Food and Chemical Toxicology 65 (2014) 227–232

Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Characterization of the binding of chrysoidine, an illegal food additive to bovine serum albumin Bingjun Yang 1, Fang Hao 1, Jiarong Li, Kai Wei, Wenyu Wang, Rutao Liu ⇑ School of Environmental Science and Engineering, Shandong University, China-America CRC for Environment & Health, Shandong Province, 27# Shanda Nanlu, Jinan 250100, PR China

a r t i c l e

i n f o

Article history: Received 10 September 2013 Accepted 27 December 2013 Available online 3 January 2014 Keywords: Proteins Dyes Spectroscopy Isothermal titration calorimetry Thermodynamics Molecular docking

a b s t r a c t Chrysoidine is an industrial azo dye and the presence of chrysoidine in water and food has become an environmental concern due to its negative effects on human beings. Binding of dyes to serum albumins significantly influence their absorption, distribution, metabolism, and excretion properties. In this work, the interactions of chrysoidine with bovine serum albumin (BSA) were explored. Isothermal titration calorimetry results reveal the binding stoichiometry of chrysoidine to BSA is 1:15.5, and van der Waals and hydrogen bonding interactions are the major driving force in the binding of chrysoidine to BSA. Molecular docking simulations show that chrysoidine binds to BSA at a cavity close to Sudlow site I in domain IIA. However, no detectable conformational change of BSA occurs in the presence of chrysoidine as revealed by UV–vis absorption, circular dichroism and fluorescence spectroscopy studies. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Food safety issues have gained considerable attention due to its effects on human health. Chrysoidine is an azo dye (structure shown in Fig. 1), which is usually used for dyeing leather, fibers and paper (Lei et al., 2011). Because chrysoidine may cause several adverse effects on human health, it is banned from being used as a food additive in China (Gui et al., 2010). However, some illegal merchants still dye bean products and yellow croaker by chrysoidine (Lu et al., 2012). In addition, since it is widely used in the dye industry, the presence of chrysoidine in water has become an environmental issue due to the adverse human health and ecological impacts (Abd El-Rahim et al., 2003; Abraham et al., 2003; Bayramoglu and Arica, 2012; Tan et al., 2011). Binding of dyes to serum albumins significantly influence their absorption, distribution, metabolism, and excretion (ADME) properties (Bolel et al., 2012). Therefore, a fundamental understanding of their interactions is essential to comprehend the biological effects of chrysoidine. However, little information is available on the interactions of chrysoidine with serum albumins. Hence, it is important to initiate studies on the interactions between chrysoidine and serum albumins in order to elucidate its relationship to human health risks. In this work, we aimed to explore the interactions of chrysoidine with bovine serum albumin (BSA) using spectroscopy, ⇑ Corresponding author. Tel./fax: +86 531 88364868. 1

E-mail address: [email protected] (R. Liu). These authors contributed equally to this work.

0278-6915/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.12.047

isothermal titration calorimetry and molecular docking methods. The binding affinity constant (K), binding stoichiometry (n), enthalpy changes (DHo), entropy changes (DSo) and Gibbs energy changes (DGo) of the interactions were obtained by means of isothermal titration calorimetry (ITC). Molecular docking simulations were performed to achieve the molecular details of the binding between chrysoidine and BSA. This study will help us apprehend the transportation mechanism and toxicity of chrysoidine in vivo.

2. Materials and methods 2.1. Materials Bovine serum albumins are biological reagent with purity of more than 98.0% by weight, which were bought from Sinopharm Chemical Reagent Shanghai Co., Ltd. Protein concentrations were measured using the BCA protein assay kit (Biocolor BioScience and Technology, Shanghai, China). All other chemicals were of analytical grade. Chrysoidine was obtained from Aladdin Reagent (Shanghai, China). NaH2PO42H2O and Na2HPO412H2O were produced by Tianjin Kermel Chemical Reagent Co., Ltd. A mixture solution of NaH2PO4 and Na2HPO4 (pH 7.4, 0.2 mol L1) was used to control the pH. All solutions were prepared with ultrapure water.

