Influence of Ternary Complexation between Bovine Serum Albumin

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/JAFC

Influence of Ternary Complexation between Bovine Serum Albumin, Sodium Phytate, and Divalent Salts on Turbidity and In Vitro Digestibility of Protein Elaine Kaspchak, Luciana Igarashi-Mafra, and Marcos R. Mafra* Chemical Engineering Department, Federal University of Paraná, Francisco H. dos Santos Street, 81531-980 Curitiba, Paraná, Brazil

J. Agric. Food Chem. Downloaded from pubs.acs.org by UNIV OF TOLEDO on 09/29/18. For personal use only.

S Supporting Information *

ABSTRACT: Phytate decreases mineral and protein availability and influences protein properties, such as solubility and stability. The binding constants and turbidity data can help with the understanding of the influence of phytate and divalent salts on protein behavior. Ternary complexes formed between bovine serum albumin, sodium phytate, and divalent salts were investigated by isothermal titration calorimetry, turbidity, and in vitro protein digestibility. Results showed a positive entropy change and a negative and small enthalpy change as a result of electrostatic binding forces and ternary and binary complex precipitation. The interaction was favored for the systems containing calcium and manganese, whereas those containing magnesium showed a low heat of interaction. Despite the high protein digestibility, the stability of divalent phytates in a wide pH range may decrease mineral bioavailability. These results can provide important insights for the study of mineral bioavailability and diverse processes that involve protein and minerals in several areas of knowledge. KEYWORDS: isothermal titration calorimetry, simplex lattice design, phytic acid, magnesium, calcium, manganese

■

cancer treatment and as an additive in the food industry.14 Thus, the understanding of the interactions of phytates with nutrients may help in better utilization of foods of plant origin. The relative importance of the interactions involving ternary complexation between phytate, protein, and minerals is difficult to establish,12 because some phytate salts are insoluble at alkaline pH.10 In this context, isothermal titration calorimetry (ITC) can be particularly valuable. According to Johnson et al.,15 ITC is an ideal technique to study metal− ligand interactions because it is not restricted by the photophysical properties of a metal.16 Studies using ITC indicate that the interaction of phytic acid and proteins in acid conditions are enthalpy-driven as a result of electrostatic and hydrogen bond interactions and are dependent upon the temperature and ionic strength of the system.17,18 Phytate presents strong negative charges on its six phosphate groups that can interact with di- and trivalent cations.19,20 Ca2+ is the most abundant divalent cation under physiological conditions and is typically one of the most prevalent minerals to interact with phytate.19 To our knowledge, no studies have yet investigated the ternary complex formation of proteins, phytates, and divalent metals by ITC. Thus, this work aimed to evaluate the binding between bovine serum albumin (BSA), sodium phytate (NaP), and the divalent metals (M2+) by ITC. Moreover, the influence of the ternary complex formation on the turbidity and in vitro digestibility of the protein was evaluated. The divalent salts used in this work are magnesium chloride, calcium chloride,

INTRODUCTION Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate) accumulates in nuts, cereal grains, and legume seeds, acting as a phosphorus storage compound.1 This substance is a strong chelator of divalent minerals2,3 and can bind to proteins and starch, altering the solubility, functionality, digestion, and absorption of these food components.4 Phytic acid can form either a simple salt with a metal or a complex salt with several metals in the same molecule depending upon the pH.5 Because humans are unable to hydrolyze phytates in their stomach, mineral chelation by these anti-nutritional compounds can lead to a mineral deficiency in the body.6 Such interactions can cause malnutrition because metal ions, such as zinc, calcium, and magnesium, are indispensable for the normal physiological maintenance of living organisms.6,7 In addition, the interaction of phytates with protein may reduce the digestibility and, consequently, amino acid bioavailability of the protein. This action can have an impact on basic physiological functions, such as body protein synthesis8 and optimal skeletal growth and maturation.9 Besides forming phytates through the direct interaction of phytic acid with salts, when polyvalent cations and proteins are present, a ternary complex is formed. The ternary complex occurs as a result of bridging between the cation, the phytate anion, and negatively charged groups found in the protein at neutral and high pH.10 This ability to form a ternary complex can influence several processes that involve proteins and minerals, such as the catalytic activity and autocatalytic degradation of trypsin,11 texture and solubility of food, protein separation,12 and stability.13 Despite presenting negative effects, the ability of phytates to interact with proteins may be beneficial in the human organism as a result of the control of various cellular functions and © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

June 15, 2018 September 13, 2018 September 18, 2018 September 18, 2018 DOI: 10.1021/acs.jafc.8b03142 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

respectively, ΔH is the molar heat of ligand binding, Xt is the bulk concentration of the ligand, Ka is the binding constant, and Q(i) is the heat released from the ith injection. The fitting involves the initial guesses of n, Ka, and ΔH, the calculation of ΔQ(i) for each injection, a comparison of these values to the experimental data, improvement in the initial values of n, Ka, and ΔH by standard Marquardt methods, and iteration of the above procedure until no further significant improvement in fit.23 Entropy change (ΔS) was calculated by MicroCal Origin software using eq 4

and manganese chloride. Calcium and magnesium can be responsible for the texture in foods, e.g., tofu, and manganese is essential for the catalytic activity of many enzymes. In the human body, these mineral play important roles, such as enzymatic reactions, muscle contraction, and bone and enzyme formation, among others.7

