Food Chemistry 133 (2012) 264–270

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Multispectroscopic studies on the interaction of maltol, a food additive, with bovine serum albumin Guowen Zhang ⇑, Yadi Ma, Lin Wang, Yepeng Zhang, Jia Zhou State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, Jiangxi, China

a r t i c l e

i n f o

Article history: Received 25 October 2011 Received in revised form 28 December 2011 Accepted 5 January 2012 Available online 18 January 2012 Keywords: Maltol Bovine serum albumin Fluorescence quenching Binding site Circular dichroism

a b s t r a c t The interaction between maltol, a food additive, and bovine serum albumin (BSA) under simulated physiological conditions was investigated by fluorescence, UV–Vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The results suggested that the fluorescence quenching of BSA by maltol was a static procedure forming a maltol–BSA complex. The positive values of enthalpy change and entropy change indicated that hydrophobic interactions played a predominant role in the interaction of maltol with BSA. The competitive experiments of site markers revealed that the binding of maltol to BSA mainly took place in subdomain IIA (Sudlow site I). The binding distance between maltol and BSA was 3.01 nm based on the Förster theory of non-radioactive energy transfer. Moreover, the results of UV–Vis, synchronous fluorescence, CD and FT-IR spectra demonstrated that the microenvironment and the secondary structure of BSA were changed in the presence of maltol. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Maltol (3-hydroxy-2-methyl-4-pyrone, Fig. 1A), a synthetic perfume, has been widely used to contribute to the fragrance of many foods, such as coffee, soybeans, cereals, breads, malt beverages, and chocolate milk (Leblanc & Akers, 1989). At the recommended concentration, maltol does not have a flavour of its own, but modifies or enhances the inherent flavours of the foods and beverages. The usual amounts of maltol added to beverages, baked food, ice creams, and candy range from 80 to 110 mg L1 (Bjeldanes & Chew, 1979). However, people have found that maltol can do harm to animals and human beings, if large amounts of this flavour enhancer are ingested. In vivo toxicity studies on male and female rats showed that treatment with maltol at a dose of 1000 mg/kg/day for 9 weeks caused significant weight loss, kidney lesions, and increased incidence of albuminuria and mortality (Gralla, Stebbins, Coleman, & Delahunt, 1969). Some researchers have revealed that maltol can enhance aluminium-induced neurofibrillary degeneration in neuronal systems (Savory, Huang, Herman, Reyes, & Wills, 1995) and exhibits weak mutagenic activity (Hayashi, Kishi, Sofuni, & Ishidate, 1988). Because of its use in the daily diet, the toxicology of maltol has been paid close attention to. The interactions between proteins and chemicals have aroused increasing research interest in recent years. Serum albumin, the most abundant soluble protein in the blood circulatory system of organisms, plays an outstanding role in the transport and ⇑ 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. doi:10.1016/j.foodchem.2012.01.014

deposition of a variety of endogenous and exogenous substances. Binding to serum albumin can significantly affect the absorption, distribution, metabolism and toxicity of chemical substances. Consequently, it is of great necessity to investigate the interaction of chemicals with serum albumin, which will help to explain the metabolism and transport process of the chemicals. Bovine serum albumin (BSA) is one of the major components in blood plasma, accounting for about 60% of the total protein, corresponding to a concentration of 43 mg mL1. It has been widely studied because of its stability and low cost, unusual ligand-binding properties and particularly its structural homology with human serum albumin (HSA) (Sulkowska et al., 2007). Fluorescence and UV–Vis absorption spectroscopy are effective techniques to study the small molecules–proteins interactions, because of their sensitivity, reproducibility and convenience. These approaches can reveal the binding affinity of small molecules with proteins and help to understand their binding mechanisms. Circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy are reliable methods for analysing the contents of secondary conformation forms of proteins, which can explain the conformational changes of proteins induced by ligands (Darwish, Abu sharkh, Abu Teir, Makharza, & Abu-hadid, 2010). In the present work, the binding of maltol to BSA was investigated under simulated physiological conditions by multispectroscopic approaches including fluorescence, UV–Vis absorption, CD and FT-IR spectroscopy. The quenching mechanism, thermodynamic parameters, the special binding site and the effect of maltol on the secondary structure of BSA were explored for the first time. The aim of this work is to clarify the binding mechanism of maltol with BSA and provide

G. Zhang et al. / Food Chemistry 133 (2012) 264–270

useful information for understanding the toxicological action at molecular level.

2. Materials and methods

265

2.3.2. UV–Vis absorption spectra The UV–Vis absorption spectra of BSA in the absence and presence of maltol, and the absorption spectra of corresponding concentration of maltol solution were measured over a wavelength range of 200–320 nm in the Tris–HCl buffer solution (pH 7.4) at room temperature.

