Formation of a Multiligand Complex of Bovine Serum Albumin with

Article pubs.acs.org/JAFC

Formation of a Multiligand Complex of Bovine Serum Albumin with Retinol, Resveratrol, and (−)-Epigallocatechin-3-gallate for the Protection of Bioactive Components Yi Wu,† Hao Cheng,† Yantao Chen,*,‡ Lingyun Chen,§ Zheng Fang,† and Li Liang*,† †

State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China ‡ Shenzhen Key Lab of Functional Polymer, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, China § Department of Agricultural, Food & Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2R3, Canada ABSTRACT: Clarification of the interaction mechanisms between proteins and bioactive components is important to develop effective carriers for encapsulation and protection of bioactive components. Bovine serum albumin (BSA), a globular protein in serum and milk, contains multiple sites to bind a variety of low-molecular-weight molecules, forming protein−monoligand complexes. In this study, the interactions of BSA with retinol, resveratrol, and/or (−)-epigallocatechin-3-gallate (EGCG) were investigated by using fluorescence, circular dichroism, and molecular docking techniques. BSA-triligand complexes were successfully formed when added in the sequence of retinol, resveratrol, and EGCG. The stability of these bioactive components was improved in the complexes relative to free ones. The complexes provided a better protective effect on retinol and resveratrol than did BSA-monoligand complexes, in which the presence of EGCG played an important role. KEYWORDS: bovine serum albumin, retinol, resveratrol, (−)-epigallocatechin-3-gallate, complex

■

INTRODUCTION Understanding of the interactions between proteins and ligands is important for the prediction of protein function, the design of drugs based on structure, and the development of lipid-free carriers.1,2 Food-associated ligands including flavoring agents, colorants, bioactive components, or preservatives can be protected and delivered to the desired sites by binding to carrier proteins.2 As natural vehicles, proteins have a potential role in the encapsulation, protection, and delivery of bioactive components through functional foods because of their ability to form protein−ligand complexes, which can possibly improve the efficiency of bioactive components by increasing their solubility, stability, bioavailability, or by controlling their release in the gastro-intestinal tract.2,3 Clarification of the underlying mechanisms of protein bindings with ligands is important for the development of effective carriers as delivery systems of food-associated ligands.4 Bovine serum albumin (BSA), found both in blood serum and in milk, is a large globular protein having a molecular weight of 66 kDa, 583 amino acids, 17 disulfide bridges, and a free thiol in a single polypeptide chain. The protein functions primarily as a transporter of endogenous and exogenous compounds in the circulatory system. BSA is made up of three homologous domains (I−III) that are divided into nine loops by 17 disulfide bonds. Each domain is composed of two subdomains (A and B). The six subdomains assemble to form a column-shaped structure.4−6 BSA contains three domains specified for metal ion, lipid, and nucleotide binding.7,8 The two main binding sites are respectively located in the hydrophobic cavities of the subdomains IIA and IIIA, namely, site I and site II.9 © XXXX American Chemical Society

BSA is able to bind a wide range of compounds including drugs, fatty acids, steroids, dyes, metals, vitamins, polyphenols, and carotenoids.6,10−14 Most studies have focused on the interaction of BSA with single ligands to form complexes. Retinol belongs to the A-group hydrophobic vitamins and could interact with BSA to form complexes with a binding constant of about 5.3 × 106 M−1 and a binding number of about 1.20.12 Resveratrol (3,5,4′-trihydroxystilbene), a natural amphiphilic polyphenolic compound, could bind to BSA with a binding constant of about 2.52 × 104 M−1 and a binding number of about 1.26. Its binding site on BSA was mainly in the vicinity of Trp134 and Trp212 located in the protein domains I and II.6 (−)-Epigallocatechin-3-gallate (EGCG), one component of catechins with good solubility in water, could bind to BSA with a binding constant of about 1.4 × 106 M−1.11,15 The galloyl group of EGCG was important for the binding to BSA and the site on BSA was possibly located within the hydrophobic pocket between subdomains IIA and IIIA.15−17 It is of industrial interest to develop the products that contain several bioactive components in the market, thus providing multiple health benefits.18,19 Moreover, the synergistic effect was reported in the combination of different bioactive components.20−22 It is thus required to develop the carriers that can simultaneously encapsulate and protect different bioactive components. β-Lactoglobulin, which contains a primary binding site in the internal cavity and multiple external Received: Revised: Accepted: Published: A

January 21, 2017 March 18, 2017 March 22, 2017 March 22, 2017 DOI: 10.1021/acs.jafc.7b00326 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

resveratrol, and EGCG as ligand models. The protective effect of the complexes on bioactive components was also investigated.

