Binding Interaction between Bovine Serum Albumin and Chicoric Acid, a Food Functional Component

Corresponding Author: Haifang Xiao and Yuanda Song Colin Ratledge Center for Microbial Lipids, School of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo, PR China Email: xiaohaifang@sdut.edu.cn; songyuanda@sdut.edu.cn Abstract: Fuorescence, FTIR and UV-Vis absorption spectroscopy were used to explore the binding between chicoric acid and Bovine Serum Albumin (BSA). Binding characteristics at various levels of temperature have been calculated. The results indicated that chicoric acid statically quenched the intrinsic fluorescence of BSA. The binding constants (Ka) were 4.14×10 L mol at 273K and 4.29×10 L mol at 298 k. The numbers of binding sites between chicoric acid and BSA were both approximately equal to 1 at the two temperatures. Furthermore, the binding distance between chicoric acid and BSA was 2.69 nm which was calculated according to the Förster’s resonance energy transfer. Thermodynamic parameters suggested that BSA bind chicoric acid spontaneously mainly via hydrophobic interaction. Results demonstrated that the conformation and microenvironment of BSA were changed after binding with chicoric acid. Moreover, chicoric acid showed stronger binding with tryptophan (Trp) residue than with tyrosine (Tyr) residue. Our results can provide scientific basis for studying availability and distribution of chicoric acid.


Introduction
Phenolic acids widely occur in plant leaves, roots and especially fruits, are aromatic acid compounds and secondary plant metabolites (Herrmann and Nagel, 1989). Hydroxybenzoic and hydroxycinnamic acids are two groups of phenolic acids that widely distribute in plants (Ghasemzadeh and Ghasemzadeh, 2011). Much attention has been paid to these natural phenolic acids because of their functional activities in intervening diabetes, inflammatory and cancer as well as antioxidative and anti-microbial properties (Chao et al., 2009;Cueva et al., 2010;Hsu et al., 2000;Maurya et al., 2010;Nayaka et al., 2010). Moreover, previous reports revealed that the binding between some phenolic acids and biomolecules such as DNA and proteins played a certain role in their biological properties (Labieniec and Gabryelak, 2005).
Binding studies of small molecules to proteins are very important in their disposition and efficacy because protein binding can influence the effective solubility, distribution and biological half-life of small molecules in vivo as well as interaction between small molecules and other endogenous or exogenous compounds. Therefore, it is of great necessity for explaining the pharmacodynamics and pharmacokinetics of small molecules to investigate the binding between them and proteins (Cui et al., 2008;Qin et al., 2007). Serum albumins, lipoproteins and alglycoprotein are proteins commonly participated in protein binding (Abdi et al., 2012). Among them, the most abundant blood proteins are serum albumins which play an important role in balancing the oncotic pressure and pH of blood (Carter and Ho, 1994). The most prominent characteristic of serum albumins is that they can act as the depot proteins and transporters for numerous endogenous and exogenous small molecules (Huang et al., 2004). BSA was used frequently in previous studies because of its advantages such as highly stability, cheap and homology with Human Serum Albumin (HSA) in structure (Carter et al., 1994;Naik et al., 2010). BSA contains two tryptophan (Trp) residues including Trp-134 and Trp-212 which possess intrinsic fluorescence. In the first domain Trp-134 is located on the surface of the molecule and in the second domain Trp-212 is located within a hydrophobic binding pocket of the protein (He and Carter, 1992;Hamdanim et al., 2009). The conformation of BSA would be changed upon interacting with small molecules.
UV-Vis absorption and fluorescence spectroscopy were used to investigate the interactions between serum albumins and small molecules for their outstanding sensitivity, selectivity, reproducibility, convenience and theoretical foundation (Zhang et al., 2012). FTIR spectroscopy is reliable method to illustrate the conformational changes of proteins after binding with small molecules (Darwish et al., 2010). Recently, several researches on the binding between phenolic acid and serum albumins have been undertaken using spectroscopic technology (Kang et al., 2004;Labieniec and Gabryelak, 2006;Meng et al., 2012;Rawel et al., 2005;Soares et al., 2007). However, no report of chicoric acid-serum albumins interaction has been found so far. Therefore, this research was carried out to explore the interaction between chicoric acid and BSA under simulated physiological conditions using fuorescence, FTIR and UV-Vis absorption spectroscopy. The mechanism of interaction between chicoric acid and BSA including quenching mechanism, binding parameters, binding distance, thermodynamic parameters and conformational change were explored.