2.2. Isothermal titration calorimetry In order to investigate the binding affinity constant, binding stoichiometry, enthalpy changes and entropy changes of interactions between chrysoidine and BSA, ITC experiments were carried out with a Microcal ITC200 microcalorimeter at 298 K. Approximately 40 lL of BSA (0.198 mM) were titrated into a buffermatched chrysoidine solution (200 lL, 0.340 mM) with stirring speed fixed at 1000 rpm. The first drop was set to 0.4 lL followed by 13 subsequent 3 lL injections. To achieve complete equilibration, the spacing time between each injection was set to 120 s.

228

B. Yang et al. / Food and Chemical Toxicology 65 (2014) 227–232

Time (min) 0

10

20

30

0.00 Fig. 1. The molecular structure of chrysoidine.

2.3. UV–vis absorption analysis UV-2450 spectrometer (Shimadzu, Japan) was applied to measure the UV–vis absorbance spectra. Using 1 cm quartz cells, UV–vis absorption spectra were recorded in the range of 190–350 nm using chrysoidine solution as references.

ucal/sec

-0.20 -0.40 -0.60

2.4. Fluorescence measurements Fluorescence spectra were recorded on a fluorescence spectrophotometer (F-4600, Hitachi, Japan) equipped with a xenon lamp light source and 1.0 cm quartz cells. Excitation and emission slit widths were set to 5 nm. With a scanning speed of 240 nm min1 and scanning voltage of 700 V, emission spectra were recorded in the wavelength range of 290–450 nm upon excitation at 280 nm and 295 nm, respectively. The synchronous fluorescence spectra, measured by scanning simultaneously both the excitation and emission monochromator with a fixed wavelength difference (Dk) between excitation and emission wavelengths, were obtained at Dk = 15 nm, and Dk = 60 nm (Dk = kem  kex, kex at 250–320 nm). In order to correct the inner filter effect caused by chrysoidine and BSA, following formula was used for correcting fluorescence intensity (Lakowicz, 2006):

F cor  F obs  10ðAexþAemÞ=2

kcal mol-1 of injectant

0.00 -0.80

2.5. Circular dichroism spectra Circular dichroism (CD) spectra were collected by a J-810 spectropolarimeter (Jasco, Japan) at room temperature under constant nitrogen flush. Using a cell of 1 cm path length, CD spectra were collected from 190 to 260 nm with a data pitch of 0.2 nm and accumulation of 3 scans. The response time, scan rate and bandwidth were 1 s, 200 nm min1 and 1 nm, respectively. The secondary structure contents of BSA from the CD spectra were estimated by the SELCON3 program in the CDPro software package, which is available at the website: . 2.6. Molecular docking study Molecular docking calculations were carried out with AutoDock 4.2. Using Gaussian 03 package (Frisch et al. 2004), the structure of chrysoidine was generated. Geometries optimized at the HF/3-21G level and the conductor-like polarizable continuum model (CPCM) was chosen to calculate aqueous solvation free energies for chrysoidine. The crystal structure of BSA (PDB code: 4F5S) was downloaded from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/) (Bujacz, 2012). To recognize the binding sites in BSA, blind molecular docking was carried out and a grid box of highest limit 126  126  126 Å grid points with 0.375 Å grid spacing was selected for calculations. Docking simulations were performed using the Lamarckian genetic algorithm method to search for the optimum binding site of chrysoidine to the protein. The molecular docking results presented in figures were prepared using Chimera (Pettersen et al., 2004).

3. Results and discussion

-6.00 H

0

-9.00

T

S

G

-5 -10

-12.00

-15

-15.00

ð1Þ

where Fcor is the correct fluorescence intensity; Fobs is the measured fluorescence intensity, and Aex and Aem are the absorbance value at the excitation and emission, respectively. Time-resolved fluorescence measurements were carried out on a FLS920 combined fluorescence lifetime and steady state spectrometer (Edinburgh, U.K.) at 298 K, with excitation and emission wavelengths at 295 and 330 nm, respectively.