2.1. Materials Bovine serum albumin (BSA) was obtained from Sino-American Biotechnology Co. (Beijing, China) and used without further purification. The stock solution of BSA (0.8 mM) was prepared with pH 7.4 Tris–HCl buffer (0.10 M Tris base, 0.10 M HCl and 0.05 M NaCl) and then diluted to the required concentrations with the buffer. Maltol was purchased from Jingchun Reagent Co. (Shanghai, China). The stock solution (7.95 mM) of maltol was initially prepared in 5% of absolute ethanol and then diluted with ultrapure water (ethanol/water mixture, i.e., 5/95). It was reported that 5% ethanol did not affect protein structure, while higher ethanol content (>50%) may alter protein structure (Lin, Li, & Wei, 2004). All other reagents and solvents were of analytical reagent grade and aqueous solutions were prepared using freshly ultrapure water obtained from a Millipore Simplicity water purification system.

2.2. Apparatus Fluorescence measurements were performed with a Hitachi spectrofluorimeter Model F-4500 equipped with a 150-W xenon lamp and a thermostat bath, using a 1.0-cm quartz cell. The widths of both the excitation slit and emission slit were set at 5.0 nm. The absorption spectra were measured on a Shimadzu (Kyoto, Japan) UV-2450 spectrophotometer using a 1.0 cm quartz cell. The CD spectra were recorded on a Bio-Logic MOS 450 CD spectrometer (Bio-Logic, Grenoble, France) using a 1.0-mm path length quartz cuvette. FT-IR spectra were measured on a Thermo-Nicolet 5700 FT-IR spectrometer (Waltham, MA) equipped with a germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector and a KBr beam splitter. pH measurements were taken with a pHS–3C digital pH-meter (Shanghai Exact Sciences Instrument Co. Ltd., Shanghai, China) with a combined glass–calomel electrode. All experiments, unless specified otherwise, were carried out at room temperature.

2.3. Procedures 2.3.1. Fluorescence measurements A 3.0 mL solution, containing 0.8 lM BSA, was titrated by successive additions of a 7.95 mM stock solution of maltol (to give a final concentration of 155.9 lM). These solutions were allowed to stand for 6 min to equilibrate. The fluorescence emission spectra were then measured at three different temperatures (298, 304 and 310 K) in the wavelength range of 300–500 nm with an excitation wavelength at 280 nm. The synchronous fluorescence spectra were obtained by setting the excitation and emission wavelength interval (Dk) at 15 and 60 nm, at which the spectrum only shows the spectroscopic behaviour of tyrosine and tryptophan residues of BSA, respectively. The competitive experiments were carried out using different site markers viz., warfarin, ibuprofen and digitoxin for sites I, II and III, respectively, by keeping the concentrations of BSA and the markers constant at 0.80 lM, and then gradually adding a certain volume of maltol solution. The appropriate blank corresponding to the buffer solution was subtracted to correct for background fluorescence. The binding constants of maltol–BSA systems in the presence of the above site markers at 298 K were determined by the fluorescence data.

2.3.3. CD studies The CD spectra were carried out on a Bio–Logic MOS 450 CD spectrometer under constant nitrogen flush using a 1.0-mm path length quartz cuvette. Each spectrum was recorded in the wavelength range of 200–250 nm and a scan speed of 60 nm min1. The CD spectra of BSA incubated with maltol at molar ratios ([maltol]/[BSA]) of 0:1, 5:1 and 10:1 were measured in a pH 7.4 Tris–HCl buffer solution at room temperature. All observed CD spectra were baseline subtracted for buffer and results were expressed as mean residue ellipticity (MRE) in deg cm2 dmol1. The contents of different secondary structures of BSA were analysed from CD spectroscopic data by the online Dichroweb software. More information about the software is available at the following website: http:// dichroweb.cryst.bbk.ac.uk/html/home.shtml. 2.3.4. FT-IR spectroscopic measurements The infrared spectra were measured on a Thermo-Nicolet 5700 FT-IR spectrometer at room temperature. All spectra were measured via the ATR method with a resolution of 4 cm1 and 64 scans. The FT-IR spectra of BSA in the absence and presence of maltol were recorded in the range of 1800–1400 cm1. The molar ratio of maltol to BSA was maintained at 1:1. 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 BSA and its maltol complex were estimated by the FT-IR spectra and curve-fitted results of amide I band. 3. Results and discussion 3.1. Fluorescence quenching studies of BSA by maltol Fluorescence measurements were carried out to investigate the binding mechanism of maltol with BSA. The fluorescence emission spectra of BSA in the absence and presence of maltol with an excitation wavelength at 280 nm are shown in Fig. 1A. As shown in Fig. 1A, BSA exhibited a strong fluorescence emission peak at 353 nm, while maltol did not show intrinsic fluorescence under the same experimental conditions. Furthermore, the fluorescence intensity of BSA remarkably decreased with increase in concentration of maltol. This indicated that maltol interacted with BSA and quenched the fluