Protein Separation Coacervation with Carboxymethyl Cellulose of

Mar 22, 2018 - Unchanged chymotrypsin inhibitory activity (CIA) indicated that BBI had negligible contribution to protein recovery and trypsin inhibit...
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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Protein Separation Coacervation with Carboxymethyl Cellulose of Different Substitution Degree: Noninteracting Behavior of Bowman− Birk Chymotrypsin Inhibitor Xingfei Li, Jie Long, Yufei Hua,* Yeming Chen, Xiangzhen Kong, and Caimeng Zhang State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, People’s Republic of China S Supporting Information *

ABSTRACT: We first observed that protein/polysaccharide interaction exhibited noninteracting behavior which makes Bowman−Birk chymotrypsin inhibitor (BBI) always free of complexation, being separated from another protein with similar isoelectric points, Kunitz trypsin inhibitor (KTI). Turbidity titrations showed that the electrostatic attractions were much stronger between KTI/BBI (KBi) and carboxymethyl cellulose of higher substitution degree. Unchanged chymotrypsin inhibitory activity (CIA) indicated that BBI had negligible contribution to protein recovery and trypsin inhibitory activity (TIA). Tricine−SDS−PAGE revealed that, at r = 20:1−2:1, unbound BBI was left in the supernatant when bound KTI transferred into precipitates, even if there was excess negative charge. Thus, purified KTI or BBI was achieved easily at the given conditions. The noninteracting behavior of BBI was further confirmed by ITC, where the binding enthalpy of BBI to CMC was negligible compared with the high binding affinity (Kb) of KTI. This work will be beneficial to protein purification based on protein− polysaccharide coacervation. KEYWORDS: protein separation, carboxymethyl cellulose, KBi, isothermal titration calorimetry, binding affinity

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tion,7,10 ionic strength, etc.6,11 A common rule is that when charge neutrality of the biopolymer system is allowed to be reached, maximum protein/polysaccharide insoluble complexes (pHmax) are generated, where the preferentially bound proteins can be easily and maximally separated. For instance, at the optimal pH, a selective coacervation was formed between lactoferrin and isoform A of β-lactoglobulin (β-LG);12 the selective complex behavior between soybean whey proteins and ι-carrageenan was used for protein separation.13 On the other hand, only soluble complexes may be obtained at a wide pH window when a system satisfies high biopolymer mass ratios or high salt concentration, where the charge of the complex moves from neutrality without intensified binding, and coacervate can redissolve without precipitation.14 This will also be helpful to protein separation since that will keep one protein free of complexation when another protein binds strongly to polysaccharide. Generally, the proteins carry heterogeneous charge patches on their surfaces, which can provide a local binding region when an opposite charged molecule is close. The onset of biopolymer coacervation occurring at pHc (soluble complex) is also due to the presence of “charge patch”, which is independent of salt concentration and pH, and has been believed to be related to binding affinity. Dubin and co-workers found that binding of β-LG with poly(diallyldimethylammonium chloride) with a lower pHc was

INTRODUCTION Proteins and polysaccharides play important roles in the food structure, texture, and stability of solutions, gels, emulsions, and foams. Interaction between proteins and polysaccharides may result in four consequences: their cosolubility, soluble complexes, incompatibility, or complex coacervation. Cosolubility exists when the bulk concentration of biopolymer is below the cosolubility threshold; while the soluble complex is formed when the pH is close to the isoelectric point of protein, which induces local electrostatic complex formation. On the other hand, protein and polysaccharide can also be incompatible: those mixtures generally lead either to phase separation through thermodynamic incompatibility (biopolymers being mutually segregating one from the other) or to complex coacervation (biopolymers associate, excluding solvent from their vicinity).1−4 Among them, the associative separation or complex coacervation is a manifestation of combined effects of several weak attractive interactions, such as hydrogen bonding, hydrophobic, electrostatic and van der Waals forces, etc.4,5 Thus, the careful regulation of phase separation between these biopolymers would allow removal or recovery of target proteins selectively without affecting their structure and function intact under either type of separation.6−8 Selective coacervation is a reversible equilibrium where only one protein participates in intermolecular interaction at a narrow complexation window, and another protein still remain in unbound supernatant, a consequence of strong binding vs weak binding. Evidence from the related system of mixed multiprotein strongly suggests that selectivity of coacervation follows very different mechanisms, with significant dependences on protein/polymer pairs,9 polymer surface charge distribu© XXXX American Chemical Society

