used in the experiment were produced by Sinopharm ChemicalReagent Co., Ltd. (Shanghai, China), and were of analytical grade. 2.2. Preparation of ovalbumin-based hydrogels The OVA-STPP conjugates were prepared as previously reported with modifications [21]. First, ovalbumin (OVA) was extracted and purified by following Geng et al. [22]. Second, OVA was dissolved at 20 g/L in 0.1 M sodium tripolyphosphate at pH values of 5.0, 7.0, and 9.0 by adjusting the pH with 1 N HCl or NaOH, followed by lyophilization. Third, the lyophilized samples were incubated at 45 ◦ C for 12 h. Finally, the dry-heated samples were dissolved and dialyzed against deionized water for 2 days to remove free pyrophosphate and then lyophilized. The phosphorylation degrees of OVAs phosphorylated by dry-heating at three different pH values (5.0, 7.0 and 9.0) were determined to be 3.373, 3.715 and 2.491 mg/g and named as P-OVA5, P-OVA7, and P-OVA9, respectively.
The hydrogels were prepared by dissolving N- and P-OVA in deionized water at concentrations of 8% and 10% (w/v). Then the protein solutions were heated in a water bath at 90 ◦ C for 30 min to form heat-induced gels. The gels were immediately cooled to room temperature by ice-water bath for 15 min, and allowed to stand at room temperature for 12 h [23]. 2.3. MALDI-TOF mass spectroscopy
13 C
nuclear magnetic resonance spectroscopy (13 C NMR)
2.5. Texture profile analysis (TPA) The gel samples were equilibrated for 12 h at room temperature (25 ◦ C) before texture analysis. The dimension of cylindrical samples was 2.5 cm in diameter and 2.5 cm in height. The texture analysis was performed using a Model TA-XT. PLUS Texture Analyzer (Stable Micro System, Surrey, UK), with the measurement parameters as follows: pretest speed of 2 mm/s, test speed of 1 mm/s, post-test speed of 1 mm/s, target distance of 4 mm. The maximum force was recorded when the penetration distance reached 4 mm. The probe used was a P/0.5 cylindrical probe [27]. The hardness (force at the peak of the first curve) and stickiness (area of the negative force curve) were analyzed from the test curves by Texture Loader software [28]. 2.6. Rheological measurements Dynamic rheological measurements and creep-recovery tests were carried out by using an AR2000ex stress controlled rheometer
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Fig. 1. MALDI spectra of typical phosphorylated peptides 264 LTEWTSSNVMEER276 at P-OVA5 (a–c), P-OVA7 (d–e) and P-OVA9 (f–g).
(TA Instruments Ltd., Crawley, UK) with a parallel-plate geometry(diameter 40 mm; gap 1 mm).The gels were formed in the rheometer and covered with a thin layer of silicone oil to prevent evaporation. The temperature of the plate was programmed
to increase from 25 to 90 ◦ C at a rate of 2.0 ◦ C/min, and was equilibrated for 10 min at 90 ◦ C, then the plate was cooled to 25 ◦ C at a rate of 5 ◦ C/min [29].
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Small-amplitude oscillatory measurements were carried out over the range of 0.1–100 Hz. The storage modulus (G ), loss modulus (G ) and loss factor (tan ␦) were all determined as functions of frequency with a constant strain of 1%, which was within the linear viscoelastic range determined by strain sweep (at constant shear frequency of 1 Hz) [9]. Creep recovery tests were conducted at 25 ◦ C after formation of gels in the rheometer. The samples were subjected to a constant stress of 0.7958 Pa within the linear viscoelastic regime for a period of 120 s (creep time), then the applied stress was suddenly removed and sample recovery was recorded for a period of 120 s (recovery time).The recovery measurements were started immediately after the creep process [30].