■

MATERIALS AND METHODS

Materials. BSA (product number A1933, purity ≥98%), BSA (product number V900933, purity ≥98%), resveratrol (trans-isomer, purity ≥99%), retinol (purity ≥97.5%), and EGCG (pharmaceutical secondary standard) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). BSA (A1933) was used for experiments except for the specific indication. Warfarin (purity ≥98%), ibuprofen (purity ≥98%), and methanol (HPLC grade) were purchased from J&K Scientific Ltd. (Shanghai, China). Other reagents were of analytical grade and purchased from SinoPharm CNCM Ltd. (Shanghai, China). Sample Preparation. Stock solution of BSA in 10 mM phosphate buffer at pH 7.4 was prepared at a concentration of 100 μM, which was determined based on absorbance around 280 nm using a molar extinction coefficient of 43,000 M−1cm−1.10 Stock solutions of resveratrol and retinol at 200 μM were prepared freshly by dissolving in 70% ethanol and absolute ethanol, respectively, at 2 mM and then diluted into phosphate buffer at pH 7.4. Stock solution of EGCG at 200 μM was prepared by dissolving directly in phosphate buffer. Mixtures of BSA and bioactive components were prepared by adding both stock solutions to phosphate buffer in varying proportions. Mixtures containing more than one bioactive components were prepared by adding stock solutions of different bioactive components sequentially to BSA phosphate buffer at 0.5 h intervals. The designations (e.g., retinol-resveratrol-EGCG or BSA-retinol-resveratrolEGCG) indicate the addition sequence. Warfarin or ibuprofen stock solution at 200 μM in phosphate buffer was added into BSA (V900933) solution before bioactive components. The highest resulting ethanol concentration in samples was 1.5%, which had no impact on the native structure of BSA (data not shown). All samples were prepared and tested at least in duplicate for each analysis. Fluorescence Measurements. Steady-state fluorescence with a spectral resolution of 2.5 nm for both excitation and emission was measured in 10 mm quartz cuvettes using a FluoroMax-4 fluorescence spectrophotometer (Horiba Jobin Yvon Inc., Edison, NJ). Protein intrinsic fluorescence emission spectra were recorded from 290 to 550 nm at an excitation wavelength of 280 nm. Backgrounds of bioactive components were subtracted from the raw spectra. Fluorescence intensity was normalized relative to that of BSA at the emission maximum (λmax) in the absence of the last ligand unless indicated otherwise. Quenching of protein fluorescence in the presence of a bioactive component is expressed using the expression [(F0 − FL)/F0 × 100%], FL and F0 are the protein fluorescence measured at λmax in the presence and absence of the component, respectively. A high percentage indicates strong binding of the ligand.25 In the site marker competitive experiments, backgrounds of warfarin and ibuprofen were subtracted from the raw spectra, and quenching of protein fluorescence in the presence of a bioactive component was expressed using the expression [(Fm0 − FmL)/Fm0 × 100%], where Fm0 and FmL are the protein fluorescence measured at λmax in the presence of a site marker and in the presence of a site marker and the component, respectively. Fluorescence emission spectra of bioactive components were recorded from 330 to 630 nm with an excitation wavelength of 320 nm. The background of BSA was subtracted from the raw spectra. Circular Dichroism (CD) Measurements. Far-UV CD spectra of BSA in the presence of retinol, resveratrol, or EGCG at 0, 2, 5, 10, 15, and 20 μM and the presence of all bioactive molecules with a concentration each of 5 μM were recorded in 1 mm quartz cuvettes with a MOS-450/AF-CD spectropolarimeter (Bio-Logic Science Instruments, Grenoble, France). Ellipiticity was recorded from 190 to 250 nm at a speed of 100 nm/min and 1.0 nm bandwidth. Molecular Modeling and Docking. The initial structure of BSA was retrieved from the RCSB Protein Data Bank (http://www.rcsb. org/pdb, ID: 4JK4) and then relaxed with molecular dynamics

Figure 1. Fluorescence spectra of 2 μM BSA in the presence of 0, 2, 5, 10, 15, and 20 μM resveratrol (RES, A), retinol (RET, B), or (−)-epigallocatechin-3-gallate (EGCG, C).

potential binding sites, was reported to bind α-tocopherol, resveratrol, and folic acid simultaneously to form proteintriligand complexes.23 A ternary ilaprazole-BSA-(+)-catechin complex might be formed, but the first formation of the (+)-catechin-BSA complex made it more difficult for ilaprazole to bind with BSA.24 This area of research is still very limited. In this study, we aim to study the interactions in protein-triligand complexes by using BSA as a protein model and retinol, B

DOI: 10.1021/acs.jafc.7b00326 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article


Figure 2. Fluorescence emission spectra of 2 μM BSA in the presence of 0, 2, 5, 10, 15, and 20 μM retinol (RET, A and D), resveratrol (RES, B and E), or (−)-epigallocatechin-3-gallate (EGCG, C and F), and of 2 μM ibuprofen (A−C) or warfarin (D−F). Inset: quenching of BSA fluorescence at λmax in the absence and presence of ibuprofen or warfarin. simulations through GROMACS software.26 The 3D structures of retinol, resveratrol, and EGCG were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and then transformed into the MOL2 format by the I-interpret software.27 The docking program AutoDock Vina was used to explore the probable binding sites of ligands on BSA.28 To carry out docking simulations, a grid box was defined to enclose the active site with dimensions of 94 × 60 × 66 Å3 and a grid spacing of 1 Å. From the docking results, the best scoring (i.e., with the lowest docking energy) docked model was chosen to represent the most favorable binding mode. After the

binding prediction of the BSA-single-ligand system, the BSA-ligand complexes were chosen as the receptor for the next docking of the BSA-multiligand system. All pictures were produced with the help of PyMol software.29 High Performance Liquid Chromatography. Contents of resveratrol, retinol, and EGCG were measured on the Alliance HPLC system equipped with a 2695 separation module and 2998 PDA detector (Waters, Milford, MA, USA). A symmetry C18 column (5 μm, 4.6 mm × 250 mm, Waters, Milford, MA) was used to separate and analyze three bioactive components by gradient elution with a C