■

MATERIALS AND METHODS

Materials. BSA (cold ethanol fraction, with a molar mass of 66 430 g/mol) and phytic acid [50% (w/w) in water, with a molar mass of 660.04 g/mol] were purchased from Sigma (St. Louis, MO, U.S.A.). Pepsin (1:10 000) was acquired from Neon (São Paulo, Brazil), and pancreatin (30 USP protease units/mg of product) was from Inlab (São Paulo, Brazil). For buffer preparation, Tris base (Norgen Biotek Corp., Thorold, Ontario, Canada) was used. The divalent metals (M2+) used were magnesium chloride hexahydrate from Neon (São Paulo, Brazil), calcium chloride dihydrate from Vetec (Rio de Janeiro, Brazil), and manganese chloride tetrahydrate from Synth (São Paulo, Brazil). All solutions were prepared with Milli-Q water (Millipore Corp., Bedford, MA, U.S.A.). Molecular Interaction of BSA, NaP, and M2+ Analyzed by ITC. Protein samples were dialyzed (12 kDa membrane, SigmaAldrich, St. Louis, MO, U.S.A.) overnight against Tris−HCl buffer (pH 7.0, 100 mM) under stirring at 5 °C. All solutions were prepared in the same dialysis buffer. The protein samples were centrifuged (10000g for 5 min) after dialysis for bubble elimination, and the protein concentration was determined.21 As a result of the difficulty in maintaining the phytic acid at pH 7.0, even at high buffer concentrations, NaP was used. NaP was prepared by titration of the phytic acid solution with NaOH (2 M), according to eq 1.

C6H18O24 P6 + 12NaOH → C6H6Na12O24 P6 + 12H 2O

ΔS =

ΔH − R ln K a T

(4)

where T is the temperature of the system and R is the ideal gas constant (8.31 J K−1 mol−1). Gibbs free energy (ΔG) was calculated using eq 5. (5)

ΔG = ΔH − T ΔS

All experiments were performed in duplicate, and the thermodynamic parameters presented are the mean of measurements. Turbidity of the Ternary Mixture Analyzed Using a Simplex Lattice Design. Determination of turbidity is a highly valuable technique because it can be correlated with the solubility of the system. To evaluate the influence of the three components of the system (BSA, NaP, and M2+), a three-factor simplex lattice design with interior points and overall centroid, according to Table 1, was established. The mixture experiments were designed and analyzed using Statistica, version 10.0 (StatSoft, Tulsa, OK, U.S.A.), which is based on the work of Scheffé.24

Table 1. Simplex Lattice Design for Turbidity Determination of Ternary Systems Varying the Concentration of BSA, NaP, and M2+

(1)

The ITC equipment measures the heat that is either released or absorbed during a molecular interaction when a ligand is sequentially titrated in a cell containing the other component (usually the macromolecule).22 In this work, one injection of 0.5 μL, followed by 18 injections of 2 μL, with 150 s intervals between each injection, was performed. The reference cell was filled with ultrapure water, and the syringe stir rate was set to 700 rpm. For ternary complex formation, each salt solution (MgCl2, CaCl2, and MnCl2) was separately titrated into NaP and BSA. The molar ratios of NaP/BSA used were 44:1, 88:1, and 200:1, which were chosen on the basis of preliminary tests. Binary systems of BSA−NaP, NaP−M2+, and BSA−M2+ were also examined. Control titrations were performed for all systems to evaluate dilution effects (Figure S1 of the Supporting Information). M2+ titration into buffer was subtracted from the curves obtained for titration of NaP into BSA and for M2+ titrations into NaP and NaP + BSA separately. Thermodynamic parameters, such as stoichiometry (n), association constant (Ka), enthalpy change (ΔH), and entropy change (ΔS), were determined by fitting the data to the one-site model (eqs 2 and 3), using Microcal Origin 7.0 software (ITC Data Analysis in Origin, MicroCal, LLC, Northampton, MA, U.S.A.)23 ÅÄÅ nM tΔHV0 ÅÅÅÅ Xt 1 ÅÅ1 + Q= + ÅÅ 2 nM t nK aM t ÅÅ ÅÇ ÉÑ ÑÑ 2 Ñ ij y X 4 X 1 zz t t Ñ ÑÑ zz − − jjj1 + + Ñ j z nM t K aM t { nM t ÑÑÑÑ k (2) ÑÖ Ä É dV ÅÅÅ Q (i) + Q (i − 1) ÑÑÑÑ ΔQ (i) = Q (i) + i ÅÅÅ ÑÑÑ − Q (i − 1) V0 ÅÅÇ 2 ÑÖ (3)

coded level

actual concentration (mg/mL)

number

BSA

NaP

M2+

BSA

NaP

M2+

1 2 3 4 5 6 7 8 9 10

1.00 0.00 0.00 0.50 0.50 0.00 0.67 0.17 0.17 0.33

0.00 1.00 0.00 0.50 0.00 0.50 0.17 0.67 0.17 0.33

0.00 0.00 1.00 0.00 0.50 0.50 0.17 0.17 0.67 0.33

1.82 0.00 0.00 0.91 0.91 0.00 1.22 0.31 0.31 0.60

0.00 1.82 0.00 0.91 0.00 0.91 0.31 1.22 0.31 0.60

0.00 0.00 1.82 0.00 0.91 0.91 0.31 0.31 1.22 0.60

The mixture designs, such as simplex lattice, can be helpful for optimization of mixtures using a reduced number of experiments in all proportions of substances (0−100%) and a possible interaction effect among variables. This design permits the evaluation of the response under study by linear (β1, β2, and β3), quadratic (β12, β13, and β23), and special cubic models (β123), as presented in eq 6.25 Y = β1X1 + β2X 2 + β3X3 + β1,2X1X 2 + β1,3X1X3 + β2,3X 2X3 + β1,2,3X1X 2X3

(6)