Received: Revised: Accepted: Published: A

January 6, 2018 March 20, 2018 March 22, 2018 March 22, 2018 DOI: 10.1021/acs.jafc.8b00091 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

(Shanghai, China). All other reagents were of analytical grade and used without purification. Preparation of Soybean Whey. Soybean whey was prepared according to our previously published work.28 Briefly, 300 g of soybean meal was ground and added to 3000 mL of deionized water, and then this suspension was adjusted to pH 7.0 and well-stirred for 30 min. After removal of undissolvable residue by gauze filter, the suspension was centrifugated (10000g, 30 min) to collect supernatant. The supernatant was adjusted to pH 4.5 with 2 M hydrochloric acid, and stirring was carried out for 30 min before centrifugation at 10000g, 30 min. Then, the supernatant was collected after removal of the protein curd and adjusted to pH 8.0 with 1.0 M NaOH before centrifugation (10000g, 30 min) again. Then, the prepared soybean whey was collected as supernatant and stored at 4 °C containing 0.02% sodium azide. Preparation of KTI/BBI Mixtures (KBi) and BBI. Preparation of KBi. KBi was prepared according to a salting-out method reported in our previous work.9 Briefly, 729 g of solid ammonium sulfate was added slowly into 3.0 L soybean whey solutions (adjusted to pH 4.5 in advance) with rapid stirring at 25 °C. After reaching saturation of 40% ammonium sulfate, the suspension was centrifuged at 10000g for 30 min to collect the precipitate. Then, the precipitate was dispersed in 100 mL of deionized water, and adjusted to pH 7.0 with stirring until it completely dissolved. Then, the above solution was dialyzed for 48 h against 0.02% sodium azide with gentle stirring at 4 °C, and lyophilized. At last, the freeze-dried sample was stored at −20 °C for further experiments. KBi extracts were found to have the following proximate composition: proteins, 94.3% (w/w); moisture, 3.3% (w/ w); and ash, 2.4% (w/w). Preparation of BBI. The BBI was prepared by our laboratory as follows.13 Briefly, 0.1% (w/v) KBi and 0.1% (w/v) ι-carrageenan (ιCG) solutions were adjusted to pH 7.0 separately; then KBi/ι-CG mixtures were obtained at a mass ratio of 5:1, pH 3.8. After centrifugation (5000g, 30 min) to remove precipitate, the supernatant was collected. The crude BBI containing solutions were then treated with ultrafiltration (100 kDa cutoff) to collect filtrate, and dialyzed for 48 h containing 0.02% sodium azide. After freeze-drying, the lyophilized powder of BBI was stored at −25 °C for the next experiment. Turbidimetric Titration. Stock solutions of protein and polysaccharide were prepared separately by dissolving 0.1% (w/w) of KBi or CMC of different DS into a corresponding volume of deionized water, and well-stirred for 2 h at room temperature (25 ± 1 °C). The stock solutions were adjusted to a pH of 6.5, separately. After mixing protein and polysaccharide stock solutions together with the desired mass ratios (20:1, 15:1, 10:1, 5:1, and 2:1), the protein concentration was 0.952, 0.937, 0.909, 0.833, and 0.667 g/mL, respectively, and the polysaccharide concentration was 0.048, 0.063, 0.091, 0.167, and 0.333 g/mL, respectively. The turbidity experiment was carried out by adding different concentrations (1.0, 0.5, and 0.25 M) of hydrochloric acid to decrease the pH values gradually to a final pH 2.5 by decrement of ∼0.1 pH, while simultaneously monitoring changes in pH using a carefully calibrated pH-meter (Mettler Toledo Delta 320, Zurich, Switzerland). At the same time, the turbidity value was recorded at 600 nm with a spectrophotometer (Unocal, UV 2000, Japan). The different critical pH points (pHc, pHφ, and pHmax) following turbidity change were determined based on Mattison et al.29 Homogeneous protein and polysaccharide solutions were used as controls. All measurements were made in triplicate. Zeta Potential. The solutions of 0.1% (w/w) CMC and 0.1% (w/ w) KBi with pH values from 7.0 to 2.5 were prepared separately. Then, the zeta potential was measured at 25.0 ± 0.1 °C using a Zetasizer Nano ZS instrument (Malvern, U.K.) equipped with a 633 nm laser. All measurements were made in triplicate. Protein Content and Protein Distribution. After coacervation, protein content was calculated by difference in protein concentration between initial solution and final supernatant, using a reported bicinchoninic acid (BCA) method.30 Briefly, 100 μL of appropriate dilute sample was added into a test tube, and then 2 mL of BCA