3. Results and discussion 3.1. MALDI-TOF/MS analysis The possible phosphorylation sites, number of phosphate groups and chemical bond connections in tryptic peptides of P-OVA were identified by MALDI-TOF/MS. The typical phosphorylation forms of the peptides 264 LTEWTSSNVMEER276 and 340 EVVGSAEAGVDAASVSEEFR359 of P-OVA5, P-OVA7 and P-OVA9 are shown in Fig. 1 and Fig. 2. From Fig. 1a, the ion peak with m/z 846.39+ [y7-P(S)]+ and 944.36+ (y7)+ in the gure matched well with the fragmentation of peptide 264 LTEWTSSNVMEER276 of P-OVA5, confirming that the phosphate group was linked to Ser270. Theoretically, if a peptide is mono-phosphorylated, the corresponding m/z peaks with 1, 2 or 3 charges will display a mass increase of ∼98.0 Da, with m/z change of ∼98.0, ∼49.0 and ∼32.7, respectively. For dualphosphorylated peptides, the mass increase will be ∼196.0 Da. The ion peak with m/z 846.39+ [y7-P(S)]+ was found to have a mass shift of 97.97 Da from the phosphorylation site (Ser270) of the peptide 264 LTEWTSSNVMEER276 with m/z 944.36+ (y7)+ , and the M of P(S) was calculated to be ∼98 Da [(y7)+ -(y7-P(S))+ = 944.36846.39 ≈ 98], equivalent to the neutral loss of H3 PO4 . Similarly, in Fig. 1b-c, the ion peak with m/z 933.41+ [y8-P(S)]+ , 1031.37+ [y8]+ , 132.45+ [y9]+ and 1034.47+ [y9-P(T)]+ in the gure matched well with the fragmentation of peptide 264 LTEWTSSNVMEER276 of P-OVA5, confirming that the phosphate group was linked to Ser269 and Thr268. Thus, the M of P(S) or P(T) was calculated to be ∼98 Da [(y8)+ -(y8-P(S))+ ≈98 or (y9)+ -(y9-P(T))+ ≈98], equivalent to the neutral loss of H3 PO4 , respectively. From Fig. 1a-c, it can be seen that there are three possible phosphorylation sites (Ser270, Ser269 and Thr268) on this peptide 264 LTEWTSSNVMEER276 of P-OVA5. Furthermore, from Fig. 1d-g, it can be observed that there are two possible phosphorylation sites (Ser270 and Ser269) on this peptide 264 LTEWTSSNVMEER276 of P-OVA7 and P-OVA9, respectively [31]. In Fig. 2a,b, a single phosphorylation site can be observed. The ion peak with m/z 623.26+ [b6]+ , 525.26+ [b6-P(S)]+ , 995.482+ {[M]-P(S)}2+ and 1044.482+ {([M]2+ )-([M]-P(S))}2+ in the gure matched well with the fragmentation of peptide 340 EVVGSAEAGVDAASVSEEFR359 of P-OVA5, confirming that the phosphate group was linked to Ser344 and Ser 353. Meanwhile, in Fig. 2c,d, there are potential double phosphorylation sites. The ion peak with m/z 525.26+ [b6-P(S)]+ , 623.26+ [b6]+ , 654.312+ [b15-2P(S)]2+ ,752.292+ [b15]2+ ,525.26+ [b6P(S)]+ ,623.26+ [b6]+ , 795.312+ [b16]2+ and 746.352+ [b16-P(S)]2+ in the gure matched well with the fragmentation of peptide 340 EVVGSAEAGVDAASVSEEFR359 of P-OVA5, confirming that the phosphate group was linked to Ser344, Ser353 and Ser344, Ser355. Wang et al. [31] reported that in natural ovalbumin, phosphorylation has been found on two of the serine residues (Ser68 and Ser344), indicating that Ser353 and Ser355 on the 340 EVVGSAEAGVDAASVSEEFR359 peptide could be considered as