-3.00

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Molar Ratio Fig. 2. Isothermal titration calorimetry for BSA interactions with chrysoidine at 298 K, pH 7.4. The upper panel of each graph shows heat flow for each injection (lcal/s) as a function of time (min); and the bottom panel shows integrated heats in each injection. The solid lines represent the least-square fit to a single set of binding site model. The inset is the thermodynamic parameters histogram of the BSAchrysoidine system.

chrysoidine solution. The ITC titrations of BSA with chrysoidine yielded negative heat deflection, which indicated that the binding was an exothermic process. With each injection, there is an increased binding of chrysoidine to BSA, and the chrysoidine becomes less saturated with BSA, hence there is a progressive lowering of endothermic peaks, which yields a typical titration isotherm. The results suggest that binding interaction occurs between chrysoidine and BSA. The binding curve analyzed with the single set of binding site model is shown in Fig. 2 (lower panel), and the derived thermodynamic parameters are summarized in Table 1. Details about the data analysis can be found in Supplementary material. The negative value of DG° means that the interaction process is spontaneous. The negative value of DH° and DS° reveal that van der Waals and hydrogen bonding interactions play a major role during the interaction. The value of binding constant Ka is (2.73 ± 0.34)  105 L mol1 at 298 K. Furthermore, the binding stoichiometry of chrysoidine to BSA is close to 1:15 (15.5, the reciprocal of n), indicating that there are fifteen binding sites in BSA for chrysoidine. This result is similar to that obtained in the binding of octylglucoside with BSA (Wasylewski and Kozik, 1979).

3.1. ITC analysis

3.2. Conformational changes of BSA investigated by UV–vis

ITC is a simple and straightforward method to characterize molecular interactions. It determines not only the binding affinity constant (Ka) and binding stoichiometry (n), but also enthalpy changes (DHo) and entropy changes (DSo) in a single experiment (Turnbull and Daranas, 2003). The raw data plot of heat flow against time for the titration of 0.198 mM BSA into 0.340 mM chrysoidine at 298 K is shown in Fig. 2 (upper panel). Each peak reflects a single injection and corresponds to the heat change associated with injection of BSA solution into the sample cell containing the

UV–vis absorption spectroscopy is a simple but effective method, which is often applied to explore the structural changes of Table 1 Thermodynamic parameters for interaction of chrysoidine with BSA at 298 K. n (102)

Ka (105 M1)

DHo (cal/mol)

DS o (cal/mol K)

DGo (cal/mol)

6.45 ± 0.14

2.73 ± 0.34

16,220 ± 545

29.5

7430

229

Absorbance

B. Yang et al. / Food and Chemical Toxicology 65 (2014) 227–232

3.0

a

2.5

e

solution increased with increasing added chrysoidine concentration. No detectable change was observed in BSA absorption spectra at around 280 nm in the presence of chrysoidine. The results indicate that the binding of chrysoidine to BSA does not drastically change the conformation of BSA.

2.0 1.5

3.3. Fluorescence quenching of BSA by chrysoidine

1.0 0.5 0.0 200

220

240

260

280

300

320

340

Wavelength (nm) Fig. 3. UV–vis absorption spectra of BSA in the different concentrations of chrysoidine. Conditions: BSA: 5.0  106 mol L1; [chrysoidine]/(105 mol L1): (a) 0, (b) 1, (c) 2, (d) 3, and (e) 4; pH 7.4.

protein and ligand–protein complex formation. BSA has two main absorption bands (Fig. 3). The strong absorption band at around 220 nm is mainly due to the peptide bond p ? p⁄ electronic transitions of the peptide backbone (Scopes, 1974). The weak absorption band around 280 nm is mainly attributed to the absorption of tryptophan and tyrosine (Pace et al., 1995). The absorption spectrum of BSA shows a hypochromic effect around 220 nm with concomitant bathochromic shift as the concentration of chrysoidine increased. The reason for this phenomenon is that the energy required for p ? p⁄ transition decreased when the polar of the BSA