Article


Figure 3. Fluorescence emission spectra of 2 μM BSA in the presence of 0−20 μM resveratrol and 5 μM retinol (A) or EGCG (B), in the presence of 0−20 μM retinol and 5 μM resveratrol (C) or EGCG (D) and in the presence of 0−20 μM EGCG and 5 μM resveratrol (E) or retinol (F). Inset: resveratrol-induced the quenching of BSA fluorescence at λmax in the absence and presence of retinol (A) and EGCG (B), as did retinol in the absence and presence of resveratrol (C) and EGCG (D), and also EGCG in the absence and presence of resveratrol (E) and retinol (F). flow rate of 1 mL/min at 35 °C using a binary mobile phase of methanol in reservoir A and water/glacial acetic acid (99.8:0.2, v/v) in reservoir B. Samples (40 μL) were injected into an HPLC system, and EGCG, resveratrol, and retinol were monitored at 274, 306, and 325 nm, respectively. Statistical Analysis. Data for each sample were presented as the mean value ± standard deviation. Duncan’s test was conducted by

using IBM SPSS Statistics 20 with the significance level determined at the 95% confidence limit (P < 0.05).

■

RESULTS AND DISCUSSION Interaction of BSA with a Single Bioactive Component. Figure 1 shows fluorescence emission spectra of BSA in the absence and presence of resveratrol, retinol, or EGCG at D


Article


consistent with the previously reported studies,6,30,31 indicating that binding of resveratrol caused the exposure of Trp residues to the aqueous milieu and consequently the unfolding of the protein tertiary structure.6 In the case of retinol and EGCG, no significant change was observed in the λmax (Figure 1B and C). At the same time, a gradual decrease in the intensity at λmax was observed as the concentrations of the three bioactive components increased. This is also consistent with the previously reported studies, and the intensity decrease was attributed to static quenching and the formation of protein−ligand complexes.12,15,32 At the same concentration of bioactive components, fluorescence quenching induced by retinol and resveratrol was similar but less than that induced by EGCG, suggesting that the affinity to BSA was in the sequence EGCG > retinol ∼ resveratrol. Warfarin and ibuprofen specifically bind to the subdomain IIA (site I) and the subdomain IIIA (site II) of BSA, respectively, and have been used as site markers to identify the binding of other ligands on serum albumins.33−35 Figure 2 shows the influence of the two site markers on the fluorescence of BSA (product number V900933) bound by retinol, resveratrol, or EGCG at pH 7.4. The change in the fluorescence spectra and quenching caused by the three bioactive components was similar in the absence (data not shown) and presence (Figure 2A−C) of ibuprofen, indicating that their binding on

Figure 4. Fluorescence emission spectra of BSA alone and in the presence of retinol (RET), resveratrol (RES), and (−)-epigallocatechin-3-gallate (EGCG). Concentrations of BSA, retinol, resveratrol, and EGCG are, respectively, 2, 5, 5, and 5 μM.

pH 7.4. BSA has an emission maximum (λmax) of 336 nm. As the concentration of resveratrol increased, a red shift was observed, with the λmax value being 352 nm at the polyphenol concentration of 20 μM (Figure 1A). These results are

Figure 5. Far-UV circular dichroic spectra of 2 μM BSA in the absence and presence of retinol (RET, A), resveratrol (RES, B), or (−)-epigallocatechin-3-gallate (EGCG, C) at various concentrations and of retinol (RET), resveratrol (RES), and EGCG at 5 μM (D). Inset table: the ellipticity at 208 and 222 nm; values with the same letter are not significantly different. E


Article


Figure 6. Fluorescence emission spectra of retinol (RET, A), (−)-epigallocatechin-3-gallate (EGCG, B), and resveratrol (RES, C) free in ethanol and free and bound to BSA in 10 mM phosphate buffer at pH 7.4 and of the (D) RET-RES-EGCG mixture, the sum of the individual ligands (RET + RES + EGCG), and the comparison between a single ligand and a triligand after binding to BSA in phosphate buffer. The concentrations of BSA, retinol, resveratrol, and EGCG are, respectively, 2, 5, 5, and 5 μM.

than did resveratrol alone. Quenching of BSA fluorescence was less (inset in Figure 3B), suggesting that EGCG reduced the affinity of resveratrol to BSA. Influence of Resveratrol or EGCG on the Binding of Retinol to BSA. Figure 3C and D shows the influence of 0−20 μM retinol on the fluorescence spectra of BSA in the presence of resveratrol or EGCG. In the presence of resveratrol, the λmax of 340 nm did not change as the concentrations of retinol increased until 10 μM and then shifted to longer wavelengths, with the value being 344 nm at 20 μM (Figure 3C). This suggests that the binding of retinol further changed the tertiary structure of BSA. The fluorescence intensity of BSA also decreased as the concentrations of retinol increased. However, retinol-induced quenching was less in the presence rather than the absence of resveratrol (inset in Figure 3C), suggesting that the polyphenol reduced the affinity of retinol to BSA. In the presence of EGCG, λmax did not change as the concentrations of retinol increased up to 20 μM (Figure 3D). The retinol induced decrease in the intensity and quenching were less, suggesting that EGCG also reduced the affinity of retinol to BSA. Influence of Resveratrol or Retinol on the Binding of EGCG to BSA. In the presence of resveratrol, the λmax of

BSA did not locate within subdomain IIIA (site II). The change in the fluorescence spectra was also similar in the absence and presence of warfarin (Figure 2D−F). The difference in the presence of EGCG in Figures 1C and 2C might due to the use of different BSA products. Fluorescence quenching caused by retinol, resveratrol, or EGCG was a bit less in the presence of warfarin (Inset in Figure 2D−F), suggesting that subdomain IIA (site I) was also not a main site for the binding of the three components. The binding sites of retinol, resveratrol, and EGCG on BSA would be further analyzed by using their own fluorescence and molecular docking. Influence of Retinol or EGCG on the Binding of Resveratrol to BSA. Figure 3A and B shows fluorescence emission spectra of BSA as the concentrations of resveratrol increased up to 20 μM in the presence of retinol or EGCG. In the absence and presence of retinol, resveratrol-induced change in the λmax (Figures 1A and 3A) and quenching of BSA fluorescence were similar (inset in Figure 3A), suggesting that addition of retinol had no impact on the affinity of resveratrol to BSA. In the presence of EGCG, the change in the λmax was greater, with the λmax value being 356 nm at 20 μM (Figure 3B), suggesting that both EGCG and resveratrol binding had a greater impact on the tertiary structure of BSA F


Article


further decreased it, as did the subsequent addition of EGCG. The fractional quenching due to EGCG was comparable in the absence and presence of retinol and resveratrol. These results suggest that BSA could bind different bioactive components simultaneously to form protein-triligand complexes when the components were added in the sequence of retinol, resveratrol, and EGCG. Influence of Bioactive Components on the Secondary Structure of BSA. The far-UV circular dichroic spectrum of native BSA exhibits two negative bands at 208 and 222 nm (Figure 5), a characteristic of α-helical structure.36 The spectral shapes were similar in the absence and presence of retinol, resveratrol, or EGCG (Figure 5A−C), indicating that the secondary structure of BSA after ligand binding was also predominantly α-helix.32 At the same time, a slight increase in the spectral intensity was observed at the concentration of retinol above 10 μM, at 20 μM of resveratrol, or at the concentration of EGCG above 2 μM, suggesting that the binding of a single bioactive component increased the content of the α-helix in the structure of BSA. However, the spectrum of BSA after the simultaneous binding of retinol, resveratrol, and EGCG remained the same as that of BSA alone, and the difference in the 208 and 222 nm ellipticities was not statistically significant (Figure 5D). These results suggest that simultaneous binding decreased the impact of a single component on the structure of BSA. Influence of BSA on Microenvironment of Bioactive Components. The surrounding microenvironment of bioactive components was analyzed by using their own fluorescence. Fluorescence emission spectra of retinol had a λmax around 496 nm in phosphate buffer at pH 7.4 (Figure 6A). The λmax shifted to 489 nm in 100% ethanol and to 482 nm upon binding to BSA in phosphate buffer, with the emission intensity, respectively, being about 9 and 16 times that of pure retinol in phosphate buffer. These results suggest that the BSA environment of the bound retinol was more hydrophobic than ethanol, consistent with a previous result that the hydrophobic environment was stronger than that of petroleum ether.37 A λmax of EGCG of ∼397 nm in phosphate buffer shifted to 381 nm in ethanol and to 368 nm upon binding to BSA, with the intensity being about 5 and 34 times that of pure EGCG in phosphate buffer (Figure 6B), indicating that the BSA environment of the bound EGCG was also more hydrophobic than that of ethanol. A λmax of resveratrol of ∼396 nm in phosphate buffer shifted to 375 nm in 100% ethanol and to 387 nm upon binding to BSA in phosphate buffer, with the intensity being about 6 and 2 times that of resveratrol in phosphate buffer (Figure 6C). These results indicate that the environment of BSA-bound resveratrol was less hydrophobic than that of ethanol, consistent with resveratrol binding to the outer site of β-lactoglobulin.38 As a whole, these results suggest that the site of resveratrol bound on BSA was different from that of retinol and EGCG. The fluorescence emission spectrum of retinol, resveratrol, and EGCG mixture overlapped the spectral sum of individual components (Figure 6D), suggesting no interaction among the three components in solution. Upon binding to BSA, two λmax around 373 and 475 nm were observed in the spectrum. The latter was mostly attributed to retinol and had an intensity similar to that of retinol bound alone to BSA, suggesting that retinol was in a similar hydrophobic environment when bound alone to BSA and when bound together with resveratrol and EGCG to BSA. The former, attributed to both resveratrol and

Figure 7. Best docked conformations for the triligand complex of BSA with retinol, resveratrol, and EGCG. (A) BSA is rendered in “cartoon” mode, and its three domains are marked in blue, yellow and orange. Retinol, resveratrol, and EGCG are rendered as “spheres” and correspondingly marked in green, violet, and red. (B−C) BSA (wheat color) was rendered in the modes of “cartoon” and “surface”, the side chain of Trp134 (blue) was shown in the mode of “sticks”, and in the short loop from Pro113 to Pro117 was labeled in skyblue. (D) EGCG marked in cyan and red represents two typical conformations when bound to BSA alone or the BSA-resveratrol complex, respectively.

