Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods

Food Chemistry 136 (2013) 442–449

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Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods Guowen Zhang ⇑, Yadi Ma State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, Jiangxi, China

a r t i c l e

i n f o

Article history: Received 17 May 2012 Received in revised form 25 August 2012 Accepted 5 September 2012 Available online 17 September 2012 Keywords: Amaranth Human serum albumin Fluorescence spectroscopy Fourier-transform infrared spectroscopy Interaction

a b s t r a c t The mechanism of interaction between food dye amaranth and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence, UV–vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Results obtained from analysis of fluorescence spectra indicated that amaranth had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The negative value of enthalpy change and positive value of entropy change elucidated that the binding of amaranth to HSA was driven mainly by hydrophobic and hydrogen bonding interactions. The surface hydrophobicity of HSA increased after binding with amaranth. The binding distance between HSA and amaranth was estimated to be 3.03 nm and subdomain IIA (Sudlow site I) was the primary binding site for amaranth on HSA. The results of CD and FT-IR spectra showed that binding of amaranth to HSA induced conformational changes of HSA. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Food dyes including natural and synthetic dyes are the most interesting group of food additives, which are applied to improve appearance, colour and texture of foodstuffs, and enhance the aesthetic demand of foodstuffs to consumers. Compared with natural dyes, synthetic colourants have more extensive application because of its high stability to light, oxygen and pH, low microbiological contamination, and relatively lower production costs (Llamas, Garrido, Nezio, & Band, 2009). Amaranth (Fig. 1A), a synthetic azo dye, has been widely used in foods with a reddish or brownish colour, including soft drinks, cake mixes, ice-creams, cereals, wines, salad dressings, and coffee (Mpountoukas et al., 2010). However, amaranth can induce allergic and asthmatic reactions in sensitive people when it contacts with some kinds of drugs (e.g., aspirin, benzoic acid) within the human body (Nevado, Cabanillas, & Salcedo, 1995). What’s more, there is ample evidence indicating that ingestible synthetic azo dyes, containing azo (N@N) functional groups and aromatic ring structure, are reductively cleaved into aromatic amines and many aromatic amines are toxic, mutagenic, and carcinogenic (King-Thom, 2000). Already in 2001, Tsuda et al. used the comet (alkaline single cell gel electrophoresis) assay to measure DNA damage induced by orally administering amaranth to pregnant and male mice, and found the assay was positive in the colon 3 h after the administration of amaranth (Tsuda et al., 2001). Moreover, the positive genotoxic effects of amaranth ⇑ Corresponding author. Tel.: +86 79188305234; fax: +86 79188304347. E-mail address: [email protected] (G. Zhang). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.09.026

have been reported in a chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line (Ishidate et al., 1984) and in application of the standard plate assay to ether extracts of aqueous solution of amaranth (Prival, Davis, Peiperl, & Bell, 1988). According to above evidences, a more widespread safety assessment of amaranth was warranted, and the use of this colourant should be strictly controlled by laws, regulations and acceptable daily intake (ADI) values. Human serum albumin (HSA) is a heart-shaped tridomain protein with each domain consisting of two identical subdomains A and B, containing 585 amino acid residues and its amino acid sequence contains 17 disulfide bridges distributed over all domains (Shaw & Pal, 2008). As the major soluble protein constituent of circulatory system, it has many physiological and pharmacological functions. For instance, it contributes to colloid osmotic blood pressure and is mainly responsible for the maintenance of blood pH. Furthermore, it can bind and transport a large number of ligands present in blood such as drugs, bilirubin, bile acids, metabolites, dyes, etc. (Qi et al., 2008). The binding of chemicals to protein will change the macromolecular conformation, and thus affect physiological function of protein. Consequently, the study of the interaction of amaranth with HSA is important, and will be helpful to shed light on the disposition, transportation and metabolism of amaranth at the molecular level. The aim of this work was to probe molecular mechanism of amaranth binding to HSA by multispectroscopic methods including fluorescence, UV–vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The binding mechanism was investigated according to the fluorescence data. The

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150 W xenon lamp and a thermostat bath. UV–vis absorption spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Shimadzu, Japan) and a quartz cell of 1.0 cm was used for the measurements. CD spectra were recorded on a Bio-Logic MOS 450 CD spectrometer (Bio-Logic, France) using a 1.0 mm path length quartz cuvette. FT-IR spectra were obtained on a Thermo Nicolet5700 FT-IR spectrometer (Thermo Nicolet Co., USA) equipped with a germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector, and a KBr beam splitter. All experiments, unless specified otherwise, were carried out at room temperature. 2.2. Materials Human serum albumin (fatty-acid free) was purchased from Sigma Chemical Company (St. Louis, USA) and dissolved in 0.05 mol L1 Tris–HCl buffer of pH 7.4 containing 0.05 mol L1 NaCl to form a 4.25  104 mol L1 stock solution, and then diluted to the required concentrations with the buffer. Amaranth was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The stock solution (1.65  103 mol L1) of amaranth was prepared in ultrapure water. All chemicals were of analytical reagent grade, and used without further purification. A Millipore Simplicity water purification system (Millipore, Molsheim, France) was applied to produce freshly ultrapure water throughout the experiment. 2.3. Procedures