Three aromatic amino acid residues (tryptophan, tyrosine, and phenylalanine) contribute to the intrinsic fluorescence of BSA. When the excitation wavelength is set to 280 nm, the emission fluorescence from BSA is dominated by tryptophan and tyrosine, while when excitation is selected at 295 nm, it minimizes the emission from tyrosine residues and is known to selectively excite tryptophan residues (Prasad et al., 1986). As shown in Fig. 4a, when the BSA was excited at 280 nm, the chrysoidine quenched the fluorescence emission spectrum of BSA. BSA contains 2 tryptophan residues: Trp-134 in domain I and Trp-213 in domain II. Trp-213 is located in a hydrophobic binding pocket, whereas Trp-134 is more exposed to solvent and it is located on the surface of molecule (Mallick et al., 2005). The fluorescence properties of tryptophan residues are exceptionally sensitive to the local environment, making it an ideal choice for the detection of conformational alterations in proteins (Lynch and Dawson, 2008). Fig. 4b shows the fluorescence quenching of BSA by chrysoidine when excitation was set to 295 nm. The maximum emission wavelength of BSA had no significant shift with the addition of chrysoidine, which suggested that chrysoidine had a slight impact on the conformation of the protein (Berger et al., 1998).

3000

10000

(b)

a

Fluorescence intensity

Fluorescence intensity

(a) 8000 6000

e

4000 2000

a

2500 2000 e

1500 1000 500 0

0 300

320

340

360

380

400

320

420

Wavelength (nm)

1800

8000

Fluorescence intensity

Fluorescence intensity

a

6000

e

4000 2000 0 250

260

270

280

290

Wavelength (nm)

360

380

400

420

Wavelength (nm)

10000

(c)

340

300

310

(d)

a

1500 1200

e

900 600 300 0 260

270

280

290

300

310

Wavelength (nm)

Fig. 4. Fluorescence emission spectra and synchronous fluorescence spectra of BSA in the different concentrations of chrysoidine. (a) kex = 280 nm; (b) kex = 295 nm; (c) Dk = 60 nm; (d) Dk = 15 nm. Conditions: BSA: 1.0  106 mol L1; [chrysoidine]/(105 mol L1): (a) 0, (b) 1, (c) 2, (d) 3, and (e) 4; pH 7.4.

230

B. Yang et al. / Food and Chemical Toxicology 65 (2014) 227–232

Table 2 Fluorescence lifetimes of BSA in different concentrations of chrysoidine. Molar ratio of BSA to chrysoidine

s (ns)

v2

1:0 1:2.5 1:5 1:10 1:15 1:25

6.20 6.01 5.82 5.56 5.42 5.08

1.006 0.989 0.992 0.980 1.016 1.019

Synchronous fluorescence spectroscopy is a useful method for the evaluation of protein conformational changes, which gives the information about the microenvironments around the fluorophore functional groups by simultaneous scanning of the excitation and emission monochromators with a constant wavelength interval. When the wavelength interval (Dk) is set to 60 nm or 15 nm, synchronous fluorescence spectra give the characteristic information of tryptophan residues or tyrosine residues, respectively (Liu and Liu, 2012). As seen in Fig. 4c and d, the intensity of the synchronous fluorescence of BSA at Dk = 60 nm and Dk = 15 nm progressively reduced with gradual addition of chrysoidine. No obvious shift in the emission maximum upon quenching was observed, indicating that the interaction of chrysoidine with BSA does not perturb the polarity around these residues significantly. 3.4. Fluorescence quenching mechanism The mechanisms of fluorescence quenching are usually classified into dynamic quenching and static quenching (Xie et al., 2010). During dynamic quenching, the quencher interacts with the excited state of the fluorophore leading to a change of the fluorescence lifetime. By contrast, during the static quenching, the formation of ground-state complex between the fluorophores and the quencher inhibits the excited state formation of the fluorophore, without affecting the fluorescence lifetime (van de Weert and Stella, 2011). Consequently, the most definitive method to distinguish static and dynamic quenching is to measure the fluorescence lifetimes in the presence and absence of quencher (Gauthier et al., 1986). The data obtained from fluorescence lifetime experiments were found to fit well to a single-exponential decay with a v2 values approached 1.00. The lifetime (s) of BSA was decreased from 6.20 to 5.08 ns with the increasing concentration of chrysoidine (Table 2), indicating that the quenching mechanism was a dynamic quenching. For the dynamic quenching, it often involves collisional quenching, photoinduced electron transfer (PET), radiative energy

transfer and Förster resonance energy transfer (FRET), which do not require a direct contact of the quencher with fluorophore. In collisional quenching, higher temperatures result in faster diffusion, and hence the quenching constants will increase with the temperature. The fluorescence quenching data of chrysoidine-BSA binding at different temperatures are analyzed by the Stern–Volmer equation:

F 0 =F ¼ 1 þ K SV ½Q 

ð2Þ

where F0 and F are the steady-state fluorescence intensities in the absence and presence of quencher, respectively; Ksv is the Stern–Volmer quenching constant; and [Q] is the concentration of the quencher (chrysoidine). The Stern–Volmer plots are shown in Fig. 5 and the Ksv values calculated from the Stern–Volmer equation are presented in Table 3. The results show the Ksv values decrease with increasing temperature, indicating that the quenching is not initiated from collisions. Since the interactions of chrysoidine with BSA induce no discernible change in the protein (results obtained from UV–vis spectra experiments), and the fluorescence emission spectrum of BSA overlaps with the UV absorption spectrum of chrysoidine (see Fig. 6), we inferred that the quenching of the BSA fluorescence caused by chrysoidine was attributable to FRET. The reason why the fluorescence of BSA is quenched to a greater extent at higher temperatures is due to the chrysoidine-BSA diffusion during the excited-state lifetime. Lakowicz also observed this phenomenon in the study of FRET between TMA and TU2D (Lakowicz et al., 1994). According to the Förster’s non-radiative energy transfer theory, the parameters related to energy transfer can be calculated based on the equation as follows:

E¼1

s R6 ¼ 6 0 s0 R0 þ r6

ð3Þ

Table 3 Stern–Volmer quenching constants for the interaction of BSA with chrysoidine at three different temperatures.

a b

pH

T (K)

KSV (104 L mol1)

Ra

S.D.b (104)

7.4

288 298 310

2.14 1.91 1.59

0.997 0.999 0.998

0.0335 0.0212 0.0212

R is the correlation coefficient. S.D. is the standard deviation for the KSV values.

2.2 288K 298K 310K

2.0

F0/F

1.8 1.6 1.4 1.2 1.0 0

1

2

[Q]

(10-5

3

mol

4

5

L-1)

Fig. 5. Stern–Volmer plots for the quenching of BSA by chrysoidine at different temperatures. Conditions: BSA: 1.0  106 mol L1; pH 7.4.

Fig. 6. The overlap of fluorescence spectra of BSA and absorption spectra of chrysoidine. Conditions: BSA: 1.0  106 mol L1; chrysoidine: 3.0  105 mol L1; pH 7.4; T = 298 K.

231

B. Yang et al. / Food and Chemical Toxicology 65 (2014) 227–232 Table 4 Förster energy transfer parameters for the BSA-chrysoidine pair in the presence of different amounts of chrysoidine. Cchrysoidine (105 M)

0.5

1

2

3

5

E (%) r (nm)

3.06 5.76

6.13 4.96

10.3 4.51

12.6 4.35

18.1 4.05

where E is the efficiency of energy transfer; s0 and s are the relative lifetimes of the donor, in the absence (s0) and presence (s) of acceptor; r is the distance between the acceptor and the donor, R0 is the distance at which transfer efficiency equals 50%, which can be calculated using the following equation:

R60 ¼ 8:79  1025 K 2 n4 /J

ð4Þ

2

where K is the spatial orientation factor of the dipole, n is the refractive index of the medium, U is the fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor:

R1 J¼

0

FðkÞeðkÞk4 dk R1 FðkÞdk 0

ð5Þ

where F(k) is the fluorescence intensity of the fluorescence donor at wavelength k, and e(k) is the molar absorption coefficient of the acceptor at wavelength k. It has been reported for BSA, that K2 = 2/3, U = 0.15 and n = 1.36 (Deepa and Mishra, 2005; Kandagal et al., 2006). Thus according to the above equations it is calculated that J = 3.78  1014 cm3 L mol1, R0 = 3.21 nm. Values of the r and E corresponding to the different concentration of chrysoidine are summarized in Table 4. With the concentration of chrysoidine increasing, the Förster distance (r) reduces from 5.76 to 4.05, and accompanied with the increase of transfer efficiency. According to Förster’s theory, the distance between the acceptor and donor should be smaller than 10 nm. r, the distance between chrysoidine and Trp residues of BSA, is in the range of 4–6 nm and 0.5R0 < r < 2R0, indicating the results are in accordance with the conditions of FRET and the energy transfer from BSA to chrysoidine occurs with high probability.