Chemicals and Reagents
Chicoric Acid and BSA was obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents used in this study were of analytical purity. Water used throughout the experiments was ultrapure.

Instrumentations
Hitachi F-4500s pectrofluorimeter (Tokyo, Japan) with a 1.0 cm quartz cell and a 150 W xenon lamp was employed in this study to record fluorescence spectra. Excitation wavelength was set at 285 nm. The widths of excitation slit and emission slit were both 10 nm. The UV-Vis absorption spectra were measured by Shimadzu UV-2550 s pectrophotometer (Kyoto, Japan) in the wavelength range 250-350 nm. FTIR spectra were measured using Thermo-Nicolet Avatar330 FTIR spectrometer (Rochester, NY, USA) using KBr pellets. The weight of samples was measured by Sartorius BP211D analytical balance with a precision of 0.1 mg (Göttingen, Germany). PHS-3Cdigital pHmeter (Shanghai, China) was used to detect pH values.

Preparation of Stock Solutions
To keep the ionic strength of solution NaCl (0.10 M) was used in Tris-HCl buffer (0.10M, pH 7.4). All BSA solutions were prepared in Tris-HCl buffer solution and kept at 0-4°C before used. The stock solution of chicoric acid was prepared in methanol.

Fluorescence Studies
Equal volumes of chicoric acid solutions with various concentrations were added to protein solutions, respectively. All solutions were mixed thoroughly. The final concentrations of chicoric acid were 0, 2, 3, 4, 5, 6, 7, 8, 9 and 10 µM. Then the mixtures of chicoric acid and BSA were equilibrated at 273 or 298 K for 20 min. The fluorescence emissions spectra were recorded in the range of 300-450 nm and the binding constants of chicoric acid-BSA systems were calculated in the base of fluorescence data. The synchronous fluorescence spectra of BSA with or without chicoric acid were recorded with the excitation and emission wavelength intervals (△λ) at 15 and 60 nm, respectively. All the experiments were carried out in triplicate and the measurement error was less than 1%.

Absorption Studies
The UV-Vis spectra were collected by Shimadzu UV-2550 spectrophotometer in the region of 200-450 nm at 298K. The final concentrations of chicoric acid were 0, 2, 3, 4 and 5µM, respectively. While that of BSA was 1 µM.

FTIR Spectroscopic Measurements
The FTIR spectra of Tris-HCl buffer, BSA in the absence and presence of chicoric acid were collected in the spectral region 1000-2000 cm −1 , respectively. Then the FTIR spectra of the sample solution were obtained by subtracting that of Tris-HCl buffer which taken as blank.

Binding Characteristics
Molecular interaction is one of the causes to decrease the fluorescence intensity of a fluorophore (Vijayabharathi et al., 2012). Therefore, the interactions between small molecules and proteins were revealed through detecting fluorescence quenching. In this study, the fluorescence spectra of BSA (λ ex = 285 nm) mixed with chicoric acid were obtained at 273 and 298 K. Figure 2 shown that chicoric acid decreased the fluorescence intensity of BSA and there was a positive correction between concentrations of chicoric acid and fluorescence intensity of BSA. These results indicated that the interaction between chicoric acid and BSA occured and the non-fluorescent complex chicoric acid-BSA formed. Moreover, the emission maximum (λem) of BSA slight red-shifted in the present of chicoric acid, indicating that Trp chromophore in BSA was located in a more hydrophilic environment because of the interaction of chicoric acid with BSA. This result was further confirmed by synchronous fluorescence spectra described below.
Dynamic and static quenching are two main mechanisms of fluorescence quenching and different in dependence on temperature and viscosity. Stern-Volmer equation (Lakowicz and Weber, 1973) (Equation 1) was usually used to analyze the quenching mechanism in the previous studies: Where: F 0 and F = The fluorescence emission intensities with and without quencher, respectively K sv = The Stern-Volmerquenching constant K q = The quenching rate constant [Q] = The concentration of quencher τ 0 = The average lifetime of the molecules without quencher and its value is about 10 −8 s Figure 3 showed the Stern-Volmer plots for BSA fluorescence quenched bychicoric acid. Satisfactory linearity of the Stern-Volmer equations was obtained in the investigated concentrations of chicoric acid. Table 1 listed the values of K sv and K q . The results suggested that with temperatures rising the values of K sv decreased, indicating that static quenching was the probable machenism of fluorescence quenching between chicoric acid and BSA. Moreover, the quenching rate constants (K q ) of BSA were determined to be 9.835×1012 and 7.454×1012L mol −1 s −1 , respectively, which were far greater than the maximum diffusion collision quenching rate constant (2.0×10 10 mol −1 Ls −1 ), further demonstrating that the dominant mechanism was static quenching in the fluorescence quenching process of BSA by chicoric acid.

Binding Constants and Binding Sites
The double-logarithm equation (Bandyopadhyay et al., 2012) (Equation 2) was used to caculate the binding constant (K a ) and the number of binding sites (n) in static quenching interaction: Figure 4 demonstrated plots of lg(F 0 -F)/F versus lg[Q] for chicoric acid-BSA. The values of K a and n can be obtained from the intercept and the slope, respectively. The calculated K a and n at different levels of temperature were summarized in Table 2. The values of n at 273 and 298 K were both equal to 1, suggesting single class of binding site in BSA for chicoric acid.

Thermodynamic Parameters and Binding Force
Generally, the non-covalent interaction of small molecules and proteins cover hydrogen-bonding forces, vander Waals forces, hydrophobic interactions and electrostatic interactions. The major evidences for determining the binding mode of small molecule-protein are thermodynamic parameters such as free energy change (∆G), enthalpy change(∆H) and entropy change (∆S). The parameters above can be estimated from Equation 3 and 4: Where: K a1 and K a2 = Binding constants at temperature T 1 and T 2 , respectively R = The gas constant The interaction researches were implymented at 273 and 298 K. Table 3 listed the thermodynamic parameters for the binding of chicoric acid and BSA. The value of ∆G was negative indicating the binding process of chicoric acid and BSA was spontaneous. The values of ∆H and ∆S were positive implied that the interaction between chicoric acid and BSA was mainly an endothermic and entropy-driven reaction. Meanwhile, the main force between chicoric acid and BSA was hydrophobic force (Zhang et al., 2012).

Binding Distance
The spectral researches revealed that a complex was formed between chicoric acid and BSA. Additionally, Fig. 5 showed the fluorescence emission spectrum of BSA and the absorption spectrum of chicoric acid.  The values of K 2 , φ and n have been reported for BSA are 2/3, 0.14 and 1.36, respectively (Zhuang et al., 2012). J, the overlap integral of the fluorescence emission spectrum of donor and absorption spectrum of the acceptor, can be obtained using Equation 7 (Pang et al., 2012): Where: F(λ) = The fluorescence intensity of fluorescent donor at wavelength λ ε(λ) = The molar absorption coefficient of the acceptor at wavelength λ Basing on Equation 5, 6 and 7, it could be obtained that J =1.07×10 −14 cm 3 ·L·mol −1 , R 0 = 2.52 nm, E = 0.40, r = 2.69 nm. The value of r is less than 8 nm, suggesting the shift of non-radiative energy between BSA and chicoric acid. Moreover, the value of r was larger than that of R 0 also suggested that the quenching mechanism of chicoric acid to BSA was static (Zhuang et al., 2012). Base on the above, the static quenching combined with non-radiative energy transfer was the quenching mechanism for chicoric acid to BSA.