Fig. 1. (A) Molecular structure of amaranth. (B) Effect of amaranth on the fluorescence spectra of HSA (pH 7.4, T = 298 K, kex = 280 nm, kem = 341 nm). c(HSA) = 2.0  106 mol L1; c(amaranth) = 0, 2.74, 5.46, 8.17, 10.86, 13.52, 16.18, 18.81, 21.43, 24.03, and 26.61  106 mol L1 for curves a ? k, respectively; Curve x shows the emission spectrum of amaranth only, c(amaranth) = 2.0  106 mol L1. (C) The Stern–Volmer plots for the fluorescence quenching of HSA by amaranth at different temperatures.

protein surface hydrophobicity was monitored using 8-anilino-1naphthalenesulfonic acid (ANS) as a fluorescence hydrophobic probe. CD and FT-IR spectroscopy were employed to explore the conformational changes of HSA in the presence of amaranth. The results reported are expected to provide some useful information for further discussing the toxicology of amaranth. 2. Materials and methods

2.3.1. Fluorescence spectroscopy A quantitative analysis of the potential interaction between amaranth and HSA was carried out by fluorimetric titration. A 3.0 mL solution containing 2.0  106 mol L1 HSA was added to a 1.0 cm quartz cuvette, and then titrated by successive addition of a 8.25  104 mol L1 of amaranth solution with a trace syringe (to give a final concentration of 26.61  106 mol L1). These solutions were allowed to stand for 6 min to equilibrate, and then the fluorescence spectra were measured at three temperatures (298, 304, and 310 K) in the wavelength range of 285–500 nm with exciting wavelength at 280 nm. The widths of both the excitation slit and emission slit were set at 5.0 nm. The appropriate blanks corresponding to the Tris–HCl buffer solution were subtracted to correct background of fluorescence. The inner-filter effect refers to the absorption of exciting and/or emitted radiation by molecule added during the fluorescence titration procedure, which caused a spurious decrease in the observed fluorescence intensity (Wu et al., 2011). In fluorescence titration experiments, amaranth has absorption at the excitation and emission wavelengths of HSA. With the gradual addition of amaranth, the excitation and emission light were absorbed, inducing factitiously reduced emission intensity. Therefore, it is necessary to subtract such effect from the fluorescence data. The extent of inner-filter effect can be eliminated using the following formula (Zhang, Zhao, & Wang, 2011):

F c ¼ F m eðA1 þA2 Þ=2

ð1Þ

where Fc and Fm are the corrected and measured fluorescence, respectively. A1 and A2 are the absorbance of amaranth at excitation and emission wavelengths, respectively. The intensity of fluorescence used in this paper was the corrected fluorescence intensity. The operative quenching mechanism in the amaranth–HSA systems can be deduced from the Stern–Volmer equation (Deng & Liu, 2012).

2.1. Apparatus

F0 ¼ 1 þ K SV ½Q  ¼ 1 þ K q s0 ½Q F

Fluorescence measurements were performed with a Hitachi spectrofluorimeter Model F-4500 (Hitachi, Japan) equipped with a

where F0 and F are the fluorescence intensities of HSA in the absence and presence of quencher, respectively. Kq is the quenching

ð2Þ

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rate constant of biomolecule, s0 is the average lifetime of biomolecule without quencher and the value of s0 of biopolymer is 108 s. KSV is the Stern–Volmer dynamic quenching constant, and [Q] is the concentration of quencher. Therefore, the KSV at different temperatures can be determined by linear regression plot of F0/F versus [Q]. Furthermore, the apparent binding constant (Kb) and number of bound amaranth to HSA (n) were determined by plotting the double logarithm regression curve using the following equation (Shahabadi, Maghsudi, Kiani, & Pourfoulad, 2011):

F0  F ¼ log K b þ n log½Q  log F

PSH ¼ ð3Þ

The association constant was estimated by the modified Stern– Volmer equation (Li, Zhu, Jin, & Yao, 2007):

F0 1 1 1 þ ¼ F 0  F fa K a ½Q fa

ð4Þ

where Ka is the modified Stern–Volmer association constant for the accessible fluorophores, and f a is the fraction of accessible fluorescence. From the plot of F0/(F0–F) versus 1/[Q], the values of Ka and fa were obtained from the values of slope and intercept, respectively. In consideration of the dependence of association constant (Ka) on temperature, a thermodynamic process was considered to be responsible for this interaction. Therefore, the thermodynamic parameters were calculated in order to further characterize the acting forces between HSA and amaranth. If the temperature does not vary significantly, the enthalpy change (DH) can be regarded as a constant, then its value and entropy change (DS) value can be calculated from the van’t Hoff equation as follows:

log K a ¼ 

DH DS þ 2:303RT 2:303R

ð5Þ

where R is the gas constant, and the temperatures used were 298, 304, and 310 K. The values of DH and DS were obtained from the slope and intercept of linear plot of log Ka versus 1/T. The value of free energy change (DG) can be calculated from the following equation:

DG ¼ DH  T DS

ð6Þ

2.3.2. Protein surface hydrophobicity (PSH) determination Surface hydrophobicity properties of HSA were assessed by fluorimetric titration experiments with the use of ANS as a fluorescence probe. A 3.0 mL solution containing 2.0  106 mol L1 HSA was transferred to a 1.0 cm quartz cuvette, and ANS was successively added to this solution until no further increase in fluorescence intensity was observed. The excitation wavelength was 390 nm and the emission wavelength was 480 nm. The data were elaborated using the Scatchard plot (Khodarahmi et al., 2012):

F F F max ¼  app þ app ½ANSfree Kd Kd

[ANS]bound = F/B and the concentration of free ANS was obtained from the difference between total and bound ANS concentrations. The Fmax and K app were calculated by standard linear regression fitd ting procedures, which could provide information about differences in the ANS binding properties of HSA in the absence and presence of amaranth. Consequently, the protein surface hydrophobicity (PSH) index can be determined using the following equation (Coi et al., 2008):

ð7Þ

where [ANS]free is the concentration of free ANS (106 mol L1), Fmax is the maximum fluorescence intensity at the saturated ANS concentration which indicates the number of surface hydrophobic sites of the protein. 1=K app is the apparent dissociation constant of HSA– d ANS complex, and 1=K app is the binding affinity of ANS to HSA. d Determination of free ANS concentration needs plotting of fluorescence intensity versus total ANS concentration. It was assumed that all ANS molecules were bound to the protein in very dilute solutions of ANS (0–5.5  107 mol L1), there was a linear relationship between fluorescence intensity (F) and ANS concentration (c) (F = Bc, where B is the proportionality coefficient between fluorescence intensity and ANS concentration). Then, the concentration of the bound ANS was calculated by the following equation:

F max ½HSAK app d

ð8Þ

2.3.3. Energy transfer between HSA and amaranth According to Förster’s non-radioactive resonance energy transfer theory (Förster & Sinanoglu, 1996), the energy transfer can occur only when the fluorescence emission spectrum of donor and the absorption spectrum of acceptor have enough overlaps and the distance between donor and acceptor is not longer than 7 nm. The energy transfer efficiency E is related to the distance R0 between donor and acceptor by the equation:

E¼

R60 R60

þ r6

¼1

F F0

ð9Þ

where r is the distance from the ligand to the tryptophan residue of protein. R0 is the critical distance when their transfer efficiency is 50%, which can be calculated based on the following equation:

R60 ¼ 8:79  1025 j2 N4 UJ

ð10Þ

2

where j is the spatial orientation factor of dipole for random orientations as in a fluid solution, N is the refractive index of medium, U is the fluorescence quantum yield of donor in the absence of acceptor, and J is the overlap integral between the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. J can be given by

P J¼

FðkÞeðkÞk4 Dk P FðkÞDk

ð11Þ

where F(k) is the fluorescence intensity of donor in the wavelength range k to k + Dk, e(k) is the molar absorption coefficient of acceptor at wavelength k. In this work, the concentrations of amaranth and HSA were kept at 2.0  106 mol L1, the UV–vis absorption spectrum of amaranth and the emission spectrum of HSA were recorded in the range of 285–500 nm. 2.3.4. Preparation of different conformers of HSA HSA is known to exist in different conformational states as native (N, pH approximately 7.0), fast moving (F, pH approximately 3.5) isomeric forms and an equilibrium intermediate state (I) in the urea induced unfolding pathway of HSA around 4.8– 5.2 mol L1 urea concentrations (Ahmad, Parveen, & Khan, 2006). For preparing the different conformers of HSA, 2.0  106 mol L1 HSA solutions were prepared in pH 7.0, pH 3.5 buffer solutions, and 5 mol L1 urea to form N, F and I conformations, respectively. Different isomers in the experimental preparation were confirmed with various spectroscopic properties of different forms viz., the emission wavelengths for N, F and I forms were 345, 340 and 350 nm, respectively, and the excitation wavelength was set at 280 nm. 2.3.5. Site marker competitive experiments The competitive experiments were carried out using four site markers (warfarin, Eosin Y, ibuprofen, and digitoxin) by keeping the concentration of HSA and the site markers constant at 2.0  106 mol L1. Amaranth was then gradually added to the site markers–HSA mixtures. The excitation wavelength of 280 nm was

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selected and the fluorescence emission spectra of above systems were recorded. 2.3.6. UV–vis absorption spectroscopy Absorption titration experiments were conducted by keeping the concentration of amaranth at 1.65  105 mol L1, while varying HSA concentrations from 0 to 12.38  106 mol L1. The absorption spectra of above solutions and free HSA solution were measured over a wavelength range of 420–640 nm. All observed absorption spectra were corrected for the buffer absorbance. For further investigating the binding properties between amaranth and HSA, the intrinsic binding constant, Kb0 was determined according to the following equation: (Ni, Du, & Kokot, 2007).

c c 1 ¼ þ jea  ef j jeb  ef j K b0 jeb  ef j

ð12Þ

where c = [HSA], the concentration of HSA; ea, ef, and eb are the apparent, free, and bound complex absorptivity, respectively. ef was determined by a calibration curve of isolated amaranth in aqueous solution, following Beer’s law. ea was determined as the ratio between the measured absorbance and amaranth concentration, Aobs/[amaranth]. The plot of [HSA]/|ea  ef| versus [HSA] gives a slope of 1/|eb  ef| and intercept equals to 1/Kb’|eb  ef|, Kb0 is obtained by the ratio of the slope to the intercept. 2.3.7. Circular dichroism (CD) spectroscopy CD measurements of free HSA and its pigment complex were made in the UV-region (200–250 nm) under constant nitrogen flush. The concentration of HSA was kept at 2.0  106 mol L1, and molar ratios of amaranth to HSA were varied as 0:1, 2:1, 4:1 and 8:1. All observed CD spectra were corrected for buffer signal, and results were expressed as mean residue ellipticity (MRE) in deg cm2 dmol1. The contents of different secondary structures of HSA, e.g., a-helix, b-sheet, b-turn, and random coil, were analyzed from CD spectroscopic data by the online SELCON3 program. More information about the program is available at the following Web site: http://dichroweb.cryst.bbk.ac.uk/html/home.shtml. 2.3.8. Fourier transform infrared spectroscopy (FT-IR) FT-IR measurements were performed on a Thermo Nicolet-5700 FT-IR spectrometer. All spectra were taken via the ATR method with a resolution of 4 cm1 and 60 scans. The FT-IR spectra of free HSA (2.0  105 mol L1) and amaranth–HSA complex (the molar ratio of amaranth to HSA was 2:1) were recorded in the range of 1800–1400 cm1 at pH 7.4 Tris–HCl buffer solution and room temperature. The corresponding spectra of buffer solution were measured under the same conditions and taken as blank, which were subtracted to obtain the FT-IR spectra of the sample solution. The secondary structure compositions of HSA and its amaranth complex were estimated by the FT-IR spectra and curve-fitted results of amide I band. 3. Result and discussion 3.1. Analysis of fluorescence quenching of HSA by amaranth Fluorescence quenching refers to any process which decreases the fluorescence intensity of a fluorophore and can be caused by a variety of molecular interactions, including excited-state reactions, molecular rearrangements, energy transfer, ground state complex formation, and collisional quenching (Zhang et al., 2011). Generally, fluorescence quenching mechanism is classified as dynamic quenching and static quenching, which can be distinguished by their different dependence on temperature. For static quenching, the quenching constant decreases with increasing temperature,

445

but the reversed effect is observed for dynamic quenching. In order to elucidate the binding of amaranth with HSA, the fluorescence spectra were recorded and shown in Fig. 1B. It was obvious that HSA had a strong fluorescence emission peak at 341 nm after being excited at a wavelength of 280 nm, while amaranth was almost non-fluorescence under the same experimental conditions. The fluorescence intensity of HSA decreased markedly with increase in amaranth concentration, which was caused by the interaction between amaranth and HSA. Meanwhile, a significant blue shift of the maximum emission wavelength from 341 to 331 nm was also observed, suggested that the polarity of the protein environment was changed in the presence of amaranth (Wang et al., 2011). Fig. 1C displays the Stern–Volmer plots of the quenching of HSA fluorescence by amaranth at different temperatures (298, 304, and 310 K). As seen in Fig. 1C, the plots of F0/F for HSA versus [amaranth], ranging from 0 to 26.61  106 mol L1, were linear. This observation may suggest that a single quenching mechanism, either static or dynamic, occurred at these concentrations (Shahabadi et al., 2011). Moreover, the corresponding KSV values for the binding of amaranth with HSA were obtained to be (2.123 ± 0.007)  104 (298 K, R = 0.9960), (1.488 ± 0.005)  104 (304 K, R = 0.9948), and (1.237 ± 0.004)  104 L mol1 (310 K, R = 0.9982), respectively. The results showed that the quenching constants decreased with increasing temperature, indicated the predominant quenching mechanism of HSA by amaranth was a static quenching procedure. According to Eq. (2), the Kq values were calculated to be (2.123 ± 0.007)  1012, (1.488 ± 0.005)  1012, and (1.237 ± 0.004)  1012 L mol1 s1 at 298, 304, and 310 K, respectively. The values of Kq were of the order of 1012 L mol1 s1, which were much greater than the maximum scatter collision quenching constant of various quenchers with biopolymers (2.0  1010 L mol1 s1). All the evidences suggested that the quenching was not initiated by dynamic collision but by the formation of the amaranth–HSA complex. In addition, the values of association constant Ka for the amaranth–HSA complex at different temperatures were obtained on the basis of Eq. (4) and presented in Table 1. As shown in Table 1, the decreasing trend of Ka with increasing temperature was in accord with KSV’s dependence on temperature as discussed above, which further confirmed that the fluorescence quenching of HSA is static quenching. 3.2. Determination of the interaction force between amaranth and HSA The molecular forces contributing to protein interactions with small molecules substrates may involve hydrophobic force, electrostatic interactions, van der Waals interactions, and multiple hydrogen bonds (Qi et al., 2008). The thermodynamic parameters, enthalpy change (DH) and entropy change (DS) of reaction are the main evidence for confirming the binding force. Based on the Eqs. (5) and (6), the thermodynamic parameters for the interaction of amaranth with HSA were calculated. The values of DH, DS, and DG are listed in Table 1. The negative sign for DG revealed the spontaneity of the binding of amaranth to HSA. According to the rules summarized by Ross and Subramanian (1981), for typical hydrophobic interactions, both DH and DS are positive, while negative DH and DS arise from the van der Waals and hydrogen bonding formation in low dielectric media. Moreover, the classic electrostatic interaction is characterized by positive DS and negative DH, and DH is usually very small, almost equal to zero. As the aqueous solution in the complex formation of amaranth with HSA, the positive value of DS is regularly regarded as an evidence of hydrophobic interaction, because the water molecules that are arranged in an orderly way around the ligand and protein acquire a more random configuration (Li et al., 2007). Besides, the negative DH value observed in the present work can be mainly attributed to hydrogen bonding, but not to electrostatic interaction (Ross &

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Table 1 Modified Stern–Volmer association constants Ka and the relative thermodynamic parameters of the amaranth–HSA system.

a b

T (K)

Ka (105 L mol1)

Ra

DH (kJ mol1)

DG (kJ mol1)

DS (J mol1 K1)

Rb

298 304 310

1.422 ± 0.062 1.203 ± 0.072 1.040 ± 0.110

0.9930 0.9955 0.9935

20.02 ± 0.013

29.40 ± 0.017 29.58 ± 0.018 29.77 ± 0.019

31.46 ± 0.103

0.9990

R is the correlation coefficient for the Ka values. R is the correlation coefficient for the van’t Hoff plot.