Fig. 7. (a) Binding site of chrysoidine on BSA. (b) Binding mode between chrysoidine and the interacting residues of BSA. The atoms of chrysoidine are color-coded as follows: C, magenta; N, blue; H, white. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.6. Molecular docking 3.5. Conformational changes of BSA monitored by circular dichroism spectroscopy To gain a better understanding of the effects of chrysoidine on the conformational change of BSA, CD spectroscopy were employed in the present study. Far UV-CD studies revealed that there were no significant changes in the ellipticity values at 208 or 222 nm, which is characteristic of an a-helical structure of the protein (Xu et al., 2012; Zhao et al., 2009) (Fig. S1, Supplementary material). Similarly, the content of secondary structural elements in BSA was nearly unchanged with increasing the concentration of chrysoidine (Table 5) indicated that the binding of chrysoidine to BSA does not lead to an apparent alteration of the secondary structure of BSA, which is consistent with the results from UV–vis spectroscopy studies.

To predict the binding sites of chrysoidine on BSA, molecular docking simulations were run with Autodock program (version 4.2), which is available free of charge from The Scripps Research Institute (http://autodock.scripps.edu/) (Morris et al., 1998). The structure of chrysoidine was optimization using Gaussian 03 package. During the docking simulation, 10 conformations were obtained (Table S1, Supplementary material), and the lowest energy solution structure of chrysoidine–BSA are shown in Fig. 7. Chrysoidine binds to BSA at a cavity close to Sudlow site I in domain IIA, where small organic compounds, including many drugs, were found to bind to serum albumin (Perry et al., 2006; Saquib et al., 2011). The distance between tryptophan (the donor) and chrysoidine (the acceptor) obtained from the docking simulation is less than 10 nm, which satisfied the condition of FRET (Gadella, 2009;

Table 5 Effect of chrysoidine on the percentage of secondary structure content in BSA. Molar ratio of BSA to chrysoidine

Secondary structural elements in BSA

a-Helix 1:0 1:100 1:400

(±2%)

b-Sheet (±1%)

b-Turn (±1%)

Unordered (±1%)