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potential novel phosphorylation sites. According to the previous study, the naturally phosphorylated peptides of OVA were considered hard to be further phosphorylated due to stronger repulsive force interaction between the natural and grafted phosphate groups, however, it was successfully phosphorylated in this study [32]. Furthermore, the effect of phosphorylation on the conformation, reaction site, and three-dimensional structure of OVA were explored.From Fig. 3a, it can be observed that the Ser269 and Ser270 were located at the random coil region of 264 LTEWTSSNVMEER276 , which were prone to a grafting reaction and resulted in phosphorylation reaction in POVA to form a phosphate ester (Fig. 1) [33]. Additionally, analysis found that phosphorylation occurred in Ser353 located on the loose structure of random coil in 340 EVVGSAEAGVDAASVSEEFR359 of P-OVA5, P-OVA7 and P-OVA9 (Fig. 3b). However, in natural Ser344, the phosphorylation site is located in the orderly ␣-helix region, and combines two glutamic acid residues of the C-terminus to form part of the recognition site for a protein kinase [1] [34]. According to the above analysis, we could exactly describe the loss process of the neutral molecules H3 PO4 of phosphorylated peptides. For example, according to the structure of the object peptides 264 LTEWTSSNVMEER276 and 340 EVVGSAEAGVDAASVSEEFR359 , a neutral loss of H3 PO4 was probably formed by loss of H in ␣-C and H2 PO4 in -C on Ser270 of 264 LTEWTSSNVMEER276 and Ser353 of 340 EVVGSAEAGVDAASVSEEFR359 .
3.2.
13 C
NMR analysis
Representative 13 C NMR spectra, the peak assignment, chemical shifts and carbon content of N- and P-OVA are shown in Fig. 4 and Table 1. A significant difference could be observed in the carbon content of OVA after the phosphorylation reaction (Table 1). Carbon contents of CH2 (Ala), ␣CH (Phe), and ␥CH2 (Lys) were increased after phosphorylation. Meanwhile, it was found that carbon contents of CH2 (Ser) and C(>C-O, >C-N,or >CH-O) were obviously decreased compared with N-OVA, which suggested the comformational change of OVA amino acid residue induced by phosphorylation reaction [12,35]. From Fig. 4, it can also be seen that the four peaks at ∼17.64, ∼21.73, ∼57.00 and ∼72.23 ppm were assigned to the chemical shift of CH2 (Ala), ␣CH (Phe),␥ CH2 (Lys) and C (>C-O, > C-N,or >CH-O) in N OVA, respectively. Similarly, the four peaks at ∼17.65, ∼21.74, ∼57.01 and ∼72.25 ppm were assigned to the chemical shift of CH2 (Ala), ␣CH (Phe), ␥ CH2 (Lys) and C (>C-O, >C-N, or >CH-O) in P-OVA, respectively, indicating that the chemical shift of the four peaks was not obviously changed by phosphorylation. Moreover, in the 13 C NMR spectrum of N-OVA and P-OVA, a peak at ∼63.00 ppm could be attributed to CH2 (Ser) group in N-OVA and P-OVA. When compared to N-OVA (63.91 ppm), the spectrum showed a chemical shift to a higher binding energy by ∼0.88 ppm in the P-OVA(63.03 ppm), indicating that the chemical environment of C atoms of CH2 (Ser) was changed after phosphorylation, further confirming the occurrence of phosphorylation reaction between the OVA and STPP, which could also support the conclusion that the major phosphate sites in OVA are CH2 group of Serine molecule [14]. The integrated results from MALDI-TOF/MS and 13 C NMR analyses indicated that the phosphate groups are introduced into OVA by phosphorylation in the presence of sodium tripolyphosphate, and the majority of phosphate sites of the ovalbumin are CH2 of serine residue side. The possible reactions between STPP and OVA are listed as follows:
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Fig. 2. MALDI spectra of typical phosphorylated peptide 340 EVVGSAEAGVDAASVSEEFR359 in P-OVA5 (a–d), P-OVA7 (e–f) and P-OVA9 (g–h).