340 nm gradually shifted to longer wavelengths as the concentrations of EGCG increased, reaching around 351 nm at 20 μM (Figure 3E). This suggests that the binding of EGCG further changed the tertiary structure of BSA. However, EGCGinduced quenching was similar in the absence and presence of resveratrol (inset in Figure 3E), suggesting that resveratrol had no substantial influence on the affinity of EGCG to BSA. EGCG had no significant influence on the λmax around 336 nm, and EGCG-induced quenching was similar in the absence and presence of retinol (Figure 3F), suggesting that retinol had no impact on the affinity of EGCG to BSA. Formation of BSA-Triligand Complexes. The results above indicate that the addition sequence of bioactive components was important for their binding to BSA. On the basis of the principle of no influence on binding, bioactive components were added into BSA solutions in the order of retinol, resveratrol, and EGCG. As shown in Figure 4, retinol decreased BSA fluorescence intensity, and adding resveratrol G


Article


Figure 8. Contents of (A) free retinol (RET) and (B−D) retinol bound to BSA at 3, 15, and 30 μM in the absence and presence of resveratrol (RES) and/or (−)-epigallocatechin-3-gallate (EGCG) during storage. Concentrations of RET, RES, and EGCG were all 30 μM.

EGCG, had greater intensity than did resveratrol bound alone to BSA, suggesting that both polyphenols transferred into more hydrophobic environments upon simultaneously binding together with retinol to BSA. However, the intensity of the former was less than that of EGCG bound alone to BSA, possibly due to the fact that the tertiary structure of the protein was more influenced by both EGCG and resveratrol than by individual components (Figure 3E). Molecular Docking. Figure 7 presents the best docked conformations for the triligand complex of BSA with retinol, resveratrol, and EGCG. BSA contains several hydrophobic cavities as the potential sites for ligand binding. First, the strongest binding sites on BSA for retinol, resveratrol, and EGCG were separated from one another and named as cavities C1, C2, and C3, respectively (Figure 7A). Cavity C1 was on the surface of domain I and surrounded by Lys20, Leu24, Phe36, Asp37, Val40, Asn44, Lys131, and Trp133; cavity C2 was in domain I and surrounded by Leu115, Pro117, Leu122, Lys136, Tyr137, Glu140, Ile141,Tyr160, Ile181, Met184, Arg185, and Val 188; cavity C3 was posed by domains I and III and surrounded by Ser109, Asp111, Leu112, Leu115, Lys116, Ile141, Arg144, His145, Glu424, and Arg458. The corresponding binding free energies (ΔG) were −7.5, −7.6, and −8.5 kcal/mol when in complex with BSA and −7.5, −7.6, and −8.6 kcal/mol when added in the sequence of retinol, resveratrol, and EGCG. Therefore, the three ligands could simultaneously bind to

BSA with enough affinities. Second, cavities C1, C2, and C3 were also the targets of other ligands. In the case of cavity C1 (Figure 7B), the ΔG for resveratrol was −7.2 kcal/mol, slightly weaker than that for retinol. The energy difference was so small that the bound resveratrol could not be easily replaced by a subsequent retinol. The affinity of retinol to BSA would thus be reduced in the presence of resveratrol (Figure 3C). Moreover, the ΔG for retinol to bind with the cavity C2 was −6.7 kcal/mol and weaker than that for resveratrol; thus, the addition of retinol did not have an obvious impact on the affinity of resveratrol to BSA (Figure 3A). Third, cavities C2 and C3 were only separated by a short loop from Pro113 to Pro117 on the surface of BSA (Figure 7C and D). This loop is so flexible that its conformation would be affected after the insertion of EGCG into cavity C3, leading to a smaller cavity C2. The last added EGCG made the complex of resveratrol with BSA more compact and stable. Compared with EGCG, resveratrol is smaller, and its binding to cavity C2 exerted very little influence on the flexible loop and cavity C3. As shown in Figure 7D, cavity C3 was large enough for EGCG to adjust its orientation after the binding of resveratrol to cavity C2. Therefore, the results from molecular docking are consistent with spectroscopic results (Figures 1, 3, and 4), supporting the fact that the charging sequence is a vital factor for the formation of a multiligand complex and that the sequence of retinol, H


Article


Figure 9. Contents of (A) free resveratrol (RES) and (B−D) resveratrol bound to BSA at 3, 15, and 30 μM in the absence and presence of retinol (RET) and/or (−)-epigallocatechin-3-gallate (EGCG) during storage. Concentrations of RET, RES, and EGCG were all 30 μM.

storage for 628 h. These results indicate that simultaneous binding together with EGCG to BSA could strengthen the protection against the loss of retinol. During light illumination of milk, green tea catechin showed stronger protective activity against the formation of hydroperoxide and the loss of retinol than did ascorbic acid, possibly due to the ability of singlet oxygen quenching.41 No protection of resveratrol when bound simultaneously to BSA might be due to the binding site of resveratrol on BSA being more hydrophilic (Figure 6). Stability of Resveratrol. Resveratrol is susceptible to isomerization and degradation dependent on light, temperature, and pH.42,43 Since the contents of cis-resveratrol were always less than 1% during storage (data not shown), only naturally occurring trans-resveratrol was analyzed in this study. Figure 9A shows the contents of free resveratrol in the absence and presence of retinol and EGCG during storage. The content of resveratrol was about 95% after 7 h and gradually decreased to about 38% after 628 h (Figure 9A). Similar results were observed in the presence of retinol. In the presence of EGCG or both retinol and EGCG, the contents of resveratrol were similar as a whole. The stability of resveratrol was improved by EGCG, with the contents being greater than that of resveratrol alone until 244 h. Figure 9B−D shows the contents of resveratrol bound to BSA in the absence and presence of retinol and EGCG during storage. Upon binding to BSA, the loss of resveratrol was faster,

resveratrol, and EGCG is the best one for the BSA-triligand complex. Stability of Retinol. Figure 8A shows the contents of free retinol in the absence and presence of resveratrol and EGCG during storage. Free retinol was susceptible to isomerization, oxidation, and degradation due to the conjugated double-bond structure. Thus, its content decreased rapidly during storage, reaching about 3% after 7 h, and complete loss was observed after 28 h.39,40 In the presence of resveratrol and/or EGCG, the loss of retinol was slower. The protective effect of resveratrol and resveratrol/EGCG was better than that of EGCG after 7 h but became similar after that, with about 2% of retinol left after storage for 52 h. Figure 8B−D shows the contents of retinol bound to BSA in the absence and presence of resveratrol and EGCG during storage. Upon binding to BSA, the stability of retinol was significantly improved over time of storage, more so as the protein concentration increased. When the concentrations of BSA were 3, 15, and 30 μM, the contents of retinol were about 23%, 50%, and 66%, respectively, after storage for 52 h and were about 1%, 6%, and 9%, respectively, after storage for 628 h. The protective effect of BSA was not dependent on the presence of resveratrol but improved in the presence of EGCG or both polyphenols. When the concentrations of BSA were 3, 15, and 30 μM, the contents of retinol were about 4%, 13%, and 23% in the presence of EGCG or both polyphenols after I


Article


Figure 10. Contents of (A) (−)-epigallocatechin-3-gallate (EGCG) and (B−D) EGCG bound to BSA at 3, 15, and 30 μM in the absence and presence of retinol (RET) and/or resveratrol (RES) during storage. Concentrations of RET, RES, and EGCG were all 30 μM.

presence of retinol and/or resveratrol had no influence on the stability of EGCG. Upon binding to BSA at 3 μM, the stability of EGCG was increased, with the contents being about 10% and 2%, respectively, after storage for 7 and 21 h, independent of the presence of retinol and/or resveratrol (Figure 10B). The stability of EGCG was further improved as the concentration of BSA increased, with the contents being 51% after 7 h and gradually decreasing to 4% after 100 h at 15 μM BSA and being 85% and 9%, respectively, after 7 and 148 h at 30 μM BSA (Figure 10C,D). The protection was decreased upon simultaneous binding of retinol and/or resveratrol, whose effect was in the following sequence: resveratrol < retinol < retinol/ resveratrol. However, when simultaneously bound to BSA together with retinol and resveratrol, the contents of EGCG were still higher than that in free EGCG (Figure 10A). In conclusion, when retinol, resveratrol, and EGCG in the sequence were added into BSA solutions, BSA could bind the three bioactive components to form protein-triligand complexes. The complex formation did not significantly affect the secondary structure of BSA but altered the protein tertiary structure. BSA-triligand complexes provided a better protective effect against the degradation of retinol and resveratrol than did BSA-monoligand complexes. The presence of EGCG in the complexes played an important role in such protection. The stability of EGCG was improved when bound to BSA, with the degradation in the BSA-triligand complexes being slower than

more so as the protein concentrations increased from 3 to 15 μM, with the contents being 26% and 10%, respectively, after 628 h (Figure 9B−C). However, no significant change in the resveratrol content was found when further increasing the protein concentration to 30 μM (Figure 9D). Simultaneous binding of retinol to BSA had no influence on the stability of resveratrol at the beginning of storage but decreased the contents of resveratrol with the starting time changing from 244 and 148 to 52 h at the protein concentrations of 3, 15, and 30 μM, respectively (Figure 9B−D). It is thus speculated that retinol increased the antagonistic effect of BSA on the stability of resveratrol. It is noted that the stability of resveratrol bound to BSA was significantly improved by EGCG, with the contents being higher than that of resveratrol alone (Figure 9A). The protective effect was strengthened as the protein concentration increased and was independent of retinol except for the cases of 628 h at 15 and 30 μM BSA (Figure 9B−D). These results suggest that simultaneous binding together with EGCG to BSA provided a better protective effect against the loss of resveratrol than that by EGCG alone. Stability of EGCG. Figure 10 shows the contents of free EGCG and EGCG bound to BSA in the absence and presence of retinol and resveratrol during storage. The content of EGCG was only 2% after storage for 7 h, and complete loss was observed after 21 h (Figure 10A), in agreement with previous studies on the stability of EGCG in neutral solutions.44,45 The J