For the preparation of the mixtures, Tris−HCl buffer (100 mM) was used in each system to maintain the pH at 7.0. The samples were homogenized in tubes of 2 mL, and the total volume of the solutions was 1.2 mL. The samples were equilibrated in a thermostat bath (model RW-1025G, Lab Companion) at 25 °C for 4 h. Transmittance (T) was analyzed at a wavelength of 420 nm (Shimadzu UV-1800 spectrophotometer, Kyoto, Japan) in triplicate, and the percentage of turbidity was defined as 100 − the transmittance of each sample.26 Effect of NaP and M2+ on In Vitro Protein Digestibility (IVPD) of BSA. For IVPD determination, different molar ratios of

where Q is the total heat content, n is the stoichiometry, Mt and [M] are bulk and free concentrations of the macromolecule in Vo, respectively, Vo and Vi are the active and injection cell volumes, B

DOI: 10.1021/acs.jafc.8b03142 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article


Figure 1. ITC curves of the interaction between BSA, NaP, and M2+ at pH 7.0 and 25 °C for binary systems: (a) NaP titration into BSA, (b) MgCl2, CaCl2, and MnCl2 titration into BSA, (c) MgCl2 titration into NaP, (d) CaCl2 titration into NaP, and (e) MnCl2 titration into NaP. Solid lines are the best fit of the data using the one-site model, and symbols are experimental data. n is the stoichiometry; Ka is the association constant; ΔH is the enthalpy change; T is the temperature of the system; ΔS is the entropy change; and ΔG is the Gibbs free energy. Raw ITC data are presented in the upper panel (μW), and the heat per injection obtained from integrating the heat flow over time is presented in the lower panel (kJ/mol) of each figure. M2+/BSA (from 25:1 to 200:1) and NaP/BSA (from 9.5:1 to 28.4:1) were used on the basis of the ITC and turbidity results. The stomach digestion simulation was conducted using pepsin with the enzyme/ substrate ratio of 1:4 (w/w) and 0.045 mM BSA (pH 3.0, adjusted with 1.0 M HCl). The samples were kept at 37 °C under stirring for 1.5 h. For intestinal digestion simulation, the samples were neutralized using NaOH (1.0 M) and pancreatin solution (0.1 M phosphate buffer at pH 7.0) was added to all samples to obtain an enzyme/ substrate ratio of 1:2 (w/w). The samples were maintained at 37 °C under stirring for 3.5 h. Sodium carbonate was added to a final concentration of 5.0 mg/mL for quenching the reaction. The protein remaining after digestion was quantified by the Bradford method.21 The IVPD was calculated according to eq 7.

Tukey’s test at a 95% significance level with the aid of Statistica, version 10.0 (StatSoft, Tulsa, OK, U.S.A.).

■

RESULTS AND DISCUSSION ITC. The formation of ternary complexes between BSA, NaP, and M2+ was examined by ITC. For the comprehension of the interaction, the ITC results were divided into two sections. Thus, the binary interactions (BSA−NaP, BSA−M2+, and NaP−M2+) were measured, followed by the ternary interaction (BSA−NaP−M2+). Interaction of BSA, NaP, and M2+ by ITC for Binary Systems. Figure 1 reveals the results of the interaction of NaP with BSA, M2+ with BSA, and NaP with M2+ performed at pH 7.0 and 25 °C. The upper panel shows the heat flow caused by the injection of the ligand into the cell. The heat of each injection was calculated by the integration of the heat flow peaks using the MicroCal Origin 7.0 software. Positive heat flow is attributed to endothermic reactions, and negative heat flow is attributed to exothermic reactions. No significant interaction between NaP and BSA was observed (Figure 1a).

protein remaining after hydrolysis zyz ji IVPD (%) = jjj1 − zz × 100 j initial protein concentration z{ k

(7) Statistical Analysis. Data in this study are expressed as the means of duplicates ± standard deviation for ITC results and means of triplicate ± standard deviation for turbidity and IVPD results. Data were analyzed through one-way analysis of variance (ANOVA) and C


Article


Figure 2. ITC curves of the interaction between BSA, NaP, and M2+ at pH 7.0 and 25 °C for ternary systems: (a) MgCl2 titration into BSA/NaP (1:44), (b) MgCl2 titration into BSA/NaP (1:88), (c) MgCl2 titration into BSA/NaP (1:200), (d) CaCl2 titration into BSA/NaP (1:44), (e) CaCl2 titration into BSA/NaP (1:88), (f) CaCl2 titration into BSA/NaP (1:200), (g) MnCl2 titration into BSA/NaP (1:44), (h) MnCl2 titration into BSA/NaP (1:88), and (i) MnCl2 titration into BSA/NaP (1:200). Solid lines are the best fit of the data using the one-site model, and symbols are experimental data. n is the stoichiometry of M2+/BSA; Ka is the association constant; ΔH is the enthalpy change; T is the temperature of the system; ΔS is the entropy change; and ΔG is the Gibbs free energy. Raw ITC data are presented in the upper panel (μW), and the heat per injection obtained from integrating the heat flow over time is presented in the lower panel (kJ/mol) of each figure.

This finding is expected because the protein interaction is governed by electrostatic forces, and at this pH, both protein and phytate are negatively charged.10,18 Similarly, Kies et al.27 showed that no protein−phytate complexes can be formed at pH values higher than 4. The salt titration into BSA resulted in positive and small enthalpy changes (Figure 1b). Although positively charged ions can reversibly associate with most of the albumins, ions,

such as Ca2+ and Mg2+, are bound to low-affinity binding sites,28 which can result in a low heat of interaction. The interactions of calcium and magnesium ions with proteins are governed by electrostatic forces occurring on negatively charged, non-specific regions of the protein,29 such as the carboxyl oxygen.30 Besides, a counterion H+/Ca2+ exchange also plays a key role in protein and M2+ interactions, while MnCl2 binds tightly to BSA through chelation by imidazole D