Synchronous Fuorescence Spectra
Synchronous fluorescence spectroscopy can be used to investigate the changes in structure and conformation of proteins. The shift in maximum emission wavelength corresponds to changes in polarity around the chromosphere molecules (Jayabharathi et al., 2012). Synchronous fluorescence spectra offer information about the characteristics of Tyrresidue and Trpresidue when ∆λ between excitation wavelength and emission wavelength is fixed at 15 and 60 nm, respectively (Shi et al., 2012). Figure  6 showed that with the increasing concentration of chicoric acid the fluorescence intensities at ∆λ = 15 and ∆λ = 60 nm were both decreased gradually. The emission maximum of Tyr residue kept unchanged at 288 nm upon addition of chicoric acid, suggesting that chicoric acid had no obvious change on the microenvironment of the Tyr residue. Whereas, it was observed that the emission maximum of Trp residue had a weak red shift by about 1 nm from 283 nm to 284 nm, indicating that Trp residue was close to chicoric acid, the hydrophobicity around the Trp residue decreased, however, the polarity increased (Zhuang et al., 2012). Additionally, we calculated fluorescence quenching ratios (R SFQ ) basing on the equation R SFQ = 1-F/F 0 . In this equation, F and F 0 are the synchronous fluorescence intensities of BSA with and without chicoric acid, respectively (Meng et al., 2012). As shown in Fig. 7, the R SFQ for ∆λ = 15 nm were smaller than the R SFQ for ∆λ = 60 nm, suggesting that chicoric acid was more accessible to Trp residue than to Tyr residue .

FTIR Measurements
Different amide bands in infrared spectra of proteins indicate different vibrations of the peptide moiety. Two amide bands related with the secondary structure of protein were the protein amide I band at 1600-1700 cm −1 (mainly C=O stretching) and II band at 1500-1600 cm −1 (C-N stretching coupled with N-H bending mode). In this study, the binding between chicoric acid and BSA was demonstrated using infrared spectroscopy to obtain more information on mechanism of this interaction and conformational changes of BSA. Figure 8 showed the FTIR spectra of BSA and chicoric acid-BSA complex. The peak position of amide I and II bands shifted from 1654 to 1649 and 1542 to 1552 cm −1 , respectively. The changes of these peak positions indicated that in the protein structural subunits C=O and C-N groups were bound with chicoric acid and then rearranged the polypeptide carbonyl hydrogen bonding pattern, finally changed the secondary structure of BSA (Tantipolphan et al., 2007).

UV-Vis Absorption Studies
For reconfirming the conformational change of BSA by the addition of chicoric acid, UV-Vis absorption spectra of BSA with varying concentrations of chicoric acid were obtained. In the present of chicoric acid, the absorption peak intensity of BSA increased as well as the peak red shifted from 278 to 286 nm (Fig. 9). It was reported that dynamic quenching did not change the absorption spectrum, but the formation of non-fluorescence ground-state complex can change it (Du et al., 2012). Thus, these results indicated that the interaction between chicoric acid and BSA caused the formation of ground sate complex and reconfirmed the static quenching mechanism of this interaction (Liu et al., 2010).

Conclusion
In this study, the interaction of chicoric acid and BSA was studied using spectroscopic analysis. The results demonstrated that static quenching process was probable the quenching mechanism of BSA by chicoric acid. It was calculated that one binding site in BSA was accessible to chicoric acid. Thermodynamic parameters revealed that the binding reaction was spontaneous and hydrophobic force played a major role during the interaction. The distance between chicoric acid and BSA was less than 8 nm base on the Förster's resonance energy transfer. Additionally, BSA undergone conformational and microenvironment changes upon binding to chicoric acid and the binding site is mainly at Trp residue.

Acknowledgement
This work was supported by Shandong Provincial Natural Science Foundation, China (No. ZR2014CQ002). Therefore, we are grateful for this funding and support of this research.

Funding Information
This work was supported by Shandong Provincial Natural Science Foundation, China (No. ZR2014CQ002).

Author's Contributions
Haifang Xiao: Took part in all experiment process as well as data analysis and manuscript preparation.
Quancai Sun: Involved in study design and data analysis.

Ethics
This article is original and contains unpublished material. The corresponding author confirms that all of the other authors have read and approved the manuscript and no ethical issues involved. Carter, D.C. and J.X. Ho, 1994