Inactivation of Soybean Trypsin Inhibitor by Epigallocatechin Gallate

Article pubs.acs.org/JAFC

Inactivation of Soybean Trypsin Inhibitor by Epigallocatechin Gallate: Stopped-Flow/Fluorescence, Thermodynamics, and Docking Studies Chun Liu,† Fenfen Cheng,† and Xiaoquan Yang*,†,‡ †

Research and Development Center of Food Proteins, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China ‡ Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou 510640, People’s Republic of China ABSTRACT: Tea is one of the most widely daily consumed beverages all over the world, and it is usually consumed with milk and/or soy milk. However, very few researches have studied the interactions between tea polyphenols (TPs) and soy milk proteins as compared with milk proteins. Here, we reported that epigallocatechin gallate (EGCG), a major component of TPs, can effectively inhibit the inhibitory activity of Kunitz trypsin inhibitor (KTI, a major antinutrient in soy milk). The mechanism of inactivation of KTI by EGCG was investigated by stopped-flow/fluorescence, thermodynamics, and docking studies. The results indicated that EGCG binds KTI via both hydrophobic and hydrophilic interactions with an association constant of 6.62 × 105 M−1 to form a 1:1 complex. Molecular docking showed the participation of amino acids includes three amino acid residues (Asn13, Pro72, and Trp117) near the reactive site of KTI, which may prevent KTI from contacting trypsin and hence inactivate KTI. KEYWORDS: soybean Kunitz trypsin inhibitor, EGCG, inactivation, interaction, spectroscopy, molecular docking

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INTRODUCTION

make a comparison between the effects of varying protein sources on these interactions. Kunitz trypsin inhibitor (KTI) has long been known as an antinutrient in humans consuming soy proteins, although the denatured inhibitors are highly nutritious, and it is high in sulfur amino acids as well as well-balanced proteins according to a dual-tracer approach to measurement (DIAAS) proposed by FAO in 2014.8 For this reason, great effort has been spent to design processing conditions for inactivating or removing trypsin inhibitors (TIs) from legumes. Most of the methodologies used in these studies are grounded in heat treatment.9 However, autoclaving and other extreme conditions applied to inactivate inhibitors may disrupt some vital essential amino acids like cysteine, lysine, arginine, etc.10 Additionally, dehydroalanine-derived bonds can be formed, and hence, lanthionine and lysinoalanine can be formed, especially at alkaline pH and high temperature. 11 Lysinoalanine is considered to be potentially harmful to human health.11 An alternative means of inactivating TIs could be provided by biochemical methods, which are economic, green, and environmentally friendly techniques and could be performed under mild conditions. Complexing soybean trypsin inhibitor with polyphenols could also be expected to be another way to inactivate its activity. TPs were found to possess an inactivation action on soybean TIs.12 Nevertheless, the mechanism of how soybean trypsin inhibitor is inactivated by TPs remains

The interactions between polyphenols and proteins may change the structural, functional, and nutritional properties as well as the digestibility of proteins.1 A typical example of protein−polyphenol interactions was described in research that shows various enzymes could be inhibited by phenolic compounds.2 It appeared that the inhibitory effect of polyphenols was ascribed to their capacity to bind to the enzyme in most of these studies.3 Polyphenols may inactivate the enzyme protein in the following three ways: precipitation, forming soluble inhibitor−enzyme complexes, and forming enzyme−substrate−inhibitor complexes.4 When the substrate of the enzyme is also a protein, namely proteases, the suppression of enzyme activity may result from polyphenol complexation with either the enzyme itself or the protein substrate.3,4 This is the reason why the interactions between protein and polyphenols lead to the loss of nutritional value of some food components and a decrease in the digestibility of proteins.1 Tea is one of the most popular daily consumed healthy beverages throughout the world. With a high antioxidant capacity, it is usually consumed with milk and/or soy milk. On the basis of the current literature, though some studies have aimed to improve our understanding of the interaction of TPs with milk proteins such as α- and β-caseins5 and β-lactoglobulin (βLG),6,7 few studies have examined the interactions of TPs with soy milk proteins such as trypsin inhibitors. From a perspective of nutritional value and digestibility, it makes great sense to conduct studies on some other food proteins like plant proteins to develop a clearer understanding of their effects and © XXXX American Chemical Society