Article

Journal of Agricultural and Food Chemistry groups.31 This explanation corroborates the results obtained by ITC for the interaction between MnCl2 and BSA that resulted in elongated peaks at the beginning of titration, which is due to a secondary and slow reaction, probably the chelation and subsequent oxidation of manganese. Panels c−e of Figure 1 show the interaction of M2+ with NaP obtained by ITC. The interaction of MgCl2 with NaP (Figure 1c) resulted in a low-intensity signal with some noise in the baseline. As such, it was not possible to adjust the onesite model to the experimental data. The interaction showed both exothermic and endothermic peaks (zoom of the peaks is presented in Figure S2 of the Supporting Information). It was observed that, when the molar ratio of salt/NaP increases, the exothermic signal rises and the endothermic signal decreases. Because exothermic peaks can be attributed to the loss of ions from the structure,32 this behavior can be associated with the ionic exchange of sodium and magnesium in the structure of phytate. For the interaction between NaP and CaCl2, the signal was predominantly exothermic (Figure 1d), with a higher intensity than that observed for systems containing MgCl2. The data presented a sigmoidal profile and well-defined peaks, demonstrating that the interaction is specific and of high affinity. Thermodynamic parameters (ΔH, TΔS, and ΔG) obtained from the interaction profiles (ITC curves) illustrated that the entropic component of the interactions is dominant (in modulus) for all systems; i.e., |ΔH| < |TΔS|. The interaction of MnCl2 with NaP presented some exothermic peaks in the salt/NaP molar ratio of around 10, probably as a result of manganese phytate precipitation. This behavior results from the competition between the mineral (in excess) and hydrogen for the negative charges of NaP, which may result in complex precipitation.33 The stoichiometry of the complex between CaCl2 and NaP and between MnCl2 and NaP was close to 6, in concurrence with Torres et al.,34 who related a 1:1 theoretical molar ratio of phosphate/divalent metal. This stoichiometry is favored when the divalent salt is in excess, producing a fully dissociated phytate anion.33 The stoichiometry and enthalpy observed in the present study are different from those of Kim et al.19 and Oh et al.,24 who recorded a Ca2+/phytate stoichiometry of 3:1 and 4:1, respectively. This difference is attributed to distinct experimental conditions because Kim et al.19 performed the experiments at pH 5.0 and 37 °C, Oh et al.24 used pH 7.0 at 30 °C, and the present study was conducted at pH 7.0 and 25 °C. Besides, the type of phytate used, such as sodium phytate and potassium phytate, can promote different profiles of interaction as a result of the dissociation tendency of each kind of anion from the structure of phytic acid. Interaction of BSA, NaP, and M2+ by ITC for Ternary Systems. At neutral and basic pH values, the interaction between NaP and protein is diminished, as described above. However, the presence of other components in the food system gives additional possibilities for protein−phytate interactions, attributed to cation bridge formation between the phytate anion and negatively charged groups of the protein.10 To evaluate the ternary complex formation, the salt solutions were titrated into NaP and the protein, thereby avoiding precipitation before the titrations. Figure 2 provides the results of the ternary complex formation between BSA, NaP, and M2+ obtained by ITC for various molar ratios of BSA/NaP (1:44, 1:88, and 1:200) based on preliminary tests. The systems

containing BSA, NaP, and M2+ presented a distinct behavior from that observed for the binary systems, indicating the formation of the ternary complex. The system containing magnesium presented a small intensity signal and the predominance of exothermic peaks during all titrations for the BSA/NaP molar ratio of 1:44 and 1:88, as shown in panels a and b of Figure 2, respectively. For the titration of MgCl2 in the samples containing 1:200 of BSA/ NaP (Figure 2c), the majority of peaks was endothermic, the signal remained small, and the thermodynamic parameters could not be determined. At the end of titration, both exothermic and endothermic peaks occurred, as also observed for the binary complex of MgCl2 and NaP (Figure 1c). For the interaction of NaP and BSA with CaCl2, the signal intensity at the beginning of the experiments was higher for BSA/NaP molar ratios of 1:44 and 1:88 (panels d and e of Figure 2, respectively) when compared to the results obtained for NaP−CaCl2 interactions (Figure 1d). As the NaP concentration increases, some small endothermic peaks are observed, possibly related to the release of substantial amounts of sodium and water from the phytate molecule as a result of precipitation of the complex. For the BSA/NaP molar ratio of 1:200 (Figure 2f), a smaller amount of heat is released when compared to the system without protein (Figure 1d) and with BSA/NaP molar ratios of 1:44 and 1:88 (panels d and e of Figure 2, respectively). All ITC curves of the systems containing CaCl2 presented similar profiles to each other. For systems containing MnCl2, when the 1:44 molar ratio was used (Figure 1g), the ITC curve had a smaller peak than that seen in the absence of protein (Figure 1e). The systems with BSA/NaP molar ratios of 1:88 (Figure 2h) and 1:200 (Figure 2i) presented initial peaks of high intensity, and the second peak was wider than the first (zoom of the figure is presented in Figure S3 of the Supporting Information). This broad signal could represent secondary reactions occurring in the system, such as oxidation and chelation, as was also observed for the interaction of MnCl2 with BSA (Figure 1b). The titration of MnCl2 into 1:200 BSA/NaP resulted in exothermic peaks that appeared simultaneously with endothermic peaks at the end of the titrations. This behavior complicated the adjustment of the one-site model to the curve and can be attributed to the higher complexity of this system relative to the others. The complexity may arise from simultaneous interactions that can be occurring, such as binary complex formation, competition between BSA and NaP for ions, ionic exchange, and release of a considerable amount of water from the structure as a result of a relatively higher insoluble complex formation. Figure 2 depicts the thermodynamic parameters (n, Ka, ΔH, ΔS, and ΔG) involved in the interactions of the systems containing CaCl2 and MnCl2. In these systems, positive ΔS (presented as TΔS) and small and negative ΔH were obtained. The interaction of protein and phytates is governed by electrostatic forces. In this way, the high and positive entropy seen in this work is related to changes in the hydrophobic residues of the protein35 as well as insoluble complex formation and loss of ions from the phytate and protein structures.32 The CaCl2 and MnCl2 ternary complexes for 1:44 and 1:88 BSA/NaP molar ratios are formed at M2+/NaP molar ratios of around 10:1 and 8:1, respectively. At 1:200 BSA/NaP molar ratio, a distinct behavior was observed for both systems, whereby the ternary complex formed at the molar ratio of E