stronger than that of bovine serum albumin (BSA), resulting from the negative “charge patch” on β-LG, absent for BSA, as visualized via computer modeling;7 while higher binding affinity of BSA was observed for coacervation with hyaluronic acid, arising from its more concentrated positive domain.15 Therefore, proteins with similar isoelectric points can also be separated from each other due to the subtle changes of their surface charge distribution at the beginning of complex formation. Carboxymethyl cellulose (CMC) is one of the most important anionic nontoxic water-soluble polysaccharides widely used in food, pharmaceutical, and medical industry.16,17 Degree of substitution (DS) of CMC reflects its charge distribution or charge density, and thereof its interaction with proteins. CMC with higher DS was found to bind strongly with protein molecules, such as CMC/lysozyme,18 CMC/ovalbumin,19 CMC/CTAB systems,20 etc. Interestingly, CMC has been used for affinity precipitation of target proteins from other components. For instance, Lali et al. found that up to 23-fold purification of the lactate dehydrogenase from crude extract was obtained after careful control of nonspecific interaction conditions.21,22 However, there is still limited research on the protein/CMC of different DS with regard to selective coacervation. Kunitz trypsin inhibitor (KTI, 20 kDa) and Bowman−Birk protease inhibitor (BBI, 7.9 kDa) belong to the serine protease inhibitor family, which always coexist in soybean whey after acid protein precipitates.23 These inhibitors are considered as antinutritional factors, due to their enrichment in soy-derived food products and ability to reducing the digestibility of crude protein by inhibiting the digestive proteases.24 However, different types of BBI, BBI concentrate and purified BBI, have been reported to have therapeutic value in both in vivo and in vitro experiments. For example, epidemiological studies indicate that BBI has a broad spectrum of cancer-protective activities, shown to be a valid suppressor of carcinogenesis in a human phase IIa clinical trial.25−27 In this work, the behavior of protein/polysaccharide coacervation and protein separation as affected by polymer degree of substitution were systematically studied using KTI/ BBI model protein pairs, as they were given the very similar pI values (4.5 vs 4.2). Meanwhile, the efficient separation of BBI can provide us a good source for the further studies of its anticarcinogenic property. During coacervation, protein recovery and trypsin/chymotrypsin inhibitory activity were monitored to check the protein distribution as a function of DS of CMC (0.7, 0.9, 1.2), pH, and biopolymer mass ratios. Isothermal titration calorimetry (ITC) was employed to compare the binding thermodynamic parameters for the coacervation of the two proteins. Examination of the CMC/ protein system can lead to a better understanding about how to control coacervation conditions to purify the target protein when binding to weak polysaccharides.

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MATERIALS AND METHODS

Materials. Hexane-defatted soybean meal was purchased from Shandong Wonderful Industrial & Commercial Co. Ltd. (Dongying, Shandong, China), containing a protein content of 52.4% (N × 6.25, dry base). Carboxymethyl cellulose (CMC, molecular weight (MW) ∼ 250 kDa) with substitution degree (DS) of 0.7, 0.9, and 1.2 was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). KTI (T2327) and xanthan gum (XG) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Arabic gum (GA) and sodium alginate (NaA) were provided by Sinopharm Chemical Reagent Co. B

DOI: 10.1021/acs.jafc.8b00091 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Turbidity curves for the KBi/CMC system (Cp = 0.10%) as a function of pH at different mass ratios for (A) KBi/CMC 0.7, (B) KBi/CMC 0.9, and (C) KBi/CMC 1.2; (D) Critical pH values (pHc, pHφ and pHmax) of the given three KBi/CMC systems. pHc and pHφ were determined graphically as the intersection of two tangents to the curve, where pHmax corresponded to the maximum optical density at 600 nm. reagent was added and mixed thoroughly. The above mixture was incubated at 37 °C for 30 min and cooled to room temperature. Changes in absorbance at 562 nm were monitored with a spectrophotometer (Unocal, UV 2000, Japan) using distilled water as a control. All measurements were made in triplicate. After coacervation with CMC, protein distribution of coacervate and supernatant was analyzed by tricine−SDS−PAGE.31 Coacervate and supernatant were dissolved in loading solution and then reduced with 2% (v/v) 2-mercaptoethanol in 100 °C boiling water for 3−5 min. 10 μL of supernatant from each sample was loaded per well. Protein molecular weight markers were included in each gel. Then, the gels were run in an electrophoresis system at a constant voltage of 30 V for the stacking gel (4%) and 100 V for the separation gel (16%). After completion, each gel was stained with 0.05% Coomassie Brilliant Blue G-250. Trypsin and Chymotrypsin Inhibitory Activity. Trypsin inhibitory activity (TIA) was determined according to the method of Liu et al. with slight modifications.32 Briefly, using 0.04% (w/v) Nαbenzoyl-L-arginine 4-nitroanilide hydrochloride (BAPA) as the substrate, the standard reaction was started by adding 2 mL of 0.01% (w/v) trypsin, while the inhibitor reaction was started by adding another 1 mL of diluted sample. 10 min later, the reaction was terminated by adding 1 mL of 30% (w/v) acetic acid. Similarly, chymotrypsin inhibitor activity (CIA) was determined by mixing αchymotrypsin (enzyme) and N-benzoyl-L-tyrosine p-nitroanilide (BTpNA, substrate) to begin standard reaction; then 1 mL of diluted sample was used for inhibitor reaction.33 The reaction absorbance at 410 nm (OD410 for TIA) and 385 nm (OD385 for CIA) was monitored using an ultraviolet−visible spectrophotometer (Shimadzu, Japan). Isothermal Titration Calorimetry (ITC). An isothermal titration calorimeter (Microcal PEAQ-ITC, Malvern, U.K.) was used to measure thermodynamics. 0.633 mM KTI (12.66 mg/mL) or BBI

(5.0 mg/mL) solution was made using pH 3.5, 20 mM citric acid− sodium citrate buffer, and 0.8 × 10−3 mM CMC 0.7, 0.9, and 1.2 were dissolved in pH 3.5, 20 mM citric acid−sodium citrate buffer. All solutions were filtered through 0.22 μm Millipore membrane, and then placed in the degassing device for 10 min. After stabilizing instrument at 25 °C, the CMC (0.7, 0.9, or 1.2) solution in the 200 μL reaction cell was titrated with 20 successive 2 μL injections of KTI or BBI solution. Each addition has an interval of 90 s between consecutive injections. The stirring rate was set at 1000 rpm throughout the experiments. The heats of dilution from the blank titration of KTI or BBI into the buffer solution were subtracted from the raw ITC data. One-site independent binding models were used to fit the binding isotherms. The best fit curves yield the thermodynamic parameters including reaction enthalpy (ΔH), binding affinity constant (K), and stoichiometry (n). The free energy change (ΔG) was calculated from the equation ΔG = −RT ln K. The entropy (ΔS) was calculated from ΔG = ΔH − TΔS. Removal of Polysaccharides by Ultrafiltration. To prepare separated KTI and BBI components, KBi/CMC 0.7 system was selected to collect the coacervate at r = 5:1 pH 3.65 by centrifugation (5000g, 20 min). At the same time, the BBI containing supernatants were collected and adjusted to pH 7.0. Ultrafiltration was carried out by using an ultrafiltration apparatus with poly(ether sulfone) membrane (100 kDa cutoff), the proteins permeated into the filtrate, and polysaccharides remained in the retentate. The contents of proteins and polysaccharides were calculated using the BCA method and the phenol−sulfuric acid method,34 respectively. SEC-HPLC Analysis. SEC-HPLC was used to analyze the protein component of KBi and the purity of separated KTI or BBI.28 Briefly, a liquid chromatography system (1260 infinity, Aglient Technologies, USA) connected to a TSK gel 2000 SWXL column (Tosoh, Tokyo, Japan) was equilibrated and run with ultrapure water containing 45% C

DOI: 10.1021/acs.jafc.8b00091 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. (A) Zeta potential of CMC solution with different DS and KBi solutions; (B) protein recovery, (C) trypsin inhibitory activity, and (D) chymotrypsin inhibitory activity of three KBi/CMC systems at different mass ratios (r). Capital letters: statistical significance in the groups. Lowercase letters: statistical significance between groups. (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid. The flow rate was 0.5 mL/min, and the chromatographic peak was monitored at 240 nm with 20 μL injections. Statistical Analysis. All the measurements were performed in triplicate and presented as the mean with standard deviation (mean ± SD) for parallel samples. Statistical significance was considered as p < 0.05 and was determined using the SPSS 13.0 statistical analysis program.

significant effect on the turbidity measurement in the pH range 4.0−5.5.38 The shift of pHφ and pHmax to lower pH values was obvious observed as decrease in r, because more protein charge was required to reach charge neutrality when excess negative charge was available. At a range of pHmax − pHφ, the charge of the complexes approaching zero, the finding that pHφ and pHmax are dependent on r can be further described by charge neutralization as given by the equation nZpr = −Zps (1),39 where n is mean number of bound proteins per polymer chain, Zpr is the net polymer charge of the protein, and Zps is the net polymer charge of polysaccharide, respectively. Neither Zpr nor Zps varied significantly with pH close to pHmax (as seen in Figure 2A), and the width of pHmax − pHφ and the slope of d[pHmax − pHφ]/d(pH) in Figure 1 corresponded to the change in n as a function of pH. For example, at pH 3.5, nearly maximum coacervates were obtained for three CMC/protein systems, where ZCMC0.7 ≈ ZCMC0.9 < ZCMC1.2, thus, the number of bound proteins (n) for CMC 0.7 and CMC 0.9 might be smaller than that for CMC 1.2. However, the quantitative relation between the biopolymer net charge and protein binding numbers is complicated because the binding affinity of KBi depends not simply on biopolymer charge density but also on other factors, such as flexibility or chain rigidity. To some extent, the maximum turbidity and pHmax for three CMC/KBi systems might be a simple measure of protein affinity. In the absence of CMC, protein solutions remained

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RESULTS AND DISCUSSION Phase Behavior of KBi/CMC Complex Coacervation. Protein−polysaccharide coacervation undergoes a critical phase transition from soluble complex to insoluble coacervate, and a subsequent maximum formation of coacervates, corresponding to the three critical pH values, pHc, pHφ, and pHmax, respectively. As seen in Figure 1, for a fixed system, such as KBi/CMC 0.7, pHc occurred around pH 5.2, higher than the isoelectric points of KTI (4.5) and BBI (4.2), a consequence of protein charge heterogeneity or called “charged patches”, opposite in sign to the net protein average (Zpr), which can provide binding sites for the polymers with the same charge sign as Zpr.35,36 The nonsignificant difference in pHc among given mass ratios, r = 20:1−2:1, indicated the independence of protein local charge at a fixed ionic strength.36,37 The degree of substitution (DS) of CMC was found to have little effect on the position of pHc (Figure 1D) in the present case. Duhoranimana et al. observed the similar trend that CMC types have no D

DOI: 10.1021/acs.jafc.8b00091 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 3. Tricine−SDS−PAGE analysis of coacervates and supernatants obtained at pHmax with different mass ratios (r) for three KBi/CMC systems: (A) KBi/CMC 0.7, pHmax was 4.27 (20:1), 4.23 (15:1), 4.05 (10:1), 3.65 (5:1), 2.85 (2:1); (B) KBi/CMC 0.9, pHmax was 4.22 (20:1), 4.12 (15:1), 3.85 (10:1), 3.77 (5:1), 2.91 (2:1); (C) KBi/CMC 1.2, pHmax was 4.38 (20:1), 4.28 (15:1), 4.23 (10:1), 3.85 (5:1), 3.27 (2:1).

coacervation was mainly derived from KTI, not BBI. Additionally, at a fixed r, the protein recovery and TIA loss were much bigger for CMC 1.2 than those of CMC 0.7 and 0.9, which might be related to the higher charge density of CMC 1.2, which enhanced the formation of complexes. Protein Distribution after Coacervation with CMC of Different DS. To study protein distribution, tricine−SDS− PAGE analysis was used to monitor the shift of KTI and BBI when binding with CMC, as presented in Figure 3. As mentioned above, the protein recovery and TIA loss reached an equilibrium at r = 5:1 and 2:1, and as a consequence, the protein bands of KTI disappeared in the supernatant, while the enriched single BBI bands were first observed at these ratios, shown in Figure 3A, lanes 5, 6; Figure 3B, lanes 4, 5; and Figure 3C, lanes 5, 6. Interestingly, coacervates contained only KTI bands; no BBI bands were enriched for all the given ratios (Figure 3, coacervates), even though the excess negative charges was available at r = 2:1. In our previous work, we found that the transfer speed of KTI into coacervates determined the occurrence ratios for purified BBI.9,40 At very low mass ratio (r = 2:1), the BBI bands entirely disappeared, partially disappeared, or did not disappear in the supernatant, depending on the polymer charge density.9 Thus, if the KTI could be fully transferred into cocacervates, the purified BBI may also be obtained for gum arabic (having the lowest charge density among studied polysaccharides). This was just the very different point of CMC, which cannot take any BBI into coacervates even though all KTI transferred into coacervates. Therefore, the surprising results of BBI shifting may reflect another very different binding behavior: noninteracting behavior of BBI when complexing with CMC, a weak polyelectrolyte. Tricine−SDS−PAGE results also indicated that the precedence for coacervation with CMC was KTI, not BBI. Thus, the purified BBI can only be obtained after complete precipitation of KTI from KBi mixture. It was found that only BBI bands remained in the supernatant at r = 5:1, 5:1, and 10:1 for CMC 0.7, 0.9, and 1.2, respectively. The transfer speed of KTI from

very low optical density in the whole pH range, indicating their good solubility in acid as well as alkali conditions; however, in the presence of CMC, pH dependent protein/polysaccharide coacervation was observed. Large values of maximum turbidity or pHmax for KBi/CMC 0.7 and KBi/CMC 1.2 (as shown in Figure 1) might be related to a higher protein affinity: the formation of coacervates was more intensive, i.e., showed higher turbidity; the required number of proteins to compensate charge neutrality were more readily available, i.e., at higher pH. Such stronger protein binding also corresponded to large values of n at a given protein/biopolymer stoichiometry. The Protein Recovery, TIA and CIA. As shown in Figure 2A, the zeta potential of CMC underwent a decrease with the pH changes, varying between ∼−5 mV and −50 mV at pH 2.0−5.0, which kept correspondence with the low dissociation constants of the carboxyl groups (−COOH, pKa ≈ 2.238). Zeta potential of KBi proteins was about −20 mV and +20 mV with the deviation of isoelectric points (around pH 4.6). Considering the similarity and difference in CMC 0.7, 0.9, and 1.2, the consequence of CMC/KBi electrostatic interaction during the given pH ranges will be accompanied by similarities and differences in protein recovery and TIA/CIA activities. As seen in Figure 2B, for the given CMC/KBi systems, protein recovery increased gradually with the decrease in mass ratio, r, reaching maximum at r = 2:1 (∼45−50%). There was no significant difference in protein recovery between r = 2:1 and r = 5:1 (p > 0.05). Here, the loss in TIA and CIA will help us to investigate distribution of KTI or BBI during coacervation. Obviously, TIA decreased significantly as r decreased (p < 0.05), until r = 5:1, where a relative equilibrium of residual TIA was obtained, corresponding to a sharp increase in protein recovery (Figure 2B). Conversely, unchanged CIA (p > 0.05) with KBi accumulation was observed for all the given r, indicating that BBI could either be out of coacervation or only form soluble complex. Therefore, although binding with CMC of different charge density, the protein contribution to E

DOI: 10.1021/acs.jafc.8b00091 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 4. Isothermal calorimetry titration raw data for KTI/CMC 0.7 (A), KTI/CMC 0.9 (B), KTI/CMC 1.2 (C), and BBI/CMC 0.7 (D). Protein titrants, concentration 0.633 mM, were added to 0.8 × 10−3 mM CMC. Incremental volume of titrant: 2 μL; with an interval of 90 s between injections.

CMC 1.2 coacervates were greater than those of CMC 0.7 coacervates. Thermodynamic parameters of KTI/CMC complex using the one-site binding model are shown in Figure 5 and Table 1. The negative enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG) suggested that KTI−CMC coacervation was a spontaneous exothermic process controlled by enthalpy changes. It was found that the affinity constant (K) increased with the increase in CMC 0.7, CMC 0.9, and CMC 1.2 (Table 1). These results were consistent with the protein recovery, TIA loss, and tricine−SDS−PAGE results (Figure 3), and further confirmed that higher polymer charge density might promote complex coacervation of the KTI/CMC. The higher binding number (n) of KTI/CMC 1.2 also satisfied the prediction described from turbidity results, since there was almost negligible contribution of BBI to turbidity curves (see Figure S1). However, a very different ITC behavior for the BBI/CMC system was observed, as shown in Figure 4D. Very limited raw endothermic isothermal curves with the injection of BBI molecules into cells were found for BBI/CMC 0.7. After integration of diluted heat, the binding enthalpy of BBI/CMC 0.7 is shown in Figure 5D. Also, the limited enthalpy changes (−ΔH) ranged from +0.1 to −0.1 kcal/mol, indicating that there was very little binding thermodynamics between BBI and CMC molecules. The same trends were also observed for CMC 0.9 and CMC 1.2 (data not shown). In our previous work, the binding behavior of BBI to sulfated polysaccharide− ι-carrageenan had been carefully studied,42