Received: October 26, 2016 Revised: January 7, 2017 Accepted: January 9, 2017

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DOI: 10.1021/acs.jafc.6b04789 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Stopped-Flow and Steady-State Fluorescence Studies. Stopped-Flow Kinetic Experiments and Data Analysis. The kinetic measurements were performed by taking advantage of a stopped-flow apparatus (MOS-450) equipped with an SFM-20 device (BioLogic Science Instruments, Claix, France) according to the method described by Pan et al.19 The fluorescence signal described for each concentration of EGCG stands for the average of 8−12 individual shots. The kinetic data were fitted by using OriginPro 8.0 to calculate the observed rate, kobs. The data fit to a single-exponential decay. Assuming a simple mechanism of KTI binding, EGCG + KTI ↔ EGCG·KTI

unknown. Among TPs, EGCG is the main (accounting for ∼50% of the total catechins present) and most potent component of green tea.13 Moreover, with the increasing number of gallol groups, the efficiency of binding in the galloyl14 D-glucose series also increases: tri < tetra < penta. Thus, EGCG tends to play a significant role in inactivating trypsin inhibitor. The crystal structure of the KTI−porcine trypsin complex has been determined and subsequently refined [Protein Data Bank (PDB) entries 1AVU and 1BA7].15,16 In spite of a lack of strong electrostatic interactions or disulfide bonds, the reactive site loop can be preserved in a well-organized conformation through a network of hydrogen bonds that involves the Nterminal loop (residues 1−14).16 The side chain of Asn13 plays a critical role in the formation of hydrogen bonds with the amide N atom of Ser60 and both the main-chain carbonyl O atoms of Ile64 and Tyr62.15 According to the findings, the backbone conformation of the reactive site loop of KTI exhibits no significant change when forming a complex with porcine pancreatic trypsin (PPT),15 which suggests that any factors affecting the backbone conformation of the reactive site loop can cause a change in the inhibitory activity of KTI. In this study, the following topics have been examined. First, the inhibitory activities of KTI and EGCG against trypsin were determined. Second, the interactions between KTI and EGCG have been studied by a stopped-flow-based fast mixing technique, steady-state fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), and FTIR to examine the mechanism of inactivation of KTI by EGCG. Third, docking studies have been performed to develop a better and clearer visualization of the residues that are involved in the interaction. Finally, a possible mechanism of inactivation of KTI by EGCG is discussed.

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kobs = kon[EGCG] + koff

(1)

in which the slope is the bimolecular association rate, kon, and the yintercept is the dissociation rate, koff.20 The KD value was determined by utilizing the kon value and the koff value with the following equation:20,21

KD = koff /kon Steady-State Fluorescence. Fluorometric assays were conducted on an F7000 fluorescence spectrophotometer (Hitachi Co.) at 25 °C. A 1 cm path length cell was used. Solutions used for fluorescence spectra contained a fixed KTI concentration (0.4 mg/mL), and the concentrations of EGCG range from 0 to 0.04 mg/mL; 1.0 mL of a KTI stock solution (2 mg/mL) and different volumes of EGCG stock solutions (2 mg/mL) were mixed and then diluted to 5 mL with 10 mM phosphate buffer (pH 7.0) to obtain final polyphenol concentrations. The EGCG solutions were kept in the dark and used as quickly as possible. The fluorescence spectra were recorded at a λexc of 280 nm and λemi values from 300 to 500 nm. The reference sample consisting of the phosphate buffer and EGCG at the same concentration was checked for fluorescence signals. The data for the tryptophan (Trp) fluorescence quenching were analyzed by adopting the Stern−Volmer equation, which can be shown as follows:22 F0/F = 1 + kqτ0[Q] = 1 + KSV[Q]

(2)

With respect to static quenching, binding constant Ka and the number of binding places (n) were calculated by utilizing a double logarithmic22,23

MATERIALS AND METHODS

Materials. Kunitz trypsin inhibitor (KTI) (>90% pure), porcine pancreatic trypsin (∼1500 units/mg, 1496 ± 23 units/mg of solid detected in this work), and Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPNA) (>98% pure) were purchased from SigmaAldrich (Shanghai, China). (−)-Epigallocatechin gallate (EGCG) (>90% pure) was purchased from Changsha Sunfull Biotech Co., Ltd. (Changsha, China). All other chemicals used in this study were of analytical grade, and all the solutions were freshly prepared. Inactivation of the KTI Inhibitory Activity Assay. The inhibitory activities of KTI and EGCG against trypsin were determined in light of the work of Smith et al.17 One trypsin inhibitory activity unit (TIU) stands for a decrease of 0.01 absorbance unit at 410 nm in 10 mL of the reaction mixture. KTI (0.1 mg/mL) and EGCG (1 mg/mL) solutions were prepared in deionized water and then diluted to different concentrations in deionized water. All these operations were performed in deionized water instead of phosphate buffer at pH 7.0, because if BAPNA was pipetted into the mixtures in a phosphate-buffered solution, the precipitate was observed. The residual activities of trypsin were measured according to the method described in ref 18. Effect of EGCG on Trypsin Inhibitory (TI) Activities in KTI− Trypsin Complexes. The experiment for examining the effects of EGCG on TI activities in KTI−trypsin complexes was performed according to the method described by Huang et al.,12 but EGCG instead of TPs was used during the measurement process. Measurement of Inhibitory Pattern of Trypsin by EGCG. To measure the inhibitory pattern of trypsin activity by EGCG, trypsin activities were tested at varying BAPNA substrate concentration (from 51.09 to 459.90 μmol) and along with 0, 0.2, and 0.4 mg/mL EGCG. The data was analyzed by Lineweaver−Burk plots.12

log[(F0 − F )/F ] = log K a + n log[Q]

(3)

The intercept of the double-logarithmic Stern−Volmer plot provides Ka, and the slope yields n. Thermodynamic Measurements. The thermodynamic measurements were taken in a Nano ITC low-volume instrument (TA Instruments, Newcastle, DE) at room temperature (25 °C).24 A 250 μL KTI solution (5 mg/mL) was inserted into the sample cell, and the syringe was filled with 50 μL of an EGCG solution (10 mg/mL) in a typical experiment. All solutions were prepared in 10 mM phosphate buffer (pH 7.0) and degassed before titration. After they have reached equilibrium, the EGCG solution was titrated into the sample cell dropwise, and the volume of each titration was 2 μL. The time delay between successive injections was 350 s. The titrations of EGCG into buffer, buffer into KTI, and buffer into buffer were control experiments. The data were analyzed according to our previous work.25 Structural Characterization of KTI−EGCG Complexes. Circular Dichroism Spectroscopy. CD spectra of KTI and KTI−EGCG complexes were scanned by a MOS-450 spectrometer (BioLogic Science Instruments) according to our previous work.8 For measurements in the far-UV region (190−250 nm), the concentration of KTI was kept constant (0.1 mg/mL) while the EGCG concentration was varied (0.001, 0.1, and 0.2 mg/mL). The secondary structure (α-helix, β-sheet, β-turn, and random coil) of KTI was calculated using CDSSTR in the CD Pro software. For near-UV CD (260−340 nm), the concentration of KTI was kept constant (1 mg/mL) while the EGCG concentration was varied (0.01, 0.1, and 0.2 mg/mL). FTIR Spectroscopic Measurements. Infrared absorption spectra were recorded with a Bruker VERTEX 70 spectrometer. A solution of B


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Figure 1. (A) Inactivation of KTI by EGCG. The concentration of KTI is 0.015 mg/mL. The inset shows the trypsin activity restored from the KTI−trypsin complex by adding EGCG. The concentration of trypsin is 0.1 mg/mL. (B) Inactivation of trypsin by EGCG. The concentration of trypsin is 0.1 mg/mL. The inset shows the inhibitory pattern exhibited by EGCG on trypsin during hydrolysis of BAPNA as shown by Lineweaver− Burk plots.

Figure 2. Stopped-flow analysis of EGCG binding KTI. (A) Kinetic traces for EGCG (at 0.005, 0.01, 0.015, 0.02, and 0.025 mg/mL) binding to KTI in 10 mM PBS buffer at pH 7.4. Each trace is the average of 8−12 independent measurements and is fitted with a single-exponential function (). (B) The observed rate constants (kobs) plotted vs EGCG concentration provided a linear fit. Each point represents the average of three individual experiments.