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Fig. 3. The cartoon diagram of the phosphorylated peptides of ovalbumin (PDB IJTI), 264 LTEWTSSNVMEER276 (a) and 340 EVVGSAEAGVDAASVSEEFR359 (b).
Table 1 Peak assignment, chemical shifts and carbon content of N -and P-OVA (P-OVA5, P-OVA7 and P-OVA9) by 13 C NMR. Sample
Peak assignment
Chemical shift(ppm)
Carbon content (%)
N OVA
CH2 (Ala) ␣CH (Phe) ␥ CH2 (Lys) -CH2 (Ser) C(>C-O,>C-N, or >CH-O) CH2 (Ala) ␣CH (Phe)
17.64 21.73 57.00 63.91 72.23 17.65 21.74
4.2 4.78 4.13 5.14 81.75 6.32 8.94
P-OVA5
␥ CH2 (Lys) -CH2 (Ser) C(>C-O,>C-N, or >CH-O) CH2 (Ala) ␣CH (Phe)
57.01 63.03 72.25 17.65 21.74
7.10 2.73 76.34 7.42 8.82
P-OVA7
␥ CH2 (Lys) CH2 (Ser) C(>C-O,>C-N, or >CH-O) CH2 (Ala) ␣CH (Phe)
57.01 63.03 72.25 17.65 21.74
7.07 0.95 75.74 6.90 7.38
P-OVA9
␥ CH2 (Lys) -CH2 (Ser) C( >C-O, >C-N, or >CH-O)
57.01 63.02 72.25
6.24 1.01 78.48
3.3. Mechanical properties of OVA-based hydrogels The texture properties of N- and P-OVA gels at concentrations of 8% and 10% are shown in Fig. 5. It can be found that hardness was positively correlated with stickiness (with the correlation coefficient of 0.994** and 0.918 at concentrations of 8% and 10%, respectively), and both hardness and stickiness were increased
with increasing concentration. As shown in Fig. 5a, P-OVA7 had the highest value of gel strength, but the P-OVA5 and P-OVA9 showed a decrease in gel strength compared with N-OVA. It can also observed that the stickiness, which attribute to the combined effect of adhesive, cohesive forces, viscosity and viscoelasticity, was improved by phosphorylation at pH 7.0 (Fig. 5b) [36,37]. The distinctive crosslinking density depended on the polymerization
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Fig. 4.
13
C NMR spectra of N-OVA (a), P-OVA5 (b), P-OVA7(c), and P-OVA9 (d).
Fig. 5. Hardness (a) and stickiness (b) of N- and P-OVA hydrogels (pH 5.0, 7.0 and 9.0) at concentrations of 8% and 10%.
mechanism at different phosphorylation pH values and resulted in the different arrangement and association of OVA molecules in the gel matrix, thereby directly contributed to variations in texture properties [38]. The storage modulus (G ) represents the stored energy of a viscoelastic material resulting from the elastic deformation, while the loss modulus (G ) represents the energy loss caused by viscous flow. Fig. 6a–d shows the G and G as a function of frequency for N- and P-OVA. It can be found that OVA-based hydrogels showed a typical gel-like behavior, with higher G than G over the entire frequency range without crossover, indicating a more solid-like behavior of network structure, and both G and G increased with an increase of frequency. The N-OVA, P-OVA5 and P-OVA9 exhib-
ited a great frequency-dependence corresponding to higher slopes of the frequency sweep curve, but P-OVA7 showed a very weak dependency with smaller slopes (which were almost parallel). This indicated that P-OVA7 gel had a strong gel network structure with more rigidity than N-OVA gel system [39]. Moreover, the viscoelastic moduli of P-OVA7 gel (G = 3952 Pa, G = 1472 Pa at 1 Hz) increased significantly relative to that of N-OVA(G = 315 Pa, G = 44 Pa at 1 Hz) at protein concentration of 8%, demonstrating that OVA phosphorylated at pH 7.0 enhanced the viscoelastic behavior of OVA hydrogel by strengthening intermolecular interaction [40,41]. Furthermore, the increasing concentration of N- and P-OVA led to an obvious increase in both G and G , thus improving viscoelastic properties of each sample significantly. The G of
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Fig. 6. Changes of G (filled symbols) and G (open symbols) in frequency sweep of N OVA,P-OVA5,P-OVA7 and P-OVA9 hydrogels (a-d) at concentrations of 8% and 10%; dynamic viscosity measurements of N- and P-OVA (pH 5.0, 7.0 and 9.0) hydrogels at concentrations of 8% (e) and 10% (f) from frequency 0.1 to 10 Hz.