Article


(13) Yang, W.; Liu, F. G.; Xu, C. Q.; Yuan, F.; Gao, Y. X. Molecular interaction between (−)-epigallocatechin-3-gallate and bovine lactoferrin using multi-spectroscopic method and isothermal titration calorimetry. Food Res. Int. 2014, 64, 141−149. (14) Chang, H. T.; Cheng, H.; Han, R. M.; Zhang, J. P.; Skibsted, L. H. Binding to bovine serum albumin protects β-carotene against oxidative degradation. J. Agric. Food Chem. 2016, 64, 5951−5957. (15) Bose, A. Interaction of tea polyphenols with serum albumins: A fluorescence spectroscopic analysis. J. Lumin. 2016, 169, 220−226. (16) Maiti, T. K.; Ghosh, K. S.; Dasgupta, S. Interaction of (−)-epigallocatechin-3-gallate with human serum albumin: Fluorescence, fourier transform infrared, circular dichroism, and docking studies. Proteins: Struct., Funct., Genet. 2006, 64, 355−362. (17) Wang, X. Y.; Ho, C. T.; Huang, Q. R. Investigation of adsorption behavior of (−)-epigallocatechin gallate on bovine serum albumin surface using quartz crystal microbalance with dissipation monitoring. J. Agric. Food Chem. 2007, 55, 4987−4992. (18) Sloan, A. E. The top 10 functional food trends 2004. Food Technol. (Chicago, IL, U.S.) 2004, 58, 28−51. (19) Conte, A.; Stasi, A.; Pilone, V.; Gammariello, D.; Padalino, L.; Bimbo, F.; Lopolito, A.; Nobile, M. A. D. Technological and economic optimization of functional ready to eat meal. J. Food Res. 2012, 1, 192− 203. (20) Liu, D. H.; Shi, J.; Ibarra, A. C.; Kakuda, Y.; Xue, S. J. The scavenging capacity and synergistic effects of lycopene, vitamin E, vitamin C, and beta-carotene mixtures on the DPPH free radical. LwtFood Science and Technology 2008, 41, 1344−1349. (21) Shin, D. M.; Amin, A. R. M.; Wang, D.; Rahman, M. A.; Nannapaneni, S.; Khuri, F. R.; Chen, Z. G. 1187 Molecular mechanism of synergistic anti-tumor activity by the combination of natural compounds green tea (−)-epigallocathetin-3-gallate (EGCG) and resveratrol for potential chemoprevention in head and neck cancer (HNC). Eur. J. Cancer 2012, 48, S286. (22) Ahmed, B. Combinations of Genistein, EGCG and/or Resveratrol Synergistically Inhibit Pre-adipocyte Differentiation by Suppressing PPARGamma/C/EBP-Alpha Pathway. Tennessee State University: Nashville, TN, 2014. (23) Zhang, J.; Liu, X. M.; Subirade, M.; Zhou, P.; Liang, L. A study of multi-ligand beta-lactoglobulin complex formation. Food Chem. 2014, 165, 256−261. (24) Zhang, Y. P.; Shi, S. Y.; Chen, X. Q.; Zhang, W.; Huang, K. L.; Peng, M. J. Investigation on the interaction between ilaprazole and bovine serum albumin without or with different C-Ring flavonoids from the viewpoint of food-drug interference. J. Agric. Food Chem. 2011, 59, 8499−8506. (25) Liang, L.; Subirade, M. Study of the acid and thermal stability of beta-lactoglobulin−ligand complexes using fluorescence quenching. Food Chem. 2012, 132, 2023−2029. (26) Pronk, S.; Pall, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M. R.; Smith, J. C.; Kasson, P. M.; Spoel, D. V. D.; Hess, B.; Lindahl, E. GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 2013, 29, 845−854. (27) Zhao, Y.; Cheng, T. J.; Wang, R. X. Automatic perception of organic molecules based on essential structural information. J. Chem. Inf. Model. 2007, 47, 1379−1385. (28) Trott, O.; Olson, A. J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 2010, 31, 455−461. (29) Seeliger, D.; de Groot, B. L. Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J. Comput.-Aided Mol. Des. 2010, 24, 417−422. (30) Xiao, J. B.; Chen, X. Q.; Jiang, X. Y.; Hilczer, M.; Tachiya, M. Probing the interaction of trans-resveratrol with bovine serum albumin: A fluorescence quenching study with Tachiya model. J. Fluoresc. 2008, 18, 671−678. (31) Cao, S. H.; Wang, D. D.; Tan, X. Y.; Chen, J. W. Interaction between trans-resveratrol and serum albumin in aqueous solution. J. Solution Chem. 2009, 38, 1193−1202.

that of the free one during storage. These results suggest the possibility and advantage of developing BSA-based carriers of a plurality of bioactive components.

■

AUTHOR INFORMATION

Corresponding Authors

*(Y.C.) Tel: +86-755-2653-5427. E-mail: [email protected]. *(L.L.) Tel: +86-510-8519-7367. E-mail: [email protected]. ORCID

Li Liang: 0000-0001-9584-6778 Funding

This work received support from the National Natural Science Foundation of China (NSFC Projects 31201291, 21174088, and 31571781) and the 2015 Innovation Project of Postgraduate cultivation in Jiangsu Province (KYLX15_1172). Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED BSA, bovine serum albumin; EGCG, (−)-epigallocatechin-3gallate; Arg, arginine; Leu, leucine; Phe, phenylalanine; Trp, tryptophan; Tyr, tyrosine; CD, circular dichroism; UV, ultraviolet; HPLC, high performance liquid chromatography