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EGCG was added dropwise into the KTI solution while it was being stirred constantly to form a homogeneous solution and realize the target that EGCG should reach at a concentration of 0.02 or 0.04 mg/ mL with a final protein concentration of 0.4 mg/mL. The samples analyzed in this study were free KTI and freeze-dried KTI−EGCG complexes. The subtractions and Fourier self-deconvolutions (FSD) employing a Lorentzian line shape were performed with the purpose of studying the amide I region of proteins by adopting Peak Fit version 4.12 (SeaSolve Software).26 Band assignments in the amide I region (1600−1700 cm−1) were made in accordance with refs 27 and 28. Docking Studies. On the basis of the results of the experiment, molecular docking was performed for the sake of visualizing the sites of binding of EGCG to KTI. To produce the KTI−EGCG complex, the crystal structure of KTI available at 2.3 Å resolution (PDB entry 1AVU) was chosen as a template and the three-dimensional structure of EGCG was obtained from PubChem entry 65064. On the basis of the results of a global search, Trp117 of KTI was chosen as a potential binding site. Docking of EGCG to KTI was performed with AUTODOCK4.1.29 The procedure included the creation of a 10 grid that took Trp117 to be the center. Ligand charges were calculated by adopting the Gasteiger method. The binding affinity of EGCG for the vicinity of Trp117 of the KTI was studied by employing a simulated annealing procedure of Accelrys Discovery Studio version 2.5. Calculations were performed by taking advantage of the CHARMm force fields PARM22.30

RESULTS Inactivation of KTI by EGCG. Figure 1A shows the trypsin inhibitor activity (TIA) of KTI when KTI was complexed with EGCG in various ratios. The curves indicate that the TIA of KTI could be inactivated significantly by such complexation. In the absence of EGCG, the TIA was 1354 units/mg, indicating the effective inhibition of trypsin activity (TA) by KTI. When KTI (3 μg/mL) at the same concentration formed a complex with EGCG (0−0.33 mg/mL) at different concentrations in deionized water, the TIA decreased as the EGCG concentration increased. It means that the TIA of KTI was reduced by the formation of a complex with EGCG. The rate of suppression of the TIA of KTI achieved a maximal value (75.30%) when the EGCG/KTI ratio (w/w) reached 40. When the EGCG/KTI ratio was >40, the TIA increased, which probably resulted from the interaction between the surplus EGCG and trypsin as it lowered its activity. Meanwhile, EGCG’s ability to inactivate KTI in trypsin−KTI complexes was also analyzed by assaying the restored TA in the system by adding an EGCG solution to the trypsin−KTI solution (Figure 1A, inset). When the EGCG solution was added to the trypsin−KTI solution, trypsin was completely inhibited, and the TA was partially recovered in a dose-dependent fashion. When 1.4 mg/mL EGCG was added, the TA increased to a maximal value of ∼73 units/mg for the C


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Figure 3. (A) Effect of EGCG on the intrinsic fluorescence of KTI (0.4 mg/mL). (B) Stern−Volmer plots describing fluorescence quenching of KTI in the presence of EGCG. The inset shows the double-logarithmic Stern−Volmer plot describing static quenching of KTI in the presence of EGCG.

from KTI, especially when EGCG was at a higher concentration. Steady-State Fluorescence. The fluorescence quenching technique was often employed to investigate the interactions of small molecules with proteins.31 Figure 3A describes the action of EGCG on the Trp intrinsic fluorescence emission spectrum of KTI. The pure KTI exhibited an emission spectrum centered around 337 nm. With an increasing concentration of EGCG (from 0.004 to 0.04 mg/mL), the fluorescence intensity of KTI decreased dramatically, indicating that EGCG interacted with KTI. The Stern−Volmer quenching constant could be acquired via fitting the data to Stern−Volmer plots. As shown in Figure 3B, the value of Kq (3.28 × 1012 M−1 s−1) is much higher than the maximal scatter collision quenching constant of a variety of quenchers (2.0 × 1010 M−1 s−1), which indicated that the fluorescence quenching induced by EGCG is static quenching.32 This result is consistent with that of previous studies,33 which suggested that EGCG could act as a direct Trp fluorescence quencher and the structure of KTI has been changed. Additionally, as the concentration of EGCG was increasing, the maximal emission wavelength underwent a red shift from 337 to 348 nm, which indicated that the Trp residues of KTI were placed in a more hydrophilic environment upon addition of EGCG. In the KTI molecule, there are two Trp residues: Trp93 is buried in the hydrophobic core of the folded polypeptide chains, and Trp117 is located in a solvent-exposed molecular surface. The interactions between KTI and EGCG probably led to partial unfolding of the protein; thereby, Trp93 is exposed to the aqueous medium, which is consistent with the stopped-flow fluorescence measurement (Figure 2). It is also in accord with previous reports about the binding of EGCG to αand β-casein.5 For the static mechanism of quenching, the doublelogarithmic Stern−Volmer equation (eq 3) could be employed to analyze the quenching data and calculate the apparent binding constants (Ka) upon the formation of EGCG−KTI complexes. As shown in the B inset of Figure 3, the Ka and the number of binding sites per KTI molecule (n) for EGCG with KTI were 6.62 × 105 L M−1 and 1.31, respectively. The great magnitude of Ka revealed strong binding between KTI and EGCG. The value of n was 1.31, demonstrating roughly one association site on KTI for EGCG. Thermodynamic Analysis of Binding of EGCG to KTI. The interaction between KTI and EGCG was further quantitatively studied by ITC, as shown in Figure 4. Upon

trypsin−KTI complex. It appeared that EGCG has removed some KTI from the active sites of trypsin; consequently, a small quantity of the TA was restored. Nevertheless, the majority of the KTI could not be inactivated by EGCG once the complexes between the trypsin inhibitor and trypsin had been formed because the TA would be ∼1500 units/mg at 100% restoration as has been expected. To confirm the ability of EGCG to inactivate trypsin, the assay of the effect of EGCG on trypsin activity was performed. Figure 1B shows that trypsin could also be inactivated by EGCG especially at relatively high concentrations. However, the inhibitory effect on trypsin was not always increased with an increase in EGCG concentration. The maximal inhibitory effect on trypsin (∼32.5%) appeared at an EGCG concentration of 1.25 mg/mL; when the EGCG concentration was increased until it was >1.25 mg/mL, the inhibitory effect on trypsin exhibited no obvious increase. These results verified that KTI could be inactivated exactly by EGCG. To measure the inhibitory pattern of trypsin by EGCG, the reaction kinetics of trypsin was investigated through different substrate (BAPNA) and EGCG concentrations. The Lineweaver−Burk plot that shows the changes in vmax and Km for trypsin inhibited by EGCG is presented in the inset of Figure 1B. As can be seen, the Km value for free trypsin was 6.76 × 10−4 mol/L BAPNA. In the presence of EGCG, the vmax value for trypsin decreased while the Km value remained unchanged. This result suggested that EGCG exhibited a pattern of noncompetitive inhibition of trypsin. Stopped-Flow and Steady-State Fluorescence Studies. Stopped-Flow Fluorescence. The tryptophan fluorescence of KTI, as an indicator of the progress of the binding-induced structural changes in KTI through the stopped-flow ultrafast mixing method, is presented in Figure 2. As shown in Figure 2A, the fluorescence of KTI dropped markedly when it was mixed with EGCG, which suggested a rapid structural change in KTI when binding took place. The amplitude of the overall fluorescence change had a direct relationship with the amount of EGCG that was added to the KTI solution. The observed rate constant, kobs, varies from ∼0.88 to ∼2.32 s−1 with EGCG concentration (Figure 2B). Fitting the relationship of kobs with EGCG concentration using eq 1 yields a kon of 32.30 μM−1 s−1 and a koff of 0.50 s−1. Therefore, the dissociation constant of EGCG with KTI, KD, is 0.02 μM. These data indicate that EGCG tended to associate with KTI rather than to dissociate D


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Figure 4. (A) Heat flows recorded upon injection of an EGCG solution (10 mg/mL) into a KTI solution (5 mg/mL) at 25 °C. (B) Molar heat values obtained through integration of the individual heat-flow signals as a function of the mixing ratio (EGCG/KTI) concentration.

Figure 5. Circular dichroism (CD) spectra of KTI and its EGCG complexes at different EGCG concentrations: (A) far-UV CD spectra and (B) near-UV CD spectra.

actions combined with hydrogen bonds.34 Similar findings for the interaction of TPs with milk proteins were observed.35 Structural Characteristics of KTI−EGCG Complexes. CD Spectroscopy. CD is an important and direct technique for protein structure studies. CD spectra of pure KTI and its EGCG complexes at different EGCG concentrations are shown in Figure 5. The fractional contents of the secondary structure of samples were calculated as shown in Table 1. As can be seen,

injection of aliquots of the EGCG solution into the KTI solution, the corrected heat trace peaks in Figure 4A demonstrated exothermic enthalpy released in the titration process, which should be caused by the binding of EGCG to KTI. On the basis of the spectroscopic measurements, we can conclude that EGCG could actually bind to the KTI cooperatively, resulting in the formation of exothermic peaks. Furthermore, a reduction in the magnitude of the exothermic peaks was seen upon addition of EGCG continuously (Figure 4A), which suggested that the number of available binding sites on the KTI was decreased. The addition of more EGCG would lead to a plateau in the exothermic peaks once the protein became saturated with EGCG. The data were best fitted to the model with one single binding site as shown in Figure 4B, where fitting parameters Ka (binding constant), ΔH (binding enthalpy), and ΔS (binding entropy) were provided. Generally, interactions with a Ka of 103 M−1 are not considered to possess a very strong affinity.30 Thus, the binding constant (Ka = 1.0 × 103 M−1) observed in this work indicated that the binding effect of EGCG with KTI was much weaker. The low level of binding could be attributed to the net negative charge that KTI bears at pH 7.0, producing electrostatic repulsions with the negatively charged molecules of EGCG. The binding of EGCG to KTI mainly occurred with the help of hydrogen bonds and hydrophobic interactions, which was based on the fact that binding was driven by entropy (ΔS > 0, and ΔG < 0), for it is universally accepted that an entropy-driven reaction mainly includes hydrophobic inter-

Table 1. Secondary Structure of the KTI Complex at Different EGCG Concentrations and pH 7.0 [EGCG] (mg/mL)

α-helix (%)

β-sheet (%)

β-turn (%)

random coil (%)

0 0.001 0.01 0.02

5.9 7.4 16.3 17.6

19.6 20.4 19.3 20.8

15.5 15.4 16.0 16.3

59.2 56.7 48.7 45.6

KTI had a CD spectrum feature of the class of β-II proteins35 with a minimal ellipticity around 201 nm. When the concentration of EGCG increased, the KTI α-helical content increased (from 5.9 to 18.2%) mostly at the expense of its random coil content (from 59.2 to 48%) (Figure 5A and Table 1), which means that EGCG induces a random coil to α-helix transition in the protein. The data in Figure 5B show that the near-UV CD spectra for the same interaction, which indicate changes at the level of tertiary structure. The maximal ellipticity of KTI is obtained E


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Journal of Agricultural and Food Chemistry because of the side-chain interactions, especially the aromatic residues that are located in the hydrophobic core of the protein. The CD spectra in the region of 260−320 nm arose from the aromatic amino acids (Phe, 260−270 nm; Tyr, 275−282 nm; Trp, 290−305 nm).37 In addition, the actual shape and magnitude of the near-UV CD spectrum of a protein would depend on the amount of aromatic amino acids in each type, their mobility, the nature of their environment, and their spatial disposition in the protein.37 As the concentration of EGCG increased, the ellipticity decreased, particularly within the range of 273−275 nm. When the concentration of EGCG reached 0.2 mg/mL, most of the tertiary structural interactions had been lost. The decrease in near-UV ellipticity that occurred under the circumstances of EGCG at such a concentration also indicated that native KTI underwent a transformation in conformation and experienced changes in the hydrophobic environment of aromatic residues. The loss of the Trp peaks in the spectrum indicates that the environment of these residues became less constrained than that of native KTI. Consequently, the protein has suffered an unfolding transition; i.e., it has experienced a change from a folded conformation (its native state) to a more expanded one (a set of equilibrium, partially unfolded conformations).38 These results are consistent with the data of stopped-flow and steady-state fluorescence studies (Figures 2 and 3). FTIR Spectra. The spectra of pure KTI and the KTI−EGCG complex are shown in Figure 6. As can be seen, there was no obvious spectral shifting for the KTI amide I band at 1656− 1652 cm−1 and the amide II band at 1544−1540 cm−1 when EGCG interaction occurred. The change in the intensity of the amide I and II bands was attributed to the binding of polyphenol to protein C−N, N−H, and CO groups. Other evidence supporting the interaction of polyphenol with C−N and N−H groups is the shift from the protein amide A band at 3290 cm−1 (N−H stretching mode) in native KTI to 3400 cm−1 upon its interaction with EGCG. Generally, the characteristic peak of intermolecular hydrogen bonds is at 3400 cm−1. Therefore, it can be inferred that hydrogen bonds may be one of the forces involved in interactions between KTI and EGCG. To obtain more information from the amide I band, Fourier self-deconvolution (FSD) curve fitting of the amide I band (1700−1600 cm−1) was performed. The deconvolved infrared spectra of pure KTI and the KTI−EGCG complex (at an EGCG concentration of 0.04 mg/mL) are illustrated in panels B and C of Figure 6, respectively. The FTIR spectra of pure KTI showed five bands at 1684 (antiparallel β-sheet), 1667 (βturn), 1651 (random coil), 1637 (β-sheet), and 1623 (antiparallel β-sheet) cm−1,27,28 while the FTIR spectra of KTI-EGCG complex exhibited six bands at 1687 cm−1 (antiparallel β-sheet/aggregated strands), 1673 cm−1 (antiparallel β-sheet), 1659 cm−1 (α-helix), 1645 cm−1 (unordered), 1632 cm−1 (β-sheet), and 1619 cm−1 (antiparallel β-sheet).27,28 The latter three bands shifted to lower wavenumbers by 4−6 cm−1 when compared to those of KTI. Such shifting from the amide I bands to lower wavenumbers may result from the decreased strength of the CO bond stretching vibration because of an increase in the extent of hydrogen bonding.39 The extended chains in partially unfolding denatured protein allow very close alignment of neighboring chains, which favors the formation of very strong intermolecular hydrogen bonds. Docking Study. On the basis of the data from experiment, molecular docking was performed to determine the binding site