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Table 2 The power-law parameters fitted by frequency sweep of N- and P-OVA hydrogels. Parameter
G0
n
R2a
G0
n
R2a
NOVA (8%) NOVA (10%) POVA5 (8%) POVA5 (10%) POVA9 (8%) POVA9 (10%)
274.74 1396.5 136.82 635.1 214.23 1881.7
0.077 0.0593 0.097 0.0642 0.0963 0.0795
0.9915 0.9878 1.00 0.994 0.9999 0.9993
39.109 306.14 23.443 133.34 36.416 421.88
0.0793 0.0468 0.1099 0.0556 0.1141 0.0707
0.9516 0.9372 0.986 0.9635 0.9809 0.9752
a
Coefficient of determiatnion.
Fig. 7. The compliance of creep phase (a) and (b), creep-recovery curves(c) and (d) of N- and P-OVA (pH 5.0,7.0 and 9.0) hydrogels at concentrations of 8% and 10% from frequency 0.1 to 10 Hz.
P-OVA7 increased from 3952 Pa to 55352 Pa as the protein concentration increased from 8% to 10%, indicating its large concentration dependence. This result demonstrated that more protein molecules were involved in the gel matrices with the increase of protein concentration and formation of stronger and firmer hydrogels [42]. According to the polymer dynamics theory, as N-OVA, P-OVA5 and P-OVA9 showed a linear frequency dependence of G and G in log—log plot, the strong power-law relation between G , G and can be fitted using the following equations: G = G0 × n
(1)
G = G0 × n
(2)
where intercepts G0 and G0 are related to the strength of intermolecular interactions of network and the slopes n and n represent the stability of the protein matrix [43]. The fitted parameter values are shown in Table 2. It can be observed that the both n and n were decreased with the protein concentration increased from 8% to 10%, indicating the decrease of frequency dependency with
the reinforcement of network structures [39,40]. At the concentration of 8%, N-OVA showed better viscoelasticity than P-OVA5 and P-OVA9 with higher G0 and G0 , but P-OVA9 had higher viscoelastic modulus than N-OVA at the concentration of 10%. Fig. 6e and f shows the curves of dynamic viscosity vs. frequency for N- and P-OVA gels at concentrations of 8% and 10%. The viscosity values of OVA hydrogels decreased with increasing frequency, indicating the N-OVA and P-OVA hydrogels were non-Newtonian fluids with shear thinning behavior [44]. The slope of log for P-OVA5, POVA7 and P-OVA9 was −0.873, −0.885 and −0.887, respectively, higher than that of N-OVA (-0.834) in the concentration of 8%. This result revealed that the STPP grafted into the OVA backbone structure could be transformed to cross-linked chain, and participate in the formation of a cross-linked polymer, thus strengthening the intermolecular entanglements and showing increased resistance to frequency initially, leading to a more obviously shear-thinning behavior [45].