Subramanian, 1981). Thus, the binding of amaranth to HSA was driven mainly by hydrophobic force and hydrogen bonds. 3.3. Determining changes in protein surface hydrophobicity (PSH) induced by amaranth ANS, a fluorescence hydrophobic probe, is known to bind to the hydrophobic portions of protein and followed by an increase in ANS fluorescence intensity. Thus, ANS fluorescence may be applied for monitoring possible changes in PSH induced upon amaranth binding. At a fixed concentration of HSA (2.0  106 mol L1) and increasing concentration of ANS, fluorescence intensity of ANS were measured in the absence and presence of 2.74  106 mol L1 amaranth and 5.46  106 mol L1 amaranth separately (Fig. 2A). It can be seen that the hyperbolic responses of ANS fluorescence in the presence of different concentrations of amaranth were slightly different in comparison to the native HSA. From the Scatchard plots (Fig. 2B), the values of K app and Fmax for ANS in the absence and d presence of amaranth with the concentrations of 2.74  106 and 5.46  106 mol L1 were (1.124 ± 0.037)  106 mol L1 and 455.13 ± 0.139, (0.902 ± 0.040)  106 mol L1 and 402.58 ± 0.134, and (0.701 ± 0.039)  106 mol L1 and 349.86 ± 0.117, respectively. Using K app and Fmax values, PSH in the absence of amaranth d was calculated to be 3044.51 ± 28.25, while PSH in the presence of amaranth with the concentrations of 2.74  106 and 5.46  106 mol L1 were 3355.78 ± 25.19 and 3752.53 ± 22.56, respectively. The surface hydrophobicity of HSA increased 10.2% and 23.3%, respectively. Consequently, it may be concluded that binding of amaranth to HSA was accompanying with increase in protein surface hydrophobicity, and the sites in protein showed tighter binding of the probe which could be appeared from the decrease of K app d in the presence of amaranth (Coi et al., 2008). 3.4. Energy transfer between HSA and amaranth The spectral studies suggested that HSA formed complex with amaranth. The distance r between HSA (donor) and the bound amaranth (acceptor) can be determined based on Förster’s non-radiative energy transfer theory. The overlap of the UV–vis absorption spectrum of amaranth with the fluorescence emission spectrum of HSA is shown in Fig. 2C. In the present case, j2 = 2/3, N = 1.336, and U = 0.118 (Zhang et al., 2011). According to Eqs. (9)–(11), the values of the parameters were calculated to be J = 6.816  1015 cm3 L mol1, R0 = 2.15 nm, E = 0.112, and r = 3.03 nm. Obviously, the distance between amaranth and HSA was less than 7 nm, and 0.5R0 < r < 1.5R0, implying that the energy transfer from HSA to amaranth occurred with high probability (Kandagal, Shaikh, Manjunatha, Seetharamappa, & Nagaralli, 2007). Fig. 2. (A) Binding of ANS to HSA in the absence and presence of amaranth (pH 7.4, T = 298 K). c(amaranth) = 0, 2.74, and 5.46  106 mol L1 for curves a, b, and c, respectively. (B) The Scatchard plots for the titration with increasing concentrations of ANS to HSA in the absence and presence of amaranth. c(amaranth) = 0, 2.74, and 5.46  106 mol L1 for linear regression equation a, b, and c, respectively. (C) The spectral overlaps of the fluorescence spectrum of HSA (a) with the absorption spectrum of amaranth (b). c(HSA) = c(amaranth) = 2.0  106 mol L1.

3.5. Identification of the binding sites of amaranth on HSA 3.5.1. The number of binding sites For static quenching interaction, if it was assumed that small molecules bound independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound mole-

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cules can be described by Eq. (3). The apparent binding constant (Kb) and the number of binding sites (n) per albumin molecule for the amaranth–HSA complex can be obtained from the double logarithm regression curve of log [(F0–F)/F] versus log [Q]. The values of Kb and n at 298 K were obtained to be (1.013 ± 0.083)  105 L mol1 and 0.989 ± 0.017 (R = 0.9972), respectively. The calculated Kb value indicated that a strong affinity existed between HSA and amaranth. The value of n was approximately equal to 1, which suggested that there was one independent class of binding site on HSA for amaranth. 3.5.2. Protein unfolding pathway The protein unfolding pathway induced by acid and urea was employed to locate the binding site for amaranth on HSA. The F isomer which predominates at pH 3.5, is characterized by unfolding and separation of domain III, while the I isomer is characterized by unfolding of domain III and partial but significant loss of the native conformation of domain I (Ahmad et al., 2006). Domain II is known to be unaffected by either N–F or N–I transitions. The number of binding sites n and Ka were obtained to be 1.057 ± 0.025 and (1.516 ± 0.028)  105, 1.008 ± 0.032 and (8.742 ± 0.041)  104, and 0.984 ± 0.030 and (2.090 ± 0.075)  104 L mol1 for N, F, I isomers at 298 K, respectively. The number of binding sites from N to F and N to I transitions changed tinily, revealed that the binding site for amaranth may be located on domain II of HSA. The decreased association constant can be deduced as the outcome of the loss of complex inter- and intradomain interactions that support the albumin structure (Khan et al., 2008). 3.5.3. The competition between specific site markers with amaranth In general, the important sub-domains IIA and IIIA of HSA, where small molecules often bind, shared a number of common features, such as the hydrophobic face, the cluster of basic amino acid residues, and praline residues at the tips of the long loops. Additionally, it is known that each sub-domain is unique and exhibits a certain degree of binding specificity. Sudlow, Birkett, and Wade (1976) have concluded that HSA has two major specific ligand-binding sites, Sudlow’s site I and site II. Site I shows affinity for bulky heterocyclic anion with a negative charge localized in the middle of the molecule, while ligands binding to site II are aromatic carboxylic acids with negatively charged acidic group at the end of the molecule (Wolf & Brett, 2000). Sjoholm et al. (1979) have reported that digitoxin binding in HSA is independent of Sudlow’s site I and II, which was nominated as site III. In order to determine the specificity of amaranth binding, competitive experiments were performed with warfarin, Eosin Y, ibuprofen, and digitoxin in the present work. Warfarin and Eosin Y are markers for site I, ibuprofen for site II (Ni, Liu, & Kokot, 2011; Qi et al., 2008), and digitoxin for site III. Then, the experiment data were analyzed by Eq. (4) and the values of Ka in the presence of warfarin, Eosin Y, ibuprofen, and digitoxin were calculated to be (4.031 ± 0.090)  104, (5.107 ± 0.083)  104, (1.302 ± 0.079)  105, and (1.113 ± 0.052)  105 L mol1 at 298 K, respectively. The results showed that the Ka values of amaranth–HSA complex decreased markedly by adding warfarin/Eosin Y, while the addition of ibuprofen and digitoxin resulted in only a lesser difference. These evidences suggested the binding site for warfarin/Eosin Y and amaranth was same on HSA, and Sudlow’s site I located in sub-domain IIA near Trp-214 was proposed to be the main binding site for amaranth on HSA. This also corroborates with the above, protein unfolding experiment placing the colourant at domain II. 3.6. Absorption spectra of amaranth interaction with HSA The UV–vis absorption spectra of amaranth in the absence and presence of HSA are shown in Fig. 3A. The maximum absorption