Figure 6. (A) FTIR spectra of pure KTI (a, 0.04 mg/mL) and FTIR spectra of the EGCG−KTI complex [EGCG concentrations of (b) 0.02 and (c) 0.04 mg/mL]. (B) Infrared spectra of pure KTI in the amide I region. (C) Infrared spectra of the KTI−EGCG complex (corresponding to spectrum c in panel A) in the amide I region. The color lines represent the best fits obtained by band decomposition.

position and the mode of binding of EGCG to KTI. The typical construction derived from the best pose with the minimal binding energy is illustrated in Figure 7. A pose of the binding of EGCG within a region near the reactive site (Arg63-Ile64) of KTI was obtained. EGCG is in the vicinity of Asn13, Glu92, Asp115, Trp117, Ser74, Pro72, and Ser60. Hydrogen bonds were observed between EGCG and these amino acid residues that stabilize polyphenol−protein complexes. Furthermore, π−π stacking (about parallel, with an interplanar separation of ∼4.1 Å measured by Pymol) between a benzene ring (Aring) of EGCG and the aromatic ring of Trp117 of KTI was F


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Figure 7. Molecular docking studies. (A) Cartoon ribbon model structure of KTI showing the binding of EGCG (green) in the vicinity of the active site (Arg63 and Ile64, indicated by purple lines) of KTI. Residues of interest and EGCG are represented as lines and sticks, respectively. Amino acid residues in the vicinity of the EGCG molecule are labeled, and hydrogen bonds are shown as dashed yellow lines. (B) Binding of EGCG to the molecular surface of KTI. The orientation from the original structure (PDB entry 1AVU) has been changed to observe the binding of EGCG to KTI clearly.

mechanism of inhibition has been shown.3 In this study, the inhibition for KTI observed in the presence of EGCG was probably of the competitive type, which was supported by the data of the effect of EGCG on TIA in KTI−trypsin complexes (Figure 1A, inset) and molecular docking (Figure 7). Interactions between polyphenol and protein, especially the noncovalent interactions, were investigated by a number of methods. However, limitations remain, though these approaches present complementary evidence, most of which is fragmentary. Therefore, it is imperative to make a combination of them to explain the phenomenon as a whole. Haslam and coworkers14,42−44 adopted global methods to conduct their research, which allowed them to identify the driving forces and determine the structure−property relationship. All previous studies of the noncovalent interactions between polyphenol and protein center on a mechanism that involves weak associations and a more accurate combination of hydrophobic interactions and hydrogen bonds. In this study, interactions between KTI and EGCG also involved noncovalent weak associations that combine hydrogen bonds with hydrophobic interactions (Figures 2−4, 6, and 7). Hydrophobic interactions would involve hydrophobic sites of proteins and aromatic rings of polyphenols, like pyrrolidine rings of prolyl residues, while hydrogen bonding takes place between H-acceptor sites of the proteins and the hydroxyl groups of the polyphenols.3 KTI is part of the substrate-like inhibitor family and has an exposed reactive site (Arg63-Ile64) loop,15 and this loop is not subjected to the secondary structure elements or disulfide bridges that could restrict its freedom of conformation, which is much different from the case for many other proteinase inhibitors.15 In KTI, the side chain of Asn13 plays a significant role in stabilizing the reactive loop conformation via a network of hydrogen bonds. The mode of interaction between KTI and porcine pancreatic trypsin (PPT) has been depicted by Song et al.15 According to them, 12 of 181 amino acid residues in KTI came into contact with PPT in the orthorhombic crystal structure, and a majority of these contacts involved five residues