Article


Figure 3. Model graph showing the effect of BSA, NaP, and M2+ on the percentage of turbidity at 25 °C and pH 7.0: (a) BSA−NaP−MgCl2 turbidity, (b) BSA−NaP−CaCl2 turbidity, and (c) BSA−NaP−MnCl2 turbidity. The model applied to surfaces was the special cubic that informs the ternary mixture influence on the result. The design points are presented inside the graphs by the small circles, and the values presented in each axis are the coded values.

red color in the turbidity graph, which represents the higher turbidity observed (panels b and c of Figure 3). For systems containing MgCl2, the turbidity values were very small (maximum of 13%), supporting those obtained by ITC that showed a low heat of interaction. Thus, the low turbidity can be attributed to the size of the complex that can be small and, therefore, does not interfere in the transmittance of the sample. Besides, the magnesium phytate complex has a high solubility in the absence of proteins,37 and the binary complex of NaP and MgCl2 does not interfere with the turbidity of the sample. The highest turbidity was observed in the low concentration of protein and high concentration of MgCl2 (sample 9 in Table S2 of the Supporting Information) probably as a result of the salting-out effect of MgCl2 on BSA. Regardless of the high solubility of magnesium phytate, it is reported that dietary phytate decreases apparent Mg 2+ absorption in the diets of growing rats and was accompanied by an increase in the hepatic lipid peroxidation and protein damage as well as a decrease in liver glutathione.3 These phenomena could be an issue of concern because phytate is typically present in feedstuffs as mineral−phytate complexes involving magnesium.38 Related to the CaCl2 and MnCl2 systems, the factors that most influenced the turbidity were the concentrations of NaP and M2+. The comparatively higher turbidity was obtained in the absence rather than the presence of BSA, indicating calcium phytate and manganese phytate formation. Previously, it was shown that phytate could considerably decrease the bioavailability of Ca2+.39 The bioavailability of phosphate and Ca2+ in foods can be increased if Ca2+−phytate salts are hydrolyzed by enzymes, which increases the nutritional quality of the food.19 In addition, the intensity and character of the interactions involving phytates can be modified by some processing aspects, such as hydration, heat treatment (e.g., baking, autoclaving, and extrusion), isolation, and separation.10 In comparison to CaCl2, MnCl2 promoted a higher turbidity range in the ternary diagram. Thus, in comparison to systems containing CaCl2, those with MnCl2 can form complexes in a wide range of concentrations of the three components, because no statistical difference (95% confidence level) was observed in samples 6−10 (Table S2 of the Supporting Information).

CaCl2/NaP of around 5:1 and the system containing MnCl2 presented a high complex curve that did not adjust to the onesite model (panels d−i of Figure 2). It was noticed that, as the BSA/NaP molar ratio increased, the stoichiometry parameter (n) rises and Ka, ΔH, TΔS, and ΔG values decrease for CaCl2 and MnCl2 ternary complexation. Furthermore, the affinity constant obtained in the absence of protein (panels d and e of Figure 1) was higher than that for CaCl2 titration into BSA/NaP of 1:200 (Figure 2f) and MnCl2 titration into BSA/NaP of 1:44 and 1:88 (panels g and h of Figure 2). The decrease in the affinity can be attributed to simultaneous complex interactions occurring when protein is added. Although ITC analysis of the ternary systems and identification of the different profiles of interaction are achievable, the formation of ternary systems is still difficult to be determined because binary interactions are also involved. In this way, the influence of the mixture of components on the turbidity was also assessed. Turbidity of BSA, NaP, and M2+ Complexes. Complexes formed by phytate can change the solubility of some substances, such as proteins and minerals, and, consequently, the properties of food during processing or consumption,36 impacting the acceptability of the product. Besides, the strong chelation ability of phytates can reduce the bioavailability of minerals in monogastric animals as a result of the formation of insoluble complexes and insufficient quantities of enzymes to hydrolyze phytate.19 The turbidity can be related to the interaction of the protein with ligands (phytates and divalent salts) to form insoluble complexes. The turbidity and composition of each component were prepared according to a simplex lattice design. Figure 3 displays the contour plot for the turbidity of the systems that contain BSA, NaP, and M2+ (MgCl2, CaCl2, and MnCl2). The estimated model parameter coefficients obtained by the special cubic model, describing the effect of different concentrations of BSA, NaP, and M2+ on turbidity, are presented in Table S1 of the Supporting Information. Except for turbidity in the system containing magnesium, good adjustments were observed. The ternary complex formation region probably occurs in the dark F


Article

Journal of Agricultural and Food Chemistry Table 2. IVPD of BSA in the Presence of NaP and M2+ under Simulated Gastrointestinal Conditionsa molar ratio of M2+/BSA

molar ratio of NaP/BSA

molar ratio of M2+/NaP

50 50 50 25 100 200

9.5 18.9 28.4 14.2 14.2 14.2

5.3 2.6 1.8 1.8 7.0 14.1

no no no no

salt salt salt salt

9.5 14.2 18.9 28.4

no no no no

IVPD of BSA−NaP−MgCl2 (%) 87.4 89.2 89.3 87.9 88.8 89.4

± ± ± ± ± ±

0.2 0.3 1.1 0.6 0.7 2.4

aB aA aA aAB aA aA

salt salt salt salt

control (no salt and no NaP)

IVPD of BSA−NaP−CaCl2 (%) 86.5 ± 0.9 bB 88.9 ± 0.7 aA 88.8 ± 0.8 aA 89.3 ± 0.7 aA 88.4 ± 0.3 aA 88.1 ± 0.7 aA IVPD of BSA−NaP (%) 87.2 ± 0.6 a 88.4 ± 0.3 a 88.9 ± 1.3 a 82.4 ± 0.3 b IVPD of BSA (%) 88.3 ± 0.7

IVPD of BSA−NaP−MnCl2 (%) 89.5 87.7 87.6 86.3 88.5 91.1

± ± ± ± ± ±

0.5 0.8 0.0 0.7 0.5 0.5

bA cB cB dB bcA aA

The stomach digestion was simulated with pepsin (enzyme/substrate ratio of 1:4, w/w) for 1.5 h at 37 °C and pH 3.0. The intestine digestion was simulated with pancreatin (enzyme/substrate ratio of 1:2, w/w) for 3.5 h at 37 °C and pH 7.0. Means followed by the same letter in columns or rows do not differ statistically (column, lowercase letters; rows, capital letters) at the 95% confidence level according to Tukey’s test. a

phytate−divalent salt complexes on mineral bioavailability. Phytate can form complexes over a wide range of pH with trivalent43 and divalent44,45 cations, meaning they can be stable along the entire gastrointestinal tract. After digestion, an insoluble complex was observed, suggesting the formation of insoluble salt phytates and strongly indicating a potential reduction in the mineral absorption of such substances. By association of the ITC, turbidity, and IVPD data, it was possible to confirm the binding of NaP, BSA, and M2+ by electrostatic forces that promoted the loss of water from the protein structure as a result of precipitation. The ternary complex formation did not have a great impact on IVPD. Despite this, the knowledge of ternary complex formation involving protein, NaP, and M2+ is important for nutrition and food technology. In nutrition, this complex formation can affect mineral bioavailability and catalytic activity of enzymes. In food technology, this complex formation can affect sensorial properties, food processing, and biochemical transformation of the food.

Considering the divalent cation effect, this is in accordance with Quiñone et al.,40 who noticed that phytate solubility increased in the following order: Mn2+, Ca2+, and Mg2+. The same tendency is observed for the salting-out effect of divalent salts on proteins, where Mg2+ is more prone to precipitate the protein, followed by Ca2+ and then Mn2+.41 Effect of NaP and M2+ on IVPD of BSA. The BSA− NaP−M2+ ternary complexes can be formed in different proportions of each component. In this way, the IVPD was performed in different molar ratios of M2+/NaP and M2+/BSA. Table 2 presents the IVPD data for BSA in the presence of NaP and M2+. Despite Tukey’s test showing a significant difference between the samples, the variation in IVPD in the presence of the three components was low (86.3−91.1%) and, probably, the protein−NaP−M2+ ternary complex formation would not impact the digestion protein in vivo. The lower IVPD was observed in the absence of M2+ and in the highest concentration of NaP. This is expected because binary protein−phytate complexes are formed at low pH, causing delayed digestion of the protein by pepsin.27 However, in the systems containing the divalent salts, there is an excess of ions that can bind to negative charges of phytate, inhibiting the formation of protein−phytate complexes, not affecting the IVPD.18 Samples containing magnesium or calcium chloride and NaP presented the same IVPD (p < 0.05), except for the system containing CaCl2 in the lower molar ratio of NaP/BSA. For samples containing MnCl2 and NaP, a slight increase in IVPD was observed with the rise in the molar ratio of Mn2+/NaP. Values of IVPD higher than the control sample (no salt and no NaP) were observed for samples containing both M2+ and NaP. The presence of cations, such as sodium, can nullify the destabilization effect of phytate on the protein structure.13 Thus, the positive effect of NaP and M2+ on protein digestibility can be due to better stabilization of the digestive enzymes. Under the simulated intestinal condition (at pH 7.0), the BSA−NaP−M2+ ternary complex can be formed. Despite this, the interaction in this environment will only occur with protein previously hydrolyzed in the stomach by pepsin, not greatly affecting the IVPD. According to Selle et al.,42 there is increasing attention on the impact of ternary protein−

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b03142. Control titrations performed for ITC experiments (Figure S1), results obtained for MgCl2 titration into NaP (Figure S2), results obtained for MnCl2 titration into BSA and NaP (Figure S3), values of p for the estimated model parameter coefficients obtained by the special cubic model (Table S1), and data of turbidity (Table S2) (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Telephone: 55-41-3361-3586. E-mail: [email protected]. ORCID

Elaine Kaspchak: 0000-0001-7885-1137 Marcos R. Mafra: 0000-0002-0018-6867 Funding

This work was supported by the Graduation Program of Food Engineering (PPGEAL) and Coordenaçaõ de AperfeiçoamenG


Article

Journal of Agricultural and Food Chemistry ́ Superior (CAPES). Marcos R. Mafra is to de Pessoal de Nivel grateful for a Brazilian National Council for Scientific and Technological Development grant (CNPq, Grant 310905/ 2015-0).

(17) Darby, S. J.; Platts, L.; Daniel, M. S.; Cowieson, A. J.; Falconer, R. J. An Isothermal Titration Calorimetry Study of Phytate Binding to Lysozyme. J. Therm. Anal. Calorim. 2017, 127 (2), 1201−1208. (18) Kaspchak, E.; Mafra, L. I.; Mafra, M. R. Effect of Heating and Ionic Strength on the Interaction of Bovine Serum Albumin and the Antinutrients Tannic and Phytic Acids, and Its Influence on in Vitro Protein Digestibility. Food Chem. 2018, 252, 1−8. (19) Kim, O. H. J.; Kim, Y. O.; Shim, J. H.; Jung, Y. S.; Jung, W. J.; Choi, W. C.; Lee, H.; Lee, S. J.; Kim, K. K.; Auh, J. H.; et al. βPropeller Phytase Hydrolyzes Insoluble Ca2+-Phytate Salts and Completely Abrogates the Ability of Phytate to Chelate Metal Ions. Biochemistry 2010, 49 (47), 10216−10227. (20) Oh, B.-C.; Chang, B. S.; Park, K.-H.; Ha, N.-C.; Kim, H.-K.; Oh, B.-H.; Oh, T.-K. Calcium-Dependent Catalytic Activity of a Novel Phytase from Bacillus amyloliquefaciens DS11. Biochemistry 2001, 40 (32), 9669−9676. (21) Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Using the Principle of Protein Dye Binding. Anal. Biochem. 1976, 72, 248−254. (22) Velázquez-Campoy, A.; Ohtaka, H.; Nezami, A.; Muzammil, S.; Freire, E. Isothermal Titration Calorimetry. In Current Protocols in Cell Biology; Bonifacino, J. S., Dasso, M., Harford, J. B., LippincottSchwartz, J., Yamada, K. M., Eds.; John Wiley & Sons, Inc.: Hoboken, NJ, 2004; pp 17.8.1−17.8.24, DOI: 10.1002/0471143030.cb1708s23. (23) MicroCal, LLC. MicroCal Tutorial Guide on ITC Data Analysis in Origin. Using Origin Scientific Plotting Software To Analyze Calorimetric Data from All MicroCal Isothermal Titration Calorimeters; MicroCal, LLC: Northampton, MA, 2004; pp 117. (24) Scheffé, H. The Simplex-Centroid Design for Experiments with Mixtures. J. R. Stat. Soc. B 1963, 25 (2), 235−263. (25) Bochi, V. C.; Barcia, M. T.; Rodrigues, D.; Speroni, C. S.; Giusti, M. M.; Godoy, H. T. Polyphenol Extraction Optimisation from Ceylon Gooseberry (Dovyalis Hebecarpa) Pulp. Food Chem. 2014, 164 (2014), 347−354. (26) Ru, Q.; Wang, Y.; Lee, J.; Ding, Y.; Huang, Q. Turbidity and Rheological Properties of Bovine Serum Albumin/pectin Coacervates: Effect of Salt Concentration and Initial Protein/polysaccharide Ratio. Carbohydr. Polym. 2012, 88 (3), 838−846. (27) Kies, A. K.; De Jonge, L. H.; Kemme, P. A.; Jongbloed, A. W. Interaction between Protein, Phytate, and Microbial Phytase. In Vitro Studies. J. Agric. Food Chem. 2006, 54 (5), 1753−1758. (28) Kragh-Hansen, U. Molecular Aspects of Ligand to Serum Albumin. Pharmacology 1981, 33 (1), 17−53. (29) Guillaume, Y. C.; Guinchard, C.; Berthelot, A. Affinity Chromatography Study of Magnesium and Calcium Binding to Human Serum Albumin: pH and Temperature Variations. Talanta 2000, 53 (3), 561−569. (30) Sun, N.; Jin, Z.; Li, D.; Yin, H.; Lin, S. An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and In Vitro Calcium Absorption Studies of Peptide−Calcium Complex. J. Agric. Food Chem. 2017, 65 (44), 9782−9789. (31) Mildvan, A. S.; Cohn, M. Magnetic Resonance Studies of the Interaction of the Manganous Ion with Bovine Serum Albumin. Biochemistry 1963, 2 (5), 910−919. (32) Poncet-Legrand, C.; Gautier, C.; Cheynier, V.; Imberty, A. Interactions between Flavan-3-Ols and Poly(L-Proline) Studied by Isothermal Titration Calorimetry: Effect of the Tannin Structure. J. Agric. Food Chem. 2007, 55 (22), 9235−9240. (33) Grynspan, F.; Cheryan, M. Calcium Phytate: Effect of pH and Molar Ratio on in Vitro Solubility. J. Am. Oil Chem. Soc. 1983, 60 (10), 1761−1764. (34) Torres, J.; Domínguez, S.; Cerdá, M. F.; Obal, G.; Mederos, A.; Irvine, R. F.; Díaz, A.; Kremer, C. Solution Behaviour of myo-Inositol Hexakisphosphate in the Presence of Multivalent Cations. Prediction of a Neutral Pentamagnesium Species under Cytosolic/nuclear Conditions. J. Inorg. Biochem. 2005, 99 (3), 828−840. (35) Li, X.; Ni, T. Probing the Binding Mechanisms of αTocopherol to Trypsin and Pepsin Using Isothermal Titration

Notes

The authors declare no competing financial interest.

■ ■ ■

ACKNOWLEDGMENTS ́ The authors thank the Central Analitica Multiusuário das Usinas Piloto (CAMUP). ABBREVIATIONS USED BSA, bovine serum albumin; M2+, divalent metal; IVPD, in vitro protein digestibility; ITC, isothermal titration calorimetry REFERENCES