Fig. 3. (A) The UV–vis absorption spectra of the amaranth–HSA system at pH 7.4. c(amaranth) = 1.65  105 mol L1, and c(HSA) = 0, 1.27, 2.53, 3.79, 5.04, 6.28, 7.51, 8.74, 9.96, 11.17, and 12.38  106 mol L1 for curves a ? k, respectively; Curve m shows the absorption spectrum of HSA only, c(HSA) = 1.65  105 mol L1. (B) The CD spectra of HSA in the presence of increasing amounts of amaranth at pH 7.4. c(HSA) = 2.0  106 mol L1, the molar ratios of amaranth to HSA were 0:1 (a), 2:1 (b), 4:1 (c), and 8:1 (d), respectively.

wavelength of amaranth was at 520 nm and HSA had no absorption in the range of 420–640 nm. The absorption peak of amaranth at 520 nm exhibited gradual decrease with the increasing concentration of HSA and a obvious red shift (from 520 to 528 nm) of the maximum wavelength was observed, indicated that amaranth could interact with HSA to form a complex. The extent of the hypochromism at 520 nm was 18.85% (curves a–k, Fig. 3A). Moreover, an isosbestic point at 564 nm provided evidence of amaranth–HSA complex formation, which further demonstrated that the fluorescence quenching mechanism was static quenching (Liu et al., 2010). On the basis of variations in the absorption spectra of amaranth upon binding to HSA, the linear regression equation of [HSA]/ |ea  ef| versus [HSA] was: [HSA]/|ea  ef| = (1.621 ± 0.068)  104 [HSA] + (0.860 ± 0.054)  109, R = 0.9931, and yielded the intrinsic binding constant, Kb’ = (1.885 ± 0.062)  105 L mol1, which agreed with the association constant (Ka) at 298 K obtained above by the fluorescence quenching data. 3.7. Conformational investigation of HSA induced by amaranth 3.7.1. Circular dichroism (CD) analysis CD spectroscopy was employed to investigate the alteration in protein secondary structure after addition of amaranth due to its sensitive prediction. In this work, the molar ratios ([amaranth]/ [HSA]) of 0:1, 2:1, 4:1, and 8:1 were used for the CD measurement. The spectrum of free HSA (Fig. 3B, curve a) exhibited two negative

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G. Zhang, Y. Ma / Food Chemistry 136 (2013) 442–449

Table 2 The contents of different secondary structures of free HSA and amaranth–HSA systems (CD spectra) at pH 7.4. Molar ratio [amaranth]:[HSA]

a-Helix (%)

b-Sheet (%)

b-Turn (%)

Random coil (%)

0:1 2:1 4:1 8:1

49.7 45.4 43.4 40.2

11.0 12.3 13.1 14.1

15.4 16.9 17.6 18.6

23.9 25.4 25.9 27.1

(A)

1658

0.0030

1650

0.0025

Absorbance

1547

0.0020

1537

0.0010

b

0.0005

0.0000

1800

1700

1600

1500

-1

Wavenumbers(cm )

(B) 0.0025

1658 1645

0.0020

1400

α-helix 50.4% random coil 22.7% β-turn 14.7% β-sheet 7.3% β-anti 4.9%

0.0015

1671

0.0010

0.0005

0.0000

1700

1688

1623

1680

1660

1640

1620

-1

1600

Wavenumbers(cm ) α-helix 45.7% random coil 24.7% β-turn 16.3% β-sheet 8.0% β-anti 5.3%