also observed. In addition, apolar hydrophobic interactions between the gallate benzene ring (D-ring) of EGCG and Pro72 of KTI existed to some extent. Among these interactions, it is noteworthy that two hydrogen bonds were formed between the side chain of Asn13 of KTI and the hydroxyl groups of the Cring of EGCG. As we know, the side chain of Asn13 plays a crucial part in stabilizing the reactive loop conformation via a network of hydrogen bonds. Thus, the interaction of EGCG with Asn13 may change the reactive loop conformation and hence change the inhibitory activity of KTI. The binding interaction between EGCG and the surface of KTI indicated that EGCG was located in a relatively hydrophobic region (Figure 7B).

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DISCUSSION In the past six decades, plant polyphenols have made great contributions to improving the taste and palatability of foodstuffs and beverages and to understanding the adverse nutritional effects in forage crops, which helps to identify more strategies of plant defense and herbal remedies. Researchers’ growing recognition of this has considerably enriched the studies of protein−polyphenol interactions.4 A deeper and clearer understanding of polyphenol−protein interactions not only could be beneficial for regulating the functional properties of proteins in food processing, transportation, and storage but also could contribute to the development of new food products with better nutritional value. In this study, EGCG, the main component of TPs, could inactivate KTI by forming soluble KTI−EGCG complexes (Figures 1A and 7). Therefore, it is important to improve the nutritional value of soy milk because KTI is the main trypsin inhibitor in soy milk, and heat treatments cannot completely inactivate all inhibitors.36 The competitive, noncompetitive, or uncompetitive character of the inhibition is highlighted by kinetic measurements. Most of the inhibition observed in the presence of polyphenols is of the uncompetitive type,40,41 except those inactivated by monomeric flavonols and their glycosides, for which a competitive G


Article


Figure 8. Possible mechanism of inactivation of KTI by EGCG.

Funding

in the reactive site loop [Pro6 (P3)−Arg65 (P2′)]. P1 residue Arg63 made the broadest hydrogen bonds with PPT and formed six hydrogen bonds in all. The side chain of Arg63 holds the position as has been expected in the primary binding pocket of PPT.15 It was also found that the backbone conformation of the reactive site loop of KTI did not show any significant change when it was forming a complex with PPT, which was consistent with similar main-chain dihedral angles for residues P4−P3′.15 In this study, interactions between KTI and EGCG involved Asn13, Pro72, and Trp117 as indicated by molecular docking (Figure 7), which may prevent KTI from making contact with trypsin and hence inactivate KTI (Figure 8). In summary, the TIA of KTI can be inactivated effectively by complexation of EGCG at an EGCG/KTI ratio of 40/1 and pH 7.0 under ambient conditions. A kinetic analysis by stoppedflow fluorescence has been performed for the first time, which revealed that EGCG tended to associate with KTI rather than to dissociate from KTI, especially at a higher concentration of EGCG. Steady-state fluorescence indicated that EGCG quenched the fluorescence intensity of KTI via a static quenching process (Kq = 3.28 × 1012 M−1 s−1; Ka = 6.62 × 105 L mol−1; n = 1.31). The changes in enthalpy (ΔH) and entropy (ΔS) of the interaction derived from the ITC data are −14.4 kJ mol−1 and 10 ± 1.68 kJ mol−1 K−1, respectively, suggesting EGCG binds KTI via both hydrophilic and hydrophobic interactions. CD spectroscopy showed that the KTI has changed from a folded conformation to a more expanded conformation in the KTI−EGCG complex, and FTIR revealed the formation of intermolecular hydrogen bonds in the KTI−EGCG complex. Molecular docking indicated that the participation of amino acids included three amino acid residues (Asn13, Pro72, and Trp117) in the vicinity of the reactive site of KTI, which may prevent KTI from making contact with trypsin and hence inactivate KTI.

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This research was supported by grants from the Project of National Key Technology Research and Development Program for the National High Technology Research and Development Program of China (863 Program, 2013AA102208). Notes

The authors declare no competing financial interest.

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Xiaoquan Yang: 0000-0002-4016-9834 Author Contributions

C.L. and F.C. contributed equally to this work. H


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Inactivation of Soybean Trypsin Inhibitor by Epigallocatechin Gallate

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