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The curves of compliance (J(t)) represents the deformation per unit stress for creep process are presented in Fig. 7a, b. As shown by the J(t) values, the softness of the material was reduced with elevated concentration, suggesting the formation of stronger hydrogel structure. From Fig. 7a, b, it can also be found that phosphorylation causes an decrease in compliance, indicative of the increased rigidity of network [46]. The creep-recovery curves of the N- and P-OVA gels are shown in Fig. 7c, d. The strain as a function of time was decreased with the increase of concentration, indicating the strong concentration dependence of hydrogel resistance against mechanical deformation. This suggested the entanglement of protein molecule was enhanced by the increase of OVA concentration, resulting in a harder three-dimensional network structure. The response of phosphorylated OVA showed the instantaneous reduction of elastic strain with the application of constant stress, and the maximum deformation (JMAX ) at 120 s was decreased from 7.49% for N-OVA to 4.74, 2.16 and 3.70% for P-OVA5, P-OVA7 and POVA9 at concentration 8%, and from 1.78% for N-OVA to 1.45, 0.57 and 1.32% for P-OVA5, P-OVA7 and P-OVA9 at concentration 10%, respectively, which means phosphorylation reinforced the elastic bonds of hydrogels, leading to stronger resistance to mechanical deformation [47]. Moreover, the residual deformation (J∞) caused by irreversible viscosity flow strain was reduced by phosphorylation compared with N-OVA, indicating that the network is more stable with a characteristic elastic behavior and the mechanical deformation of P-OVA hydrogels is mostly reversible. These results demonstrate that by grafting electronegative phosphate groups, the cross-linking of gels network could be enhanced via non-covalent intermolecular interaction, thus improving their mechanical properties by promoting the formation of hydrogen bond and intermolecular bridge bond between grafted phosphate groups and water molecules [45,48].
4. Conclusions A facile one-step grafting approach was developed to modify the OVA by phosphorylation using STPP. The site-specific phosphorylation was investigated by 13 C NMR and MALDI-TOF/MS, and it was found that the specific derivatization of OVA could be directed to Ser and Thr residues, and Ser residue could be more readily phosphorylated owing to the higher steric hindrance of methyl group in -C of Thr. As a result, CH2 of Ser acted as a hydroxyl donor to react with STPP and form phosphoester bonds, and the P-OVA5 with flexible protein loops had more potential phosphorylation sites. The phosphorylation showed a significant effect on molecular dynamics of OVA-STPP conjugates, specially the mechanical properties of hydrogels. The P-OVA7 formed a quasisolid viscoelastic hydrogel (G and G exceeding 104 Pa) with the highest value of hardness and stickiness (corresponding to highest viscosity), which represents a weak frequency-dependence. This result suggests that grafting electronegative phosphate groups at an appropriate level could enhance the cross-linking of hydrogels via non-covalent intermolecular interaction, thus forming a strong and firm three-dimensional network structure. However, the P-OVA5 showed lower hardness and stickiness than N-OVA with weaker viscoelastic behavior (102 < G and G < 103 ), and the increased slopes of dynamic rheological curves demonstrated a strong frequency-dependence, indicating the formation of more flexible hydrogels. Consistent with our previous study [21], the P-OVA5 had high surface hydrophobicity, resulting in a strong hydrophobic interaction between protein molecules, thus hindering their entanglement with gel matrix and causing the formation of more heterogeneous gels. Although the distinctively crosslinking density at a different phosphorylation pH could display the form
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of homogeneous or heterogeneous network, however, dynamic viscosity measurements demonstrated that all the phosphorylated OVAs had a more obviously shear-thinning behavior than N-OVA, which confirmed that phosphorylation strengthens the inter-molecular cross-linking. It can also be concluded from creeprecovery test results that phosphorylation caused a decrease in compliance and mechanical deformation to enhance the resistance against constant mechanical stress. This study demonstrates that the mechanical properties of polymeric hydrogels could be improved by phosphorylation as a natural protein-based soft material which offering a promising practical route to applicate in the food and biomedical field.
Acknowledgment This work was funded by the Modern Agro-industry Technology Research System (Project code no. nycytx-41-g22) and the National Natural Science Foundation of China (grant no. 31571784).
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