■

REFERENCES

(1) Shortridge, M. D.; Bokemper, M.; Copeland, J. C.; Stark, J. L.; Powers, R. Correlation between protein function and ligand binding profiles. J. Proteome Res. 2011, 10, 2538−2545. (2) De Wolf, F. A.; Brett, G. M. Ligand-binding proteins: Their potential for application in systems for controlled delivery and uptake of ligands. Pharmacol. Rev. 2000, 52, 207−236. (3) Tavares, G. M.; Croguennec, T.; Carvalho, A. F.; Bouhallab, S. Milk proteins as encapsulation devices and delivery vehicles: Applications and trends. Trends Food Sci. Technol. 2014, 37, 5−20. (4) Liang, L.; Subirade, M. Beta-lactoglobulin: Bioactive Nutrients Delivery. In Encyclopedia of Biomedical Polymers and Polymeric Biomaterials; Mishra, M., Ed.; Taylor and Francis: Now York, 2016; pp 729−739. (5) Zhang, A. M.; Jia, L. P. Spectroscopic study of the interaction between folic acid and bovine serum albumin. Spectrosc. Lett. 2006, 39, 285−298. (6) Bourassa, P.; Kanakis, C. D.; Tarantilis, P.; Pollissiou, M. G.; Tajmir-Riahi, H. A. Resveratrol, genistein, and curcumin bind bovine serum albumin. J. Phys. Chem. B 2010, 114, 3348−3354. (7) Li, J.; Yao, P. Self-assembly of ibuprofen and bovine serum albumin-dextran conjugates leading to effective loading of the drug. Langmuir 2009, 25, 6385−6391. (8) Livney, Y. D. Biopolymeric Amphiphiles and Their Assemblies as Functional Food Ingredients and Nutraceutical Delivery Systems. In Encapsulation Technologies and Delivery Systems for Food Ingredients and Nutraceuticals; Garti, N., McClements, D. J., Eds.; Woodhead Publishing: Cambridge, UK, 2012; pp 252−286. (9) Sudlow, G.; Birkett, D. J.; Wade, D. N. The characterization of two specific drug binding sites on human serum albumin. Mol. Pharmacol. 1975, 11, 824−832. (10) Roy, A. S.; Tripathy, D. R.; Chatterjee, A.; Dasgupta, S. A spectroscopic study of the interaction of the antioxidant naringin with bovine serum albumin. J. Biophys. Chem. 2010, 1, 141−152. (11) Skrt, M.; Benedik, E.; Podlipnik, C.; Ulrih, N. P. Interactions of different polyphenols with bovine serum albumin using fluorescence quenching and molecular docking. Food Chem. 2012, 135, 2418−2424. (12) Belatik, A.; Hotchandani, S.; Bariyanga, J.; Tajmir-Riahi, H. A. Binding sites of retinol and retinoic acid with serum albumins. Eur. J. Med. Chem. 2012, 48, 114−123. K


Article

Journal of Agricultural and Food Chemistry (32) Gorji, E. G.; Rocchi, E.; Schleining, G.; Bender-Bojalil, D.; Furtmueller, P. G.; Piazza, L.; Iturri, J. J.; Toca-Herrera, J. L. Characterization of resveratrol-milk protein interaction. J. Food Eng. 2015, 167, 217−225. (33) Yuan, L. X.; Liu, M.; Liu, G. Q.; Li, D. C.; Wang, Z. P.; Wang, B. Q.; Han, J.; Zhang, M. Competitive binding of (−)-epigallocatechin-3gallate and 5-fluorouracil to human serum albumin: A fluorescence and circular dichroism study. Spectrochim. Acta, Part A 2017, 173, 584− 592. (34) Zhang, Y. Z.; Zhou, B.; Zhang, X. P.; Huang, P.; Li, C. H.; Liu, Y. Interaction of malachite green with bovine serum albumin: Determination of the binding mechanism and binding site by spectroscopic methods. J. Hazard. Mater. 2009, 163, 1345−1352. (35) Uppuluri, K. B.; Ahmed, K. B. A.; Jothi, A.; Veerappan, A. Spectrofluorimetric and molecular docking investigation on the interaction of 6-azauridine, a pyrimidine nucleoside antimetabolite, with serum protein. J. Mol. Liq. 2016, 219, 602−607. (36) Kelly, S. M.; Jess, T. J.; Price, N. C. How to study proteins by circular dichroism. Biochim. Biophys. Acta, Proteins Proteomics 2005, 1751, 119−139. (37) Futterman, S.; Heller, J. The enhancement of fluorescence and the decreased susceptibility to enzymatic oxidation of retinol complexed with bovine serum albumin, beta-lactoglobulin, and the retinol-binding protein of human plasma. J. Biol. Chem. 1972, 247, 5168−5172. (38) Liang, L.; Tajmir-Riahi, H. A.; Subirade, M. Interaction of betaLactoglobulin with resveratrol and its biological implications. Biomacromolecules 2008, 9, 50−56. (39) Lee, S. C.; Yuk, H. G.; Lee, D. H.; Lee, K. E.; Hwang, Y. I.; Ludescher, R. D. Stabilization of retinol through incorporation into liposomes. J. Biochem. Mol. Biol. 2002, 35, 358−363. (40) Semenova, E. M.; Cooper, A.; Wilson, C. G.; Converse, C. A. Stabilization of all-trans-retinol by cyclodextrins: A comparative study using HPLC and fluorescence spectroscopy. J. Inclusion Phenom. Mol. Recognit. Chem. 2002, 44, 155−158. (41) Jung, M. Y. Effects of green tea catechin on the lipid oxidation, volatile compound formation, and losses of retinol and alphatocopherol in whole milk during light illumination as compared with ascorbic acid. Food Sci. Biotechnol. 2011, 20, 1425−1434. (42) Zupancic, S.; Lavric, Z.; Kristl, J. Stability and solubility of transresveratrol are strongly influenced by pH and temperature. Eur. J. Pharm. Biopharm. 2015, 93, 196−204. (43) Figueiras, T. S.; Neves-Petersen, M. T.; Petersen, S. B. Activation energy of light induced isomerization of resveratrol. J. Fluoresc. 2011, 21, 1897−1906. (44) Li, N.; Taylor, L. S.; Ferruzzi, M. G.; Mauer, L. J. Kinetic study of catechin stability: Effects of pH, concentration, and temperature. J. Agric. Food Chem. 2012, 60, 12531−12539. (45) Su, Y. L.; Leung, L. K.; Huang, Y.; Chen, Z. Y. Stability of tea theaflavins and catechins. Food Chem. 2003, 83, 189−195.

L


Formation of a Multiligand Complex of Bovine Serum Albumin with

Recommend Documents