KBi became faster with the increase in DS, justifying the contribution of KTI to protein recovery and TIA activity loss, and the contribution of BBI to unchanged CIA activity. Conversely, purified KTI was easily attained at each given r (20:1−2:1, coacervates seen in Figures 3A, 3B, and 3C), since the BBI was always out of coacervation. Isothermal Titration Calorimetry (ITC). ITC measurements were conducted to quantitatively compare the thermodynamics of CMC binding of KTI versus BBI. Raw curves from ITC measurements resulting from injections of KTI (or BBI) solution into CMC solution at pH 3.5 are shown in Figure 4. The raw vertical peaks corresponded to the energy change in the cell containing CMC at each protein injection. Obviously, the injection profiles in the sample cells for KTI with CMC of different DS were exothermic and underwent regular decrease in heat energy to a thermodynamic equilibrium state (about zero) after the 11th, 9th, and 9th injections for CMC 0.7, CMC 0.9, and CMC 1.2, respectively. Exothermicity is considered as a reflection of an enthalpic contribution to complex coacervation when the nonspecific electrostatic neutralization occurred between the two oppositely charged biopolymers.41 In addition, it was found that enthalpy change (−ΔH) was stronger with the increase in DS of CMC and might be associated with a stronger binding behavior of KTI to CMC. Xiong et al.19 found a similar trend when studing complex coacervation between ovalbumin and CMC: the binding was derived from spontaneous exothermic reaction, and the binding enthalpy and stoichiometric properties of F

DOI: 10.1021/acs.jafc.8b00091 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 5. Isothermal calorimetry titration corresponding plots after integration of peaks area and normalization for KTI/CMC 0.7 (A), KTI/CMC 0.9 (B), KTI/CMC 1.2 (C), and BBI/CMC 0.7 (D). One-site independent models were employed to fit the thermodynamic isotherms.

Table 1. Thermodynamic Properties Obtained from One-Site Model for KTI/CMC Interactions protein/polymer

K (M−1) × 106

n

ΔH (kcal/mol)

TΔS (kcal/mol)

ΔG (kcal/mol)

KTI/CMC 0.7 KTI/CMC 0.9 KTI/CMC 1.2

2.14 ± 0.43 4.06 ± 0.33 5.50 ± 0.59

58.8 ± 2.93 49.5 ± 1.62 63.7 ± 0.22

−7.88 ± 0.92 −9.57 ± 0.86 −11.3 ± 1.39

−0.619 ± 0.05 −1.92 ± 0.16 −3.52 ± 0.33

−7.26 ± 0.87 −7.65 ± 0.70 −7.83 ± 1.06

noninteracting behavior of BBI was first observed during the investigated protein/polysaccharide systems. Finally, we can note that the different binding thermodynamics of KTI vs BBI just explained the reason why two proteins can separate from each other for coacervation with CMC. Removal of Polysaccharides by Ultrafiltration. For the KBi/CMC 0.7 system, the coacervates and supernatant obtained at r = 5:1, pH 3.65, were further treated with ultrafiltration to remove the polysaccharide. After ultrafiltration, it was found that more than 96% of polysaccharides was kept in retentate, while most of proteins (KTI ∼ 93.5%, BBI ∼ 94.6%) transferred into the filtrates, indicating that ultrafiltration was a good method to remove polysaccharide from protein/ polysaccharide complexes. Then, the prepared proteins were further analyzed using SEC-HPLC, as shown in Figure 7. Although two main proteins (KTI and BBI) present in the KBi mixture, KTI and BBI isolated from each other after coacervation (r = 5:1, pH 3.65), and as a consequence, a purified KTI (∼89.4%) and a purified BBI (∼92.1%) were both obtained (Figure 7). With regard to purification yield, it was found that, after coacervation with CMC 0.7, about 83% of KTI and 92% of BBI were recovered from KBi mixtures; the detailed results are summarized in a purification table (Table S1).