64.3 64.2 64.3

3.0 3.0 3.4

11.4 11.4 11.7

20.4 20.4 20.6

232

B. Yang et al. / Food and Chemical Toxicology 65 (2014) 227–232

Lakowicz, 2006) and verified the conclusion obtained from fluorescence quenching analysis. Calculation of the binding energy from docking simulations revealed that van der Waals and hydrogen bonding interactions (chrysoidine with Arg 256) play a major role in the binding of chrysoidine to BSA, in agreement with the thermodynamic results obtained from ITC. 4. Conclusions Using spectroscopy, isothermal titration calorimetry and molecular docking methods, this work establishes a strategy to investigate the binding mechanisms of chrysoidine at the molecular level, which will help us apprehend the human health risks caused by chrysoidine exposure. Since the binding stoichiometry of chrysoidine to BSA is 1:15.5 and sudlow site I in domain IIA is identified as the most possible binding site for chrysoidine, the biological effects of chrysoidine in human deserve to be given more attention, and its use should be limited according to the legal regulations. The methods used in this paper could also be applied to explore the molecular mechanisms of other organic pollutants toxicity. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency document associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct. 2013.12.047. Acknowledgements This work is supported by NSFC (21277081), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (708058). Independent innovation program of Jinan (201202083) and Independent innovation foundation of Shandong University natural science projects (2012DX002) are also acknowledged. The authors thank the Theoretical Chemistry Institute of Shandong University for helping with the quantum chemical calculations. We sincerely thank reviewers for constructive suggestions to enhance the quality of the article. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct.2013.12.047. References Abd El-Rahim, W.M., Moawad, H., Khalafallah, M., 2003. Microflora involved in textile dye waste removal. J. Basic Microbiol. 43, 167–174. Abraham, T.E., Senan, R.C., Shaffiqu, T.S., Roy, J.J., Poulose, T.P., Thomas, P.P., 2003. Bioremediation of textile azo dyes by an aerobic bacterial consortium using a rotating biological contactor. Biotechnol. Progr. 19, 1372–1376. Bayramoglu, G., Arica, M.Y., 2012. Preparation of comb-type magnetic beads by surface-initiated ATRP: modification with nitrilotriacetate groups for removal of basic dyes. Ind. Eng. Chem. Res. 51, 10629–10640. Berger, I., Winston, W., Manoharan, R., Schwartz, T., Alfken, J., Kim, Y.G., Lowenhaupt, K., Herbert, A., Rich, A., 1998. Spectroscopic characterization of a DNA-binding domain, Z alpha, from the editing enzyme, dsRNA adenosine deaminase: evidence for left-handed Z-DNA in the Z alpha-DNA complex. Biochemistry 37, 13313–13321. Bolel, P., Mahapatra, N., Halder, M., 2012. Optical spectroscopic exploration of binding of Cochineal Red A with two homologous serum albumins. J. Agric. Food Chem. 60, 3727–3734. Bujacz, A., 2012. Structures of bovine, equine and leporine serum albumin. Acta Crystallogr. Sect. D 68, 1278–1289. Deepa, S., Mishra, A.K., 2005. Fluorescence spectroscopic study of serum albumin– bromadiolone interaction: fluorimetric determination of bromadiolone. J. Pharm. Biomed. Anal. 38, 556–563. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., MontgomeryJr J.A., Vreven, T., Kudin, K.N., Burant, J.C., Millam, J.M., Iyengar, S.S.,