(1) Michaelsen, K. F.; Hoppe, C.; Roos, N.; Kaestel, P.; Stougaard, M.; Lauritzen, L.; Mølgaard, C.; Girma, T.; Friis, H. Choice of Foods and Ingredients for Moderately Malnourished Children 6 Months to 5 Years of Age. Food Nutr. Bull. 2009, 30, S343−S404. (2) Ma, G.; Li, Y.; Jin, Y.; Zhai, F.; Kok, F. J.; Yang, X. Phytate Intake and Molar Ratios of Phytate to Zinc, Iron and Calcium in the Diets of People in China. Eur. J. Clin. Nutr. 2007, 61, 368−374. (3) Rimbach, G.; Pallauf, J. Effect of Dietary Phytate on Magnesium Bioavailability and Liver Oxidant Status in Growing Rats. Food Chem. Toxicol. 1999, 37 (1), 37−45. (4) Oatway, L.; Vasanthan, T.; Helm, J. H. Phytic acid. Food Rev. Int. 2001, 17, 419−431. (5) Ferrando, R. Natural Antinutritional Factors Present in European Plant Proteins. Qual. Plant.Plant Foods Hum. Nutr. 1983, 32 (3−4), 455−467. (6) Gharibzahedi, S. M. T.; Jafari, S. M. The Importance of Minerals in Human Nutrition: Bioavailability, Food Fortification, Processing Effects and Nanoencapsulation. Trends Food Sci. Technol. 2017, 62, 119−132. (7) Stein, A. J. Global Impacts of Human Mineral Malnutrition. Plant Soil 2010, 335 (1), 133−154. (8) Moughan, P. J. Protein: Digestion, Absorption and Metabolism. In Encyclopedia of Food and Health; Elsevier: Amsterdam, Netherlands, 2016; pp 524−529, DOI: 10.1016/B978-0-12-3849472.00572-9. (9) Bihuniak, J. D.; Insogna, K. L. The Effects of Dietary Protein and Amino Acids on Skeletal Metabolism. Mol. Cell. Endocrinol. 2015, 410, 78−86. (10) Hídvégi, M.; Lásztity, R. Phytic Acid Content of Cereals and Legumes and Interaction with Protein. Period. Polytech., Chem. Eng. 2002, 46 (1−2), 59−64. (11) Caldwell, R. A. Effect of Calcium and Phytic Acid on the Activation of Trypsinogen and the Stability of Trypsin. J. Agric. Food Chem. 1992, 40 (1), 43−46. (12) Ali, F.; Ippersiel, D.; Lamarche, F.; Mondor, M. Characterization of Low-Phytate Soy Protein Isolates Produced by Membrane Technologies. Innovative Food Sci. Emerging Technol. 2010, 11 (1), 162−168. (13) Bye, J. W.; Cowieson, N. P.; Cowieson, A. J.; Selle, P. H.; Falconer, R. J. Dual Effects of Sodium Phytate on the Structural Stability and Solubility of Proteins. J. Agric. Food Chem. 2013, 61 (2), 290−295. (14) Silva, E. O.; Bracarense, A. P. F. R. L. Phytic Acid: From Antinutritional to Multiple Protection Factor of Organic Systems. J. Food Sci. 2016, 81 (6), R1357−R1362. (15) Johnson, R. A.; Manley, O. M.; Spuches, A. M.; Grossoehme, N. E. Dissecting ITC Data of Metal Ions Binding to Ligands and Proteins. Biochim. Biophys. Acta, Gen. Subj. 2016, 1860 (5), 892−901. (16) Quinn, C. F.; Carpenter, M. C.; Croteau, M. L.; Wilcox, D. E. Isothermal titration calorimetry measurements of metal ions binding to proteins. Methods Enzymol. 2016, 567, 3−21. H


Article

Journal of Agricultural and Food Chemistry Calorimetry, Spectroscopic, and Molecular Modeling Methods. J. Biol. Phys. 2016, 42 (3), 415−434. (36) Cheryan, M.; Rackis, J. J. Phytic Acid Interactions in Food Systems. C R C Crit. Rev. Food Sci. Nutr. 1980, 13 (4), 297−335. (37) Grynspan, F. F.; Cheryan, M. Phytate-Calcium Interactions with Soy Protein. J. Am. Oil Chem. Soc. 1989, 66 (1), 93−97. (38) Selle, P. H.; Cowieson, A. J.; Ravindran, V. Consequences of Calcium Interactions with Phytate and Phytase for Poultry and Pigs. Livest. Sci. 2009, 124 (1−3), 126−141. (39) Fredlund, K.; Isaksson, M.; Rossander-Hulthén, L.; Almgren, A.; Sandberg, A. S. Absorption of Zinc and Retention of Calcium: Dose-Dependent Inhibition by Phytate. J. Trace Elem. Med. Biol. 2006, 20 (1), 49−57. (40) Quiñone, D.; Veiga, N.; Torres, J.; Bazzicalupi, C.; Bianchi, A.; Kremer, C. Self-Assembly of Manganese(II)−Phytate Coordination Polymers: Synthesis, Crystal Structure, and Physicochemical Properties. ChemPlusChem 2017, 82 (5), 721−731. (41) Arakawa, T.; Timasheff, S. N. Mechanism of Protein Salting in and Salting out by Divalent Cation Salts: Balance between Hydration and Salt Binding. Biochemistry 1984, 23 (25), 5912−5923. (42) Selle, P. H.; Cowieson, A. J.; Cowieson, N. P.; Ravindran, V. Protein−phytate Interactions in Pig and Poultry Nutrition: A Reappraisal. Nutr. Res. Rev. 2012, 25 (1), 1−17. (43) Bretti, C.; Cigala, R. M.; Lando, G.; Milea, D.; Sammartano, S. Sequestering Ability of Phytate toward Biologically and Environmentally Relevant Trivalent Metal Cations. J. Agric. Food Chem. 2012, 60 (33), 8075−8082. (44) De Stefano, C.; Milea, D.; Porcino, N.; Sammartano, S. Speciation of Phytate Ion in Aqueous Solution. Sequestering Ability toward mercury(II) Cation in NaClaq at Different Ionic Strengths. J. Agric. Food Chem. 2006, 54 (4), 1459−1466. (45) Crea, F.; De Stefano, C.; Milea, D.; Sammartano, S. Formation and Stability of Phytate Complexes in Solution. Coord. Chem. Rev. 2008, 252 (10−11), 1108−1120.

I


Influence of Ternary Complexation between Bovine Serum Albumin

Recommend Documents