and a two-step thermodynamic behavior was found with different BBI concentration, ionic strength, and temperatures; very similar results were observed for other sulfated polysaccharides, such as κ- and ι-carrageenan, dextran sulfate, and chondroitin sulfate (unpublished data). All the above results showed that BBI binding to sulfated polysaccharide belongs to a strong thermodynamic phenomenon. In the present case, the thermodynamic binding of BBI to the carboxyl polysaccharides (CPs) of different charge density, such as sodium alginate (high), xanthan gum (medium), and gum arabic (weak),9 were also investigated to probe the difference from binding with CMC. As shown in Figure 6, for all three BBI/CP systems, both the exothermic enthalpy and an equilibrium state of heat were gained after successive injection of BBI molecules. Specifically, the binding enthalpy (−ΔH) of BBI/sodium alginate was around −6.0 kcal/mol, and −ΔH of BBI/xanthan gum was about −3.0 kcal/mol, whereas the −ΔH of BBI/gum arabic was only around −0.9 kcal/mol, being consistent with their charge density orders. Thus, the weak binding affinity of gum arabic made it bind with smaller amounts of proteins than the former two. However, with regard to BBI, the CMC seemed to be too weak a polyelectrolyte to form an electrostatically driven thermodynamic reaction; and G

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Figure 6. Isothermal calorimetry titration raw data (upper) and corresponding plot after integration of peaks area and normalization (lower) for different BBI/carboxyl polysaccharide systems: (A) sodium alginate; (B) xanthan gum; (C) gum arabic.

at coacervates, and single BBI was left in supernatant after complete precipitation of KTI. The noninteracting behavior of BBI when complexing with CMC was further confirmed by ITC, where the thermodynamic enthalpy was negligible compared to the strong binding affnity of KTI (by orders of magnitude, ×106 M−1). Investigation of other carboxyl polysaccharides, such as NaA, XG, and GA, shows that they cannot perform similar binding behavior, suggesting that CMC might be a relatively very weak polyelectrolyte for BBI. However, the relationship between protein selectivity and polymer charge density still requires more work. Given the high therapeutic value of BBI, the building of a simple and renewable purifying method may promote the further study of its anticarcinogenic properties.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b00091. Purification of KTI and BBI by complex coacervation method and turbidity curves for the BBI/CMC 1.2 system (Cp = 0.10%) as a function of pH at different mass ratios (PDF)

Figure 7. SEC-HPLC analysis of KBi mixture, purified KTI, and BBI.

In summary, remarkable differences were found in the properties of coacervation of CMC with KTI as compared with BBI, which were probably due to their different effective charge density and structure of proteins. Although given with the very similar pIs of KTI vs BBI (4.5 vs 4.2), the priority binding of KTI to CMC provided the opportunity to be separated from BBI once the maximum coacervation occurred. Critical pH values were found to shift to lower pH values to satisfy the charge neutralization, and the maximum turbidity became greater with the high DS of CMC. At pHmax, the increased protein recovery and TIA loss with decrease in r were related to the KTI involvement; while the unchanged CIA was mainly derived from the uncomplexed BBI. Tricine−SDS−PAGE results justified the separation of KTI and BBI, where the former kept moving into coacervate, and the latter was always free of coacervation. As a result, enriched single KTI was found

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AUTHOR INFORMATION

Corresponding Author

*Phone: 0510-85917812. Fax: 0510-85329091. E-mail: [email protected]. ORCID

Xingfei Li: 0000-0003-2730-9123 Funding

This study was supported by the National Natural Science Foundation of China (No. 31601413 and No. 21276107), the Nature Science Foundation of Jiangsu Province (No. BK20160168), the National Great Project of Scientific and Technical Supporting Programs (No. 2012BAD34B04-1), and H

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Hi-tech Research and Development Program of China (No. 2013AA102204-3). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED KTI, Kunitz trypsin inhibitor; BBI, Bowman−Birk chymotrypsin inhibitor; KBi, KTI/BBI mixtures; CMC, carboxymethyl cellulose; DS, substitution degree; CIA, chymotrypsin inhibitory activity; TIA, trypsin inhibitory activity; ITC, isothermal titration calorimetry; XG, xanthan gum; NaA, sodium alginate; GA, arabic gum; BCA, bicinchoninic acid; BTpNA, N-benzoyl-L-tyrosine p-nitroanilide; BAPA, Nαbenzoyl-L-arginine 4-nitroanilide hydrochloride; CPs, carboxyl polysaccharides

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