Tomasi, J., Barone, V., Mennucci, B., Cossi, M., Scalmani, G., Rega, N., Petersson, G.A., Nakatsuji, H., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Klene, M., Li, X., Knox, J.E., Hratchian, H.P., Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Ayala, P.Y., Morokuma, K., Voth, G.A., Salvator, P., Dannenberg, J.J., Zakrzewski, V.G., Dapprich, S., Daniels, A.D., Strain, M.C., Farkas, O., Malick, D.K., Rabuck, A.D., Raghavachari, K., Foresman, J.B., Ortiz, J.V., Cui, Q., Baboul, A.G., Clifford, S., Ciomslowski, J., Stefanov, B.B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I., Martin, R.L., Fox, D.J., Keith, T., Al-Laham, M.A., Peng, C.Y., Nanayakkara, A., Challacombe, M., Gill, P.M.W., Johnson, B., Chen, W., Wong, M.W., Gonzalez, C., Pople, J.A., 2004. Gaussian 03, Revision D.01, Gaussian Inc, Wallingford CT. Gadella, T.W.J., 2009. Laboratory Techniques in Biochemistry and Molecular Biology. Academic Press, Burlington. Gauthier, T.D., Shane, E.C., Guerin, W.F., Seitz, W.R., Grant, C.L., 1986. Fluorescence quenching method for determining equilibrium constants for polycyclic aromatic hydrocarbons binding to dissolved humic materials. Environ. Sci. Technol. 20, 1162–1166. Gui, W., Xu, Y., Shou, L., Zhu, G., Ren, Y., 2010. Liquid chromatography–tandem mass spectrometry for the determination of chrysoidine in yellow-fin tuna. Food Chem. 122, 1230–1234. Kandagal, P.B., Ashoka, S., Seetharamappa, J., Shaikh, S.M.T., Jadegoud, Y., Ijare, O.B., 2006. Study of the interaction of an anticancer drug with human and bovine serum albumin: spectroscopic approach. J. Pharm. Biomed. Anal. 41, 393–399. Lakowicz, J.R., 2006. Principles of Fluorescence Spectroscopy, third ed. Springer ScienceþBusiness Media, New York. Lakowicz, J.R., Gryczynski, I., Kusba, J., Wiczk, W., Szmacinski, H., Johnson, M.L., 1994. Site-to-site diffusion in proteins as observed by energy transfer and frequency-domain fluorometry. Photochem. Photobiol. 59, 16–29. Lei, H.T., Liu, J., Song, L.J., Shen, Y.D., Haughey, S.A., Guo, H.X., Yang, J.Y., Xu, Z.L., Jiang, Y.M., Sun, Y.M., 2011. Development of a highly sensitive and specific immunoassay for determining chrysoidine, a banned dye, in soybean milk film. Molecules 16, 7043–7057. Liu, Y., Liu, R.T., 2012. The interaction of a-chymotrypsin with one persistent organic pollutant (dicofol): Spectroscope and molecular modeling identification. Food Chem. Toxicol. 50, 3298–3305. Lu, F.G., Sun, M., Fan, L.L., Qiu, H.M., Li, X.J., Luo, C.N., 2012. Flow injection chemiluminescence sensor based on core–shell magnetic molecularly imprinted nanoparticles for determination of chrysoidine in food samples. Sensor Actuator B 173, 591–598. Lynch, I., Dawson, K.A., 2008. Protein–nanoparticle interactions. Nano Today 3, 40– 47. Mallick, A., Haldar, B., Chattopadhyay, N., 2005. Spectroscopic investigation on the interaction of ICT probe 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine with serum albumins. J. Phys. Chem. B 109, 14683–14690. Morris, G.M., Goodsell, D.S., Halliday, R.S., Huey, R., Hart, W.E., Belew, R.K., Olson, A.J., 1998. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 19, 1639–1662. Pace, C.N., Vajdos, F., Fee, L., Grimsley, G., Gray, T., 1995. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423. Perry, J.L., Goldsmith, M.R., Williams, T.R., Radack, K.P., Christensen, T., Gorham, J., Pasquinelli, M.A., Toone, E.J., Beratan, D.N., Simon, J.D., 2006. Binding of warfarin influences the acid-base equilibrium of H242 in sudlow site I of human serum albumin. Photochem. Photobiol. 82, 1365–1369. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Ferrin, T.E., 2004. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612. Prasad, A., Luduena, R.F., Horowitz, P.M., 1986. Detection of energy transfer between tryptophan residues in the tubulin molecule and bound bis (8anilinonaphthalene-1-sulfonate), an inhibitor of microtubule assembly, that binds to a flexible region on tubulin. Biochemistry 25, 3536–3540. Saquib, Q., Al-Khedhairy, A.A., Siddiqui, M.A., Roy, A.S., Dasgupta, S., Musarrat, J., 2011. Preferential binding of insecticide phorate with sub-domain IIA of human serum albumin induces protein damage and its toxicological significance. Food Chem. Toxicol. 49, 1787–1795. Scopes, R., 1974. Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 59, 277–282. Tan, C.Y., Li, M., Lin, Y.M., Lu, X.Q., Chen, Z.L., 2011. Biosorption of basic orange from aqueous solution onto dried A. filiculoides biomass: equilibrium, kinetic and FTIR studies. Desalination 266, 56–62. Turnbull, W.B., Daranas, A.H., 2003. On the value of c: can low affinity systems be studied by isothermal titration calorimetry? J. Am. Chem. Soc. 125, 14859– 14866. van de Weert, M., Stella, L., 2011. Fluorescence quenching and ligand binding: a critical discussion of a popular methodology. J. Mol. Struct. 998, 144–150. Wasylewski, Z., Kozik, A., 1979. Protein–Non-ionic detergent interaction. Eur. J. Biochem. 95, 121–126. Xie, X.Y., Wang, X.R., Xu, X.M., Sun, H.J., Chen, X.G., 2010. Investigation of the interaction between endocrine disruptor bisphenol A and human serum albumin. Chemosphere 80, 1075–1080. Xu, T.C., Guo, X.J., Zhang, L., Pan, F., Lv, J.N., Zhang, Y.Y., Jin, H.J., 2012. Multiple spectroscopic studies on the interaction between olaquindox, a feed additive, and bovine serum albumin. Food Chem. Toxicol. 50, 2540–2546. Zhao, L.Z., Liu, R.T., Zhao, X.C., Yang, B.J., Gao, C.Z., Hao, X.P., Wu, Y.Z., 2009. New strategy for the evaluation of CdTe quantum dot toxicity targeted to bovine serum albumin. Sci. Total Environ. 407, 5019–5023.