Multi-spectroscopic and computational evaluation on the binding of sinapic acid and its Cu(II) complex with bovine serum albumin

Food Chemistry 301 (2019) 125254

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Multi-spectroscopic and computational evaluation on the binding of sinapic acid and its Cu(II) complex with bovine serum albumin

T

Priti Senguptaa, Uttam Palb, Prasenjit Mondala, Adity Bosea,

⁎

a b

Department of Chemistry, Presidency University, 86/1 College Street, Kolkata 700073, India Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India

ARTICLE INFO

ABSTRACT

Keywords: Sinapic acid Metal complex BSA Fluorescence quenching Molecular docking

Researches based on metal complexes of plant-derived phenolic acids have attracted much attention due to their beneficial applications in the development of functional food products, dietary supplements and pharmacology. Binding of phenolic acids with serum proteins greatly influences their pharmacological properties. In this context, interactions of a naturally occurring phenolic acid, sinapic acid (SA) and its Cu2+ complex with a model transport protein, bovine serum albumin (BSA), have been explored by means of different spectroscopic and theoretical tools. Spectroscopic studies revealed that the interaction of SA and its Cu2+ complex with BSA occurred through quenching of intrinsic fluorescence of BSA. Site-specific experimental and docking studies were performed to predict the binding site. The geometies of bound Cu2+ and interacting residues of protein were predicted from a solution dynamics study. Interestingly, the complexation of SA with Cu2+ enhanced the antioxidant activity of SA.

1. Introduction Phenolic acids, aromatic secondary metabolites of plants, are frequently included in human diets and are often utilized as significant food additives in Asian countries due to their beneficial biological activities. At present, different types of phenolic acids have attracted much attention, due to their wide applications in the development of functional food products, dietary supplements, and pharmacological materials. Sinapic acid (SA, inset of Fig. 1a), one of the most common members of the hydroxycinnamic acid group. It is a naturally occurring and widely utilized phenolic acid, most commonly found in various fruits, vegetables, cereal grains, some spices, and medicinal plants (Niciforovic & Abramovic, 2014; Andreasen, Landbo, Christensen, Hansen, & Meyer, 2001). SA is thus common in human diet and recognized to possess several biological activities, e.g antioxidant (Natella, Nardini, Felice, & Scaccini, 1999), anti-inflammatory (Yun et al., 2008), anti-bacterial (Maddox, Laur, & Tian, 2010), anti-cancer (Hudson, Dinh, Kokubun, Simmonds, & Gescher, 2000) and anti-anxiety (Yoon et al., 2007). The antioxidant activity of SA is considered to be superior to that of ferulic acid, another member of the hydroxycinnamic acid group already used as a natural antioxidant in food and beverage industries (Cuvelier, Richard, & Berset, 1992). Due to such beneficial roles, SA has been suggested for use in food processing and pharmaceutical industries ⁎

(Niciforovic & Abramovic, 2014). However, the antioxidant property of naturally occurring compounds is also correlated with their ability to chelate transition metal ions, which are directly involved in the free radical generation process (Roy, Tripathy, Ghosh, & Dasgupta, 2012). Generally, metal ion chelation alters the structure of the compounds, and significantly affects their biological activities and mode of interaction with serum albumins (Andjelkovic et al., 2006; Leopoldini, Russo, Chiodo, & Toscano, 2006; Liu, Qi, & Li, 2010). In some cases, metal complexes are often more active than are the free compounds (Zhang, Shi, Sun, Xiong, & Peng, 2011). Copper, the third most abundant transition metal after iron and zinc, is essential for some cellular activities, such as respiration, angiogenesis, immune responses and many more (Hamza & Gitlin, 2002; Finney, Vogt, Fukai, & Glense, 2009). Apart from the effectiveness, the Cu2+ ion also participates in free radical-generating reactions which cause oxidative stress in potential biological targets (Shi, Zhang, Chen, & Peng, 2011). The SA-Cu2+ complex can thus limit the bioavailability of the Cu2+ ion and reduce the participation of the Cu2+ ion in free radical generation. In our previous article, we showed SA to be an excellent colorimetric Cu2+ ion sensor which can recognize Cu2+ by forming a pinkcoloured 1:2 charge-transfer complex between SA and Cu2+ ion (Sengupta, Ganguly, & Bose, 2018). We have also established the structure of the complex [SA(Cu2+)2, inset of Fig. 1b] from 1H NMR

Corresponding author. E-mail address: [email protected] (A. Bose).

https://doi.org/10.1016/j.foodchem.2019.125254 Received 28 January 2019; Received in revised form 23 July 2019; Accepted 24 July 2019 Available online 30 July 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Variation in the fluorescence quenching spectra of BSA in the presence of various concentrations of (a) SA and (b) SA(Cu2+)2 complex (inset: structure of SA and SA(Cu2+)2 complex). Corresponding Stern-Volmer plots for quenching of BSA fluorescence by (c) SA and (d) SA(Cu2+)2 complex; [BSA] = 3 µM; λex = 295 nm; pH = 7.4; T = 298 K; excitation band pass = 5 nm and emission band pass = 5 nm.