(C) 1650 0.0008 0.0006

Absorbance

3.7.2. Fourier transform infrared (FT-IR) analysis FT-IR spectroscopy is a powerful technique for detecting H-bonded components of conformer mixtures, which has recently become very popular for quantitatively analyzing the secondary structure of proteins without the restriction of special amino acids (Jiang et al., 2004). In the IR region, proteins exhibit a number of amide bands, which represent different vibrations of the peptide moiety. Among these amide bands of protein, amide I and amide II bands of IR spectra, which severally occur in the region 1700– 1600 cm1 (mainly C = O stretch) and 1600–1500 cm1 (C–N stretch coupled with N–H bending mode), have been extensively used in characterization of chemical composition and conformational studies of proteins (Shahabadi et al., 2011). Fig. 4A shows the FT-IR spectrum of free HSA (subtracting Tris–HCl buffer) and difference spectrum of HSA ([(amaranth–HSA)–amaranth solution]) after binding with amaranth. It can be seen that the peak position of amide I band was shifted from 1658 to 1650 cm1, while that of amide II band was moved from 1547 cm1 to 1537 cm1 upon the addition of amaranth to HSA. The changes of these peak positions suggested that amaranth interacted with the C = O and C–N groups in the protein structural subunits, which induced the rearrangement of the polypeptide carbonyl hydrogen bonding pattern (Khan et al., 2008). In general, amide I band is the most widely used in studies of protein secondary structures as it is more sensitive to the changes than amide II band, and its sub-peaks ascriptions are more mature than that of amide II band (Deng and Liu, 2012). According to the well-established assignment criterion (Wang et al., 2011), the spectral ranges from 1610 to 1640 cm1, 1640–1650 cm1, 1650– 1660 cm1, 1660–1680 cm1, and 1680–1692 cm1 are assigned to b-sheet, random coil, a-helix, b-turn, and b-antiparallel, respectively. The percentages of each secondary structure of HSA were calculated from the integrated areas of component bands in amide I band. Based on Fig. 4B, the free HSA contained major amounts of a-helix 50.4%, random coil 22.7%, b-turn 14.7%, b-sheet 7.3%, and

a

0.0015

Absorbance

bands at 209 and 222 nm, which are characteristic of a-helix of protein, and both contribute to n-k⁄ transfer for the peptide bond of the a-helical structure (Ahmad et al., 2006). As shown in Fig. 3B, first titration of amaranth resulted in deduction of peak intensity (shifting to zero levels), which further decreased upon continuous addition of the amaranth without any significant shift of the peaks. The above results revealed that the a-helical content of HSA decreased, because the intensities of the two negative peaks reflect the amount of protein helicity. Furthermore, the online SELCON3 program was employed to quantificationally analyze the contents of different secondary structures of HSA and summarized in Table 2. A decreasing tendency of a-helical content and an increasing tendency of bsheet, b-turn, and random coil structure contents were observed with increasing concentration of amaranth. The result implied that amaranth bound with the amino acid residues of main polypeptide chain of the protein and destroyed their hydrogen bonding networks (Zhang et al., 2011). Besides, the loss of a-helical content also indicated that the binding of amaranth to HSA induced a little unfolding of the polypeptides of protein, which resulted in the exposure of some hydrophobic regions that were previously buried increasing (Zhang, Dai, Xiang, Li, & Liu, 2010).

1640

0.0004

1676

0.0002 0.0000

1700

1616

1684 1680

1660

1640

-1

Wavenumbers(cm )

1620

1600

Fig. 4. (A) The FT-IR spectra of free HSA (a) and difference spectra [(amaranth– HSA)–amaranth solution] (b) at pH 7.4 in the region of 1800–1400 cm1. c(HSA) = 2.0  105 mol L1, c(amaranth) = 4.0  105 mol L1. The curve-fitted amide I region of free HSA (B) and its amaranth complex (C).

b-antiparallel 4.9%. Upon the interaction with amaranth, the content of a-helix decreased accompanied by an increase in random coil, b-turn, b-sheet, and b-antiparallel. When the molar ratio of amaranth to HSA was 2:1, the content of a-helix decreased from 50.4% to 45.7%, while the contents of random coil and b-turn increased from 22.7% to 24.7% and from 14.7% to 16.3%, respectively, and b-sheet and b-antiparallel increased slightly from 7.3% to 8.0% and from 4.9% to 5.3% (Fig. 4C). The considerable alterations of protein secondary structures were due to a partial protein unfolding in the presence of amaranth (Deng and Liu, 2012).

G. Zhang, Y. Ma / Food Chemistry 136 (2013) 442–449

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