and ESI-MS studies in that work (data not shown here). In the present work, we have utilized this complex-forming ability of SA, to investigate its interaction with BSA in the presence of Cu2+ and observe the effect of this complexation on the antioxidant activity of free SA. Previously, some researchers have studied the nature of the interaction of SA with BSA, using different spectroscopic tools (Smyk & Drabent, 1989; Liu, Xie, Jiang, & Wang, 2005; Trnkova, Bousova, Kubícek, & Drsata, 2010; Jin et al., 2012). But no such studies have yet been done to observe the effect of metal ion complexation on the interaction of serum albumins with SA. Hence we have endeavoured to focus on two major concerns: (i) to observe whether the presence of Cu2+ ion altered the mode of binding of SA to serum albumin and (ii) to investigate any improvement of the antioxidant activity of SA upon complexation with Cu2+ ion. In the SA(Cu2+)2 complex, the chelation of SA with Cu2+ makes the OH groups much less involved in the interaction with protein, which may affect the overall interaction process. Bioavailability, distribution, and metabolism of biologically active compounds in the mammalian body are associated with their ability to bind to transport proteins present in the blood. Thus, in the human system, pharmacological activities of a molecule highly depend on interaction with plasma proteins, and more specifically serum albumins (Feroz, Mohamad, Bujang, Malek, & Tayyab, 2012). Serum albumins are the most abundant circulatory proteins in blood plasma and play a crucial role in the transport and disposition of various exogenous and endogenous compounds at target sites. Bovine serum albumin (BSA) is often utilized as a human serum albumin (HSA)-replacing model for different biophysical and biochemical studies due to its high structural

homology (76% resemblance). BSA is composed of three structurally homologous α-helical domains (I, II and III), each divided into two subdomains, A and B (Sengupta, SahaSardar, Roy, Dasgupta, & Bose, 2018). Crystal structure analysis has also revealed that BSA is a heartshaped molecule containing 582 amino acid residues with 20 tyrosyl residues (Tyr) and two tryptophan (Trp) residues, Trp-159, and Trp-237 (UniProt KB: ALBU_BOVIN). Trp-237 is located in the hydrophobic subdomain IIA, whereas Trp-159 is located on the surface of BSA in the hydrophilic subdomain IB (Shi, Wang, Zhu, & Chen, 2014). The intrinsic fluorescence of BSA arises mainly from the presence of Trp residues (Trp-159 and Trp-237). Thus, selective excitation of Trp (at ∼295 nm) is often used as a probe to investigate the binding properties of drugs with BSA. The binding mode of SA and SA(Cu2+)2, with BSA, was predicted with the help of UV–vis, fluorescence spectroscopy, and time-resolved fluorescence studies. We have also confirmed their binding site through site-marker experiments and molecular docking studies. Importantly, an elaborate theoretical study has been performed in order to complete understanding of the involved forces, complex geometry and interacting residues of the protein molecule. Solution dynamic studies of the SA(Cu2+)2 complex have been performed to observe the geometry of bound Cu2+ while interacting with the protein molecule. To attain such results, we have allowed SA to form a complex with Cu2+ first, and then observed its interaction with BSA.

2

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2. Materials and methods

by a non-linear iterative fitting procedure based on the Marquardt algorithm. Intensity decay curves were fitted as a sum of exponential terms:

2.1. Materials

F=

BSA (≥98%, agarose gel electrophoresis), Sinapic acid (SA) (≥98%, powder), tris(hydroxymethyl)aminomethane (Tris-buffer), Warfarin, Ibuprofen (≥98%, GC), were purchased from Sigma-Aldrich, USA. Copper chloride hydrate (CuCl2·2H2O) was purchased from Merck Millipore (EMPLURA grade). BSA was dissolved in 10 mM tris-HCl buffer (pH 7.4). Sinapic acid (SA) was initially dissolved in an ethanolbuffer mixture and further diluted with buffer to obtain the experimental concentration. The ethanol percentage was kept < 0.5% so that the spectral behaviour of BSA could not be affected by ethanol. Triple distilled water was used throughout all the experiments. 6-Hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was purchased from TCI, Japan. 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from SRL, India and potassium persulfate (K2S2O8) was obtained from Merck.

i exp(

/ i)

(1)

where αi represents the pre-exponential factor of the time-resolved decay of the component with a lifetime τi. The decay parameters were recovered using a nonlinear iterative fitting procedure based on the Marquardt algorithm. The quality of each fitting was evaluated from χ2 values. The average lifetime 〈τ〉 was calculated using the equation:

=

i i i

(2)

2.2.4. Molecular docking AutoDock 4.2 of The Scripps Research Institute was used for docking (Morris et al., 2009). The structure information of SA was obtained from ChemSpider (CSID: 553361) whereas, for its Cu complex, the previously reported DFT optimized geometry was used (Sengupta, Ganguly, et al., 2018). Prior to docking, the ligand structures were geometry-optimized in vacuum using the steepest descent, followed by conjugate gradient algorithms in Avogadro (Hanwell et al., 2012). In the case of the Cu complex of SA, Cu – O bond distances were restrained during geometry optimization in Avogadro. BSA (PDB: 3 V03) structural information was obtained from the Protein Data Bank. BSA and SA were prepared for docking in AutoDockTools (Morris et al., 2009), using the previously published protocol (Chakraborty, Sengupta, Pal, & Basu, 2017; Pal, Pramanik, Bhattacharya, Banerji, & Maiti, 2015; Pal, Pramanik, Bhattacharya, Banerji, & Maiti, 2016). Model 3 V03 for BSA has a resolution of 2.7 Å and it is the best model so far according to the validation report available at wwPDB (Gore, Velankar, & Kleywegt, 2012). However, it showed alternate atom positions for some amino acid residues and contained many missing side chain atoms. The alternate atoms were removed and the missing side chains were modelled at PDB_hydro-server (Azuara, Lindahl, Koehl, Orland, & Delarue, 2006). All the hetero-atoms, water, and the extra subunits were removed from the protein PDB structure. Polar hydrogen atoms and Gasteiger charges were added to the protein, as well as to the ligands. The custom parameter for Cu atoms was added, following the AutoDock 4.2 user guide (Ray, Koley Seth, Pal, & Basu, 2012). Partial charges on the Cu ions were standardized, following previously published protocols (Rudra, Pal, Maiti, Reiter, & Swarnakar, 2013). All the rotatable bonds in the ligand were set free. A search space was defined surrounding the drug site I of BSA. The box dimension was 40 × 40 × 40 cubic points with a grid point spacing of 0.375 Å. Docking was performed by AutoDock 4.2, using a genetic algorithm, which was run 100 times at random seed and a cut off of 25 million energy evaluations each time. Predicted poses were clustered using 2 Å RMSD tolerance (Pal, 2016). For docked Cu complex of SA, residues within 5 Å of ligand were subjected to energy minimization in Schrodinger Maestro, followed by re-scoring of binding interaction in AutoDock 4.2. Molecular docking outputs were rendered in MGLTools (Morris et al., 2009) and PyMOL. Ligand interaction diagrams were generated in Schrodinger Maestro 11.0. UniProt residue numbering was used for BSA sequences (UniProt KB: ALBU_BOVIN).

2.2. Methods 2.2.1. UV–vis spectroscopy All the UV–vis spectra were recorded on a Hitachi U-2910 spectrophotometer. The absorption spectra of SA and SA(Cu2+)2 in the absence and presence of an increasing concentrations of BSA were recorded in the range of 300–500 nm and 300–600 nm, respectively. 2.2.2. Fluorescence spectroscopy The emission spectra were measured on a F-7000 Hitachi fluorescence spectrophotometer equipped with a circulating water bath. The excitation wavelength for BSA was 295 nm. All the measurements were done at the micromolar range to avoid aggregation and inner filter effect. Four sets of fluorescence measurements were performed: A. The fluorescence emission spectra of BSA (3 µM) were recorded when the BSA solution was titrated with gradual addition of SA (0–35 µM), referred to as the BSA-SA system. B. SA (1 equivalent) was incubated with Cu2+ (2 equivalents), so that the complex, SA(Cu2+)2, could be formed. The formation of the complex was confirmed by the generation of a dark pink colour and the formed complex was gradually added to the solution of BSA (3 µM), referred to as BSA-[SA(Cu2+)2] system. C. BSA (3 µM) was incubated with Cu2+ (6 µM) and to this solution, SA was gradually added. D. The buffered solution of BSA (3 µM) was titrated with gradual addition of Cu2+ solution (0–55 µM). The site-specific studies were performed at 298 K, using warfarin and ibuprofen as site I and site II markers, respectively. The concentrations of BSA and site markers were fixed at 3 µM. Warfarin and ibuprofen were incubated with BSA for 1 h. SA was gradually added to the incubated solution and the corresponding binding constant values were evaluated. 2.2.3. Time-resolved fluorescence spectroscopy Time-resolved spectra were measured by a Time Master fluorimeter from Photon Technology International (PTI, USA), equipped with a pulsed laser driver of a PDL series (PDL-800-B from Picoquant, Germany) with interchangeable sub-nanosecond pulsed LEDs and picodiode lasers (Picoquant, Germany) with a TCSPC set up (PTI, USA). The signals were collected at the magic angle of 54.75° to eliminate any interference from fluorescence anisotropy and no detectable differences between the fitted τ values and those obtained from normal decay measurements were observed. A buffered solution of BSA with gradual addition of SA and its Cu2+ complex was excited using PLS-290 at a repetition frequency of 10 MHz. The decay of the sample was analyzed

2.2.5. Molecular dynamics Molecular dynamics (MD) analysis was carried out in Schrodinger Maestro Molecular Modeling environment (academic release 2017-4), using the Desmond (Bowers et al., 2006) molecular dynamics programme. The solvation dynamics of SA(Cu2+)2 complex was monitored in water (TIP4P) and acetonitrile for 480 ps under a OPLS (optimized potentials for liquid simulations) 2005 force field (Harder et al., 2016). Docked complex SA(Cu2+)2 with BSA was subjected to MD simulation for 12 ns in SPC (simple point charge) water environment under the same force field. The protein, the complexes or the ligand were placed 3

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in the centre of the orthorhombic simulation box with periodic boundary conditions and the distance from each side was at least 10 Å. All systems were charge-neutralized, using appropriate counter ions (Na+ or Cl-). A five step relaxation protocol (Pal et al., 2016) was used, starting with Brownian dynamics for 100 ps with restraints on solute heavy atoms at NVT (with T = 10 K) followed by 12 ps of dynamics with restraints at NVT (T = 10 K) and then at NPT (T = 10 K), using the Berendsen method. Then the temperature was raised to 300 K for 12 ps, followed by a 24 ps relaxation step without restraints on the solute heavy atoms. The production MD was run at NPT with T = 300 K. The molecular dynamics output was rendered in Schrodinger Maestro Suite.

collision between the fluorophore and the quencher, whereas, static quenching is caused by the formation of a non-fluorescent ground state fluorophore-quencher complex. To differentiate the aforementioned quenching mechanism, we need (a) a temperature variation study and (b) a fluorescence lifetime study. In the temperature variation study, if the quenching rate constant values decrease with increasing temperature, the operative mechanism is static quenching and the reverse effect is observed in the case of dynamic quenching (Lakowicz, 2006). In this context, to get a generalized picture of the quenching mechanism involved, a fluorescence titration experiment was performed at three different temperatures for the BSA-SA system only. In the introductory part, we have already mentioned that SA forms a chargetransfer complex with Cu2+. Generally, a weak association between the molecule and metal ion results in the formation of charge-transfer complexes. This type of weak association does not lead to a strong bonding and hence, is subject to significant temperature dependencies (Jain, 2012). Thus a temperature study for the BSA-SA(Cu2+)2 system was not performed. Fig. 1a and b portray the effects of increasing concentrations of SA and SA(Cu2+)2 complex, respectively, on the fluorescence spectra of BSA at 298 K. The emission maxima of BSA appeared at ∼342 nm when excited with a wavelength of 295 nm. BSA solutions excited at 295 nm emit fluorescence attributable to Trp residues (Sengupta, Sahasardar, et al., 2018). The emission intensity of BSA solution decreased regularly with gradual addition of SA and its Cu2+ complex. As calculated from Fig. 1, the fluorescence intensity of BSA was quenched about ∼70% and ∼85% by adding ∼30 µM, of SA and its Cu2+ complex, respectively. Thus the quenching efficiency of SA was lower than that of the SA(Cu2+)2 complex. This observation clearly indicated that Cu2+ chelation of SA altered its structure and thus affected the quenching of BSA fluorescence. Interestingly, a gradual increase in the emission spectra of BSA with an increasing amount of SA was observed in the region of 450–470 nm. This may be attributed to the presence of free SA which has emission maxima in that particular region. No such observation was found in the case of the SA-Cu2+ complex. Additionally, a significant red shift of ∼8 nm (from 342 nm to 350 nm) of the emission maxima was observed when SA was added to the solution of BSA. This bathochromic shift indicates a decrease in hydrophobicity around the Trp microenvironment upon the addition of SA. The observation was quite different in the case of the BSASA(Cu2+)2 system. The presence of a 35 µM concentration of SA(Cu2+)2 complex resulted in a blue shift of ∼ 4 nm (from 342 nm to 338 nm) of the emission maxima of BSA. This hypsochromic shift symbolizes an increase in hydrophobicity of the Trp microenvironment upon the addition of SA(Cu2+)2 complex (Lakowicz, 2006). Thus, again, a difference in the interaction mode could be discerned on chelation of SA by Cu2+. Further, the well-known Stern-Volmer equation (Eq. (3)) has been utilized to describe the quenching mechanism involved in BSA-SA and BSA-SA(Cu2+)2 interaction (Lakowicz, 2006).

2.2.6. ABTS assay The ABTS assay was performed according to the method of Re et al. (1999). In this assay trolox (0–150 µM) was used as an anti-oxidant standard. The stock solutions include 7.4 mM ABTS and 2.6 mM potassium persulfate. The stock solutions were then mixed in equal quantities and then allowed to react for 12–16 h in the dark at room temperature to produce ABTS radical cation (ABTS•+). The solution was then diluted with 60 ml of deionized water to acquire an absorbance of < 1 at 734 nm. Next, the experimental sample sets were prepared by mixing 100 µl solutions of SA and its Cu2+ complex or trolox of different concentrations, separately with 1900 µl of ABTS•+ solution. The resulting solution was allowed to react for 2 h in the dark. The absorbances of all the solutions were recorded at 734 nm with the help of a UV–vis spectrophotometer. A blank reading with an appropriate solvent was also taken. All the solutions were freshly prepared on the day of the experiment and all determinations were carried out in triplicate. The results were recorded in terms of trolox equivalent antioxidant capacity [TEAC (µM)]. 3. Result and discussion 3.1. Complex formation between SA and Cu2+ The appearance of a new peak in UV–vis spectra symbolizes the formation of a new complex. The absorbance spectrum of free SA consists of two peaks at ∼236 nm and ∼320 nm. In our earlier manuscript, the formation of SA(Cu2+)2 complex was described in terms of the appearance of a new peak at ∼512 nm with two shoulder bands at ∼446 nm and ∼479 nm (Sengupta, Ganguly, et al., 2018). As mentioned in the introduction section, we have already characterized the complex formation of SA with Cu2+ via ESI-MS and 1H NMR spectra. Here, in this report we offer 13C NMR spectra as further evidence of complex formation (Fig. S7 in the Supporting information). 3.2. Steady-state fluorimetric studies Fluorescence spectroscopy reflects the changes in the fluorophore environment upon binding with small molecules, and is thus widely employed to investigate any interaction. Fluorescence of BSA arises from the presence of tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) residues. When an excitation wavelength of 295 nm is applied, aromatic Trp residues, Trp 237 and Trp 159 for BSA (UniProt KB: ALBU_BOVIN) (Shi et al., 2014) alone contribute most to the fluorescence because of the substantially low quantum yield of phenylalanine and a negligible molar absorption of Tyr. Generally, small molecules result in quenching of the BSA fluorescence. In the present study, when SA (in the absence and presence of Cu2+) was added to the solution of BSA, the fluorescence spectra of BSA were observed to be quenched. Fluorescence quenching of a molecule may arise from different molecular interactions, e.g. molecular rearrangements, energy transfer, excited state reactions, ground state complex formation or collisional quenching interactions. The mechanisms of fluorescence quenching are usually classified as dynamic quenching or static quenching or staticdynamic both. Dynamic quenching originates from excited state

F0 = 1 + kq 0 [Q] = 1 + K SV [Q] F

(3)

where, F0 and F represent the corrected fluorescence intensities of BSA in the absence and presence of different concentrations of quencher; respectively, kq is quenching constant of the biomolecule, τ0 is the fluorophore lifetime in the absence of quencher, [Q] is the quencher concentration and KSV is the Stern-Volmer quenching constant. Fig. 1c and d depict the plot of F0/F versus [Q] of BSA-SA (three different temperatures) and the BSA-SA(Cu2+)2 system, respectively. The straight line obtained from the Stern-Volmer plot of the BSA-SA system signifies the predominant presence of one type of quenching mechanism, either static or dynamic, while the same for the BSASA(Cu2+)2 system depicted a perceptible deviation from linearity (upward curvature). This can be due to two reasons. It can either be ascribed to the presence of a combined quenching mechanism (static and 4

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dynamic) or to the presence of free Cu2+ in equilibrium with the complex, which can induce dynamic quenching (Lakowicz, 2006). To confirm whether the upward curvature was due to the presence of free Cu2+ or due to the combined mechanism, we performed a fluorescence titration experiment by monitoring the BSA fluorescence with gradual addition of Cu2+ ion at three different temperatures and calculated the KSV values (Table S1 and Fig. S1 in the Supporting information). As is evident from Table S1, the KSV values increased with an increase in temperature which convincingly justified the presence of dynamic quenching. Hence the nonlinear nature of the Stern-Volmer plot of the BSA-SA(Cu2+)2 system may arise from the presence of free Cu2+ ion in equilibrium. Additionally, the time-resolved fluorescence studies have also been utilized to further confirm the dynamic quenching by Cu2+ (cf. Section 3.3). To evaluate the Stern-Volmer quenching constant (KSV) value for the BSA-[SA(Cu2+)2] system, the lower concentration range of the plot of F0/F vs. [Q] was considered (Fig. 1d). The KSV values of the BSA-SA and BSA-[SA(Cu2+)2] systems were obtained from the slope of the regression curves and the corresponding bimolecular quenching constant (kq) values were also calculated using the equation, KSV = kqτ0 (Eq. (3)) and lifetime of native BSA. The values of KSV and kq along with the correlation coefficient (R2) are listed in Table 1. A steady decrease in KSV value with an increase in temperature of the BSA-SA system justified the presence of a static quenching mechanism. Additionally, the kq values listed in Table 1 were much greater than 2 × 1010 M−1 s−1, exceeding the largest possible value of dynamic collisional quenching constant (Sengupta et al., 2018a). This further showed that fluorescence quenching of BSA with SA was static, which was also in accordance with previous reports of BSA-SA interaction (Liu et al., 2005; Trnkova et al., 2010; Jin et al., 2012) and the UV–vis study discussed later (cf. Section 3.8). Again, the data accumulated in Table 1 clearly show that the KSV value for the interaction of BSA with SA(Cu2+)2 complex was much greater than that of free SA. In this case, also the kq value was much greater than 2 × 1010 M−1 s−1. Accordingly, the static quenching mechanism can be considered to be playing an extensive role in the overall quenching process of BSA with SA(Cu2+)2. This phenomenon of static quenching of the BSA-SA(Cu2+)2 system was also evident from the UV–vis study discussed later (cf. section 3.8). We have also performed similar experiments in the high concentration range of SA and SA(Cu2+)2 complex (up to 120 µM) and obtained similar natures of the Stern-Volmer curve (cf. Fig. S2 in the Supporting information). A comparative study was also carried out by gradual addition of SA to a solution of BSA previously incubated with Cu2+, and the value of KSV was calculated for this system (Fig. S3 and Table 1). As is evident from Table 1, the values of KSV for the systems BSA-SA and (BSA + Cu2+)-SA were almost comparable. This important observation convincingly suggested that the binding of BSA with SA was altered only when Cu2+ was allowed to form a complex with SA, but not with BSA; i.e. to say, BSA structure was not perhaps so much altered in the presence of Cu2+ as to affect binding. Hence the binding of Cu2+ incubated BSA with SA, also remained the same. A similar conclusion could also be drawn from the binding experiments (cf. Section 3.4).

3.3. Time-resolved study of BSA-SA and BSA-Cu2+systems To achieve a further understanding of the interaction of the BSA and SA(Cu2+)2 system we have resorted to lifetime studies. The phenomenon of dynamic quenching can be well recognized by time-resolved fluorescence measurements. Static quenching deals with the formation of ground‐state complexes and thus the decay time of the native fluorophores (here, BSA) remains unaffected upon increasing quencher concentration. In contrast, dynamic quenching influences the excited state and thus decreases the mean decay time of the entire excited‐state population (Lakowicz, 2006; Saha Sardar, Samanta, Maity, Dasgupta, & Ghosh, 2008). As is evident from Fig. 2a and b, BSA exhibits biexponential fit in the presence of both SA and free Cu2+ ion, separately, with a favourable χ2 values. The relevant decay parameters are assembled in Table S2 in the Supporting information. The bi-exponential fluorescence decay of the free native BSA was in the range of 5.04–5.65 ns, which is compatible with numerous literature reports (Saha Sardar et al., 2008). The data of decay parameters collected in Table S2 did not reflect any significant change in the average fluorescence lifetime (τavg) of BSA as a function of SA, indicating a static quenching mechanism. In contrast, the interaction of the free Cu2+ ion with BSA occurred through a perceptible decrease in the τavg value which convincingly suggested the presence of dynamic quenching in the operative quenching mechanism. Now, the Stern-Volmer equation (Eq. (4)) was employed to evaluate the dynamic quenching constant, KD (Lakowicz, 2006) 0

pH

Temperature

KSV (M−1)

kq (M−1 s−1)

R2

BSA-SA

7.4

BSA-[SA(Cu2+)2] (BSA + Cu2+)-SA

7.4 7.4

298 K 303 K 308 K 298 K 298 K

3.7 × 104 3.5 × 104 3.0 × 104 1.1 × 105 1.7 × 104

6.6 × 1012 6.1 × 1012 5.4 × 1012 2.2 × 1013 3.4 × 1012

0.99 0.99 0.99 0.99 0.99

(4)

where, 〈τ0〉 and 〈τ〉 are the average lifetimes of BSA in the absence and presence of the quencher molecule (SA and free Cu2+ion), respectively. Fig. 2c and d represent the plots of 〈 τ0〉/〈τ 〉 vs [Q] for the BSA-SA and BSA-Cu2+ systems, respectively. From the slope of the linear plot, the dynamic quenching constant (KD) was evaluated as 4.84 × 102 M−1 for the BSA-SA system, which was much lower than that for the potential dynamic quenching agents. Again, the inset of Fig. 2c clearly indicates that the time-resolved Stern-Volmer curve was far below than that of the Stern-Volmer curve for fluorescence quenching in the BSA-SA system, which accounts for the predominant presence of a static quenching process. In contrast, the value of KD was found to be 3.70 × 103 M−1 for the BSA-Cu2+ system, which supports the presence of dynamic quenching in the operative quenching mechanism. Additionally, the inset of Fig. 2d displays almost similar patterns of both time-resolved and SternVolmer curves for fluorescence quenching, which also indicates a dynamic quenching mechanism. Thus, based on the above analysis, it can be concluded that the upward curvature of the Stern-Volmer plot for the interaction of BSA with SA(Cu2+)2 (Fig. 1d), as mentioned earlier, was due to the presence of free Cu2+ ion in the solution, which induced dynamic quenching. Consequently, the presence of simultaneous static and dynamic quenching in the operative quenching mechanism for the BSASA(Cu2+)2 system can be ruled out. We also performed time-resolved fluorescence studies for the BSA-SA(Cu2+)2 system but the experimental results were hampered by the presence of free Cu2+ in solution and thus the data have not been presented here. But in keeping with the observations found with SA alone, where the static mechanism was the predominant one, we can safely associate a static mechanism with the copper complex too.

Table 1 List of Stern-Volmer quenching constants (KSV) and bimolecular quenching rate constants (kq) for the BSA-SA, BSA-[SA(Cu2+)2] and (BSA + Cu2+)-SA systems. System

= 1 + KD [Q]

3.4. Identification of binding parameters With an objective to see the binding interactions of BSA with SA and its Cu2+ complex, the binding constant values were calculated using the following equation (Eq. (5)) (Lakowicz, 2006) 5

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Fig. 2. Fluorescence decay of (a) BSA-SA system and (b) BSA-Cu2+ system and the corresponding Stern-Volmer curves of (c) BSA-SA and (d) BSA-Cu2+ system originating from lifetime study (inset: The Stern–Volmer curves of both lifetime and quenching data of the system; excitation and emission band passes are 10 nm each).

log

(F0

F) F

= log kb + n log[Q]

3.5. Thermodynamic parameters and nature of binding forces

(5)

Thermodynamic measurements have long been recognized to identify the major binding forces between drug and serum albumins. Hydrogen bonding, hydrophobic forces, van der Waals forces, and electrostatic interactions are the four principal forms of interaction between drug and proteins. Thermodynamic parameters, such as enthalpy change (ΔH), entropy change (ΔS) and free energy change (ΔG), were used to analyze these forces of interactions. The ΔH, ΔS, ΔG values were evaluated from the van’t Hoff equation:

where F0 and F represent the corrected fluorescence intensities of BSA in the absence and presence of quencher molecule, respectively, kb being the binding constant and n the number of binding sites. The double logarithmic plots of log [(F0-F)/F] vs. log [Q] of the BSA-SA and BSA-SA(Cu2+)2 systems are portrayed in Fig. S4 in the Supporting information. The values of kb and n were obtained from the intercept and slope of the linear curve and the corresponding values are listed in Table S3 in the Supporting information. A gradual decrease in kb values was observed for the BSA-SA system with an increase in temperature confirming the destabilization of the system at a higher temperature. The value of the binding constant of the BSA-SA(Cu2+)2 system was almost comparable to that of the BSA-SA system. The number of binding sites (n) was found to be approximately 1, indicating that there was one binding site in BSA to interact with SA or SA(Cu2+)2 complex. Further, when BSA was incubated with Cu2+ and SA was gradually added to this solution, the binding constant for this system was also found to be comparable with the previous two systems (Fig. S5 and Table S3 in the Supporting information). Thus a further proof of the inability of Cu2+ to affect BSA structure in its interaction with SA has been obtained.

log kb = G=

H S + 2.303RT 2.303R H

(6) (7)

T S −1

−1

where, kb represents the binding constant and R (8.314 J K mol ) is the gas constant. Thus Eqs. (6) and (7) were utilized to establish the nature of operative forces for the interaction of BSA with SA. Since we were unable to perform the temperature variation studies of the BSASA(Cu2+)2 system, van’t Hoff plots could not be obtained. The acting forces for the BSA-SA(Cu2+)2 system were recognized with the help of theoretical modelling discussed in Section 3.7. The van’t Hoff plot of log Kb vs. 1/T for the BSA-SA system is illustrated in Fig. S6 (Supporting information). The values of ΔH and ΔS were calculated from the slope and intercept of the plot, respectively, 6

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and are summarized in Table S4 in the Supporting information. Ross and Subramanian (1981) proposed three categories of binding forces, depending upon the magnitude of thermodynamic parameters:

complex was prepared in acetonitrile and then added to the buffer solution containing BSA. Solvation dynamics of SA(Cu2+)2 complex in water and acetonitrile showed that both the Cu ions prefer octahedral geometries in solution. However, due to the strain imposed by the two ortho oxygen atoms in the SA, the coordination number may fluctuate between 6 and 7, thus transiently assuming capped prismatic geometry. SA(Cu2+)2 complexes in solution with immobilized solvent atoms are shown in Video S8 and Video S9. Within the protein complex, as well, the Cu ions prefer octahedral geometry, where the solvent is replaced by the side chains of BSA, as depicted in Fig. 3b. One copper centre recruits the side chain of Glu176 and three water molecules to form an octahedral arrangement. The other copper centre recruits the side chain of Glu315 and the backbone carbonyl groups of Ser310, His311 and Ile313, to form a capped octahedron. A snapshot of interaction from MD simulation after 10 ns is also shown in Fig. 3b. Apart from the interactions through copper, several other interactions were observed as we have also observed for free SA. For example, Tyr173 forms a pistacking interaction with the benzene ring of SA; Arg222, Ser238 and Arg241 form hydrogen bonds with the carboxylic acid group; Arg280 maintains contact with the phenolic oxygen. Other interacting residues involved are His265, Leu283, Ala284 and Ala314.

(a) ΔH > 0, ΔS > 0 symbolize hydrophobic forces. (b) ΔH < 0, ΔS < 0 symbolize van der Waals interaction, hydrogen bond formation. (c) ΔH < 0, ΔS > 0 symbolize electrostatic interactions. The data collected in Table S4 in the Supporting information reflect that the values of both ΔH (−273.17 kJ mol−1) and ΔS (−811.40 J mol−1 K−1) are negative, which suggests that the forces of interactions between BSA and SA include hydrogen bond formation and weak van der Waals forces (Ross & Subramanian, 1981). A negative value of ΔG accounted for the spontaneously BSA-SA binding process. 3.6. Site-specific experiment In order to comprehend the efficacy of a biologically active molecule to function as a therapeutic agent, recognition of its binding location in the model transport protein is very crucial and important. Here, we have explored the binding site of SA and SA(Cu2+)2 in BSA on the basis of a site-specific experiment. The principal binding sites of BSA are located at subdomain IIA (site I) and IIIA (site II). Site I is the warfarin binding site, whereas, site II is the ibuprofen-binding site (Sudlow, Birkett, & Wade, 1976). The site selectivity experiment was performed with the help of warfarin (site I maker) and ibuprofen (site II maker). The binding constant values (kb) of BSA with SA/SA(Cu2+)2 in the absence and presence of warfarin and ibuprofen were evaluated using Eq. (5). Fig. S7 in the Supporting information represents the plots of binding constants for the BSA-SA and BSA-SA(Cu2+)2 systems, respectively, in the presence of warfarin and ibuprofen. The kb values of both SA and its Cu2+ complex with BSA in the presence of warfarin and ibuprofen are summarised in Table S5 in the Supporting information. The data collected in Table S5 reflect a considerable decrease of the kb value in the presence of warfarin, which signifies a similarity of binding site between warfarin and SA/SA(Cu2+)2 to BSA. Furthermore, the almost unaltered kb value in both the systems, in the presence of ibuprofen, accounted for the low affinity of SA towards site II. Hence from the results, we can assume that the principal binding site of both SA and its Cu2+ complex to BSA is site I (subdomain IIA).

Video S8.

Video S9.

3.7. Molecular docking and dynamics

3.8. UV–vis studies

After a series of spectroscopic experiments, molecular docking has been employed to compare the interaction patterns of SA and its Cu2+ complex with BSA. Experimental results suggested that SA and its Cu2+ complex bind to the major drug binding site, the drug site I (Petitpas, Bhattacharya, Twine, East, & Curry, 2001), on BSA. Therefore, molecular docking simulations were performed to find out the most favourable binding pose of SA and SA(Cu2+)2 complex inside the drug site I of BSA. The binding free energies for the interactions of these two ligands were −24.56 and −21.30 kJ/mol, respectively. The binding energies are favourable, as well as comparable, for both the SA and the SA(Cu2+)2 complex. Fig. 3a depicts the interaction of SA with BSA. Hydrogen bonding, pi-stacking, and hydrophobic interactions are the major forces involved in this interaction. Arg222, Arg241 and Ser238 form hydrogen bonds with the carboxylic acid group of SA, whereas Arg280 forms a hydrogen bond with the phenolic OH group. Tyr 173 forms pi stacking with the benzene ring of SA. Other hydrophobic amino acids, Trp237, Leu234, Leu261, Leu283, and Ala314, provide solvent exclusion. Binding of SA(Cu2+)2 complex with BSA is depicted in Fig. 3b, where Cu2+ plays an important role. To find the coordination number of these bound coppers, we studied the solution dynamics of the SA(Cu2+)2 complex in both water and acetonitrile as the SA(Cu2+)2

UV–vis absorption spectroscopy is an efficient tool for identifying the ground state complex formation between drug molecules and protein. Thus we have utilized this method to observe the changes in the absorption spectra of SA and SA(Cu2+)2 complex in the presence of BSA. Fig. 4 portrays the effect of addition of BSA on the absorption spectra of SA and SA(Cu2+)2 complex. As already mentioned in section 3.1, the absorption spectra of free SA consist of two characteristics peaks at ∼236 nm and ∼ 320 nm, while a new peak at ∼512 nm, along with the characteristic peak of ∼320 nm, was observed in the case of the SA(Cu2+)2 complex. In this experiment, we have monitored the changes in the absorption region of 300–600 nm in order to exclude any interference from BSA in the range 250–280 nm. Thus any changes in the absorption spectra will be associated with the ground state complexation. As is evident from Fig. 4a, the absorption band of SA at ∼320 nm undergoes a slight but steady decrease with no apparent peak shift with gradual addition of BSA. Thus a probability of ground state BSA-SA complex formation was evident from UV–vis studies. Further, gradual addition of BSA to the solution of SA(Cu2+)2 complex resulted in a significant decrease of the absorption peak at ∼512 nm, along with a slight increase of the peak at ∼320 nm. Additionally, an abrupt red shift of the peak at ∼320 nm on addition of BSA was observed. 7

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Fig. 3. (a) Binding of SA in the domain IIA of BSA (Protein: cartoon representation with the rainbow colour scheme; Ligand: stick and interacting side chains in lines). Standard colour representation is used for the elements. The details of the interacting residues are shown in the bottom panel. Hydrophobic residues and the interactions are shown in green. Positively charged residues are shown in blue and the negatively charged residues in orange. Polar residues are highlighted in stacking interaction as shown in solid green line; hydrogen bonding with pink arrow; (b) Binding of SA-Cu complex with BSA as obtained by docking and molecular dynamics simulation. Interacting residues through copper are highlighted in the top right figure. Distances of the coordination bonds are shown. The bottom figure shows a snapshot of interactions after 10 ns of MD simulation. Types of residues and the interactions are colour-coded as follows: hydrophobic residues, green; positively charged residues, blue; negatively charged residues, red; polar residues, cyan; hydrogen bond, pink arrow from donor to acceptor; pi-stacking, solid green line; salt bridge, red-blue lines. Cu, LIG d: 22 and LIG d: 33.

Interestingly an isosbestic point at ∼438 nm was identified which suggested that the SA(Cu2+)2 complex was in equilibrium with the newly formed ground state complex, BSA-SA(Cu2+)2. Such alterations in the absorption spectra convincingly suggested the presence of ground state complexation in the interaction of SA(Cu2+)2 complex with BSA (Ray et al., 2012). It is notable that the variation in the absorbance spectra was much more prominent in the case of the Cu2+ complex, which may arise from the exchange of ligand from coordination sphere (Mann, Heinisch, Ward, & Borovik, 2018). In the case of free SA, not

much change occurs in the electronic structure and thus changes in the absorption were not prominent. 3.9. Anti-oxidant activity: ABTS assay The theory of ABTS assay is based on the capacity of an antioxidant (here, SA and its Cu2+ complex) to reduce ABTS radical cation (ABTS• +). During the process, the solution of ABTS•+ changes its colour from blue to colourless. As mentioned earlier, trolox has been utilized as a

Fig. 4. UV–vis spectra of (a) SA (inset: zoomed view of the squared region) and (b) SA(Cu2+)2 in the absence and presence of BSA (0–25 µM); [SA] = [SA(Cu2+)2] = 10 µM; pH = 7.4 (tris-HCl buffer). 8

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Acknowledgment AB gratefully acknowledges the financial support of DST for Project no.YSS/2014/000403 and also the FRPDF grant from Presidency University. PS thanks WBDST, West Bengal for her fellowship (Project no. 546(sanc.)/ST/P/S&T/4G-13/2014). PS would like to acknowledge Dr. Biplab Biswas, Dr. Pinki Saha Sardar, Dr. Aniruddha Ganguly and Mr. Tapendu Samanta for their valuable suggestions and Mr. Ajay Das from Saha Institute of Nuclear Physics (SINP) for lifetime experiments. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125254. References Andjelkovic, M., Camp, J. V., Meulenaer, B. D., Depaemelaere, G., Socaciu, C., Verloo, M., & Verhe, R. (2006). Iron-chelation properties of phenolic acids bearing catechol and galloyl groups. Food Chemistry, 98, 23–31. Andreasen, M. F., Landbo, A. K., Christensen, L. P., Hansen, A., & Meyer, A. S. (2001). Antioxidant effects of phenolic rye (Secale cereale L.) extracts, monomeric hydroxycinnamates, and ferulic acid dehydrodimers on human low-density lipoproteins. Journal of Agriculture and Food Chemistry, 49, 4090–4096. Azuara, C., Lindahl, E., Koehl, P., Orland, H., & Delarue, M. (2006). PDB_Hydro: incorporating dipolar solvents with variable density in the Poisson-Boltzmann treatment of macromolecule electrostatics. Nucleic Acids Research, 34, W38–W42. Bowers, K. J., Chow, E., Xu, H., Dror, R. O., Eastwood, M. P., Gregersen, B. A., ... Shaw, D. E. (2006). Scalable Algorithms for Molecular Dynamics Simulations on Commodity Clusters. SC ' 06: Proceedings of the 2006 ACM/IEEE Conference on Supercomputing (pp. 43). . Chakraborty, B., Sengupta, C., Pal, U., & Basu, S. (2017). Acridone in a biological nanocavity: detailed spectroscopic and docking analyses of probing both the tryptophan residues of bovine serum albumin. New Journal of Chemistry, 41, 12520–12534. Cuvelier, M. E., Richard, H., & Berset, C. (1992). Comparison of the antioxidative activity of some acid-phenols: Structure–activity relationship. Bioscience, Biotechnology, and Biochemistry, 56, 324–325. Feroz, S. R., Mohamad, S. B., Bujang, N., Malek, Sri N. A., & Tayyab, S. (2012). Multispectroscopic and molecular modeling approach to investigate the interaction of flavokawain B with human serum albumin. Journal of Agriculture and Food Chemistry, 60, 5899–5908. Finney, L., Vogt, S., Fukai, T., & Glense, D. (2009). Copper and angiogenesis: Unravelling a relationship key to cancer progression. Clinical and Experimental Pharmacology and Physiology, 36, 88–94. Gore, S., Velankar, S., & Kleywegt, G. J. (2012). Implementing an X-ray validation pipeline for the Protein Data Bank. Acta Crystallographica. Section D, Biological Crystallography, 68, 478–483. Hamza, I., & Gitlin, J. D. (2002). Copper chaperones for cytochrome c oxidase and human disease. Journal of Bioenergetics and Biomembranes, 34, 381. 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Coordination chemistry within a protein host: Regulation of the secondary coordination sphere. Chemical Communications, 54, 4413–4416. Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D. S., & Olson, A. J. (2009). AutoDock4 and AutoDockTools4: Automated docking with

Fig. 5. Plot of TEAC (μM) vs. varying concentrations of SA and SA(Cu2+)2.

standard for this experiment and a plot of absorbance at 734 nm vs. concentration of trolox was constructed (data not shown) in order to calculate the trolox equivalent anti-oxidant capacity [TEAC (μΜ)]. The standard curve with trolox (0–150 µM) was observed to be linear. The obtained result with SA and its Cu2+ complex is portrayed in Fig. 5. As is evident from Fig. 5, the TEAC (μΜ) of SA(Cu2+)2 complex was significantly higher than that of free SA, which convincingly indicates the predominant anti-oxidant activity of the chelate complex, SA(Cu2+)2, over free SA. 4. Conclusions The present work demonstrates the spectroscopic and detailed theoretical studies of the interaction of a naturally occurring phenolic acid, SA and its Cu2+ complex with BSA. Such a study is crucial as metal chelation of phenolic acid is reported to increase its beneficial role, as also has been found in our case. The presence of Cu2+ did not alter the nature of binding of SA to BSA. Both SA and its Cu2+ complex quenched the intrinsic fluorescence of BSA via a static quenching mechanism, though the extent of quenching was greater in the case of SA(Cu2+)2. Meanwhile, a comparative study by incubation of BSA with Cu2+ suggested that the presence of Cu2+ altered the quenching effect only when it was in complexation with SA. The site-marker competitive experiment revealed that the binding site was site I (subdomain IIA) for both the systems, which was also supported by molecular docking studies. Molecular dynamics suggested that the preferred geometry of Cu2+ during its interaction with BSA was octahedral. Finally, ABTS assay revealed a better anti-oxidant activity for SA(Cu2+)2 than SA. Being a more potent free radical-scavenger than the parent molecule, SA(Cu2+)2 is believed to play a prominent role in protecting against oxidative stress. We believe that the present work provides a significant insight into the interacting nature of SA with serum albumin, in both the complexed and free state. Moreover, the elevated antioxidant activity of SA in the presence of Cu2+ provides a significant observation of the biological behaviour of SA which could be further extended to other naturally occurring phenolic acids to understand the biological applications of the drug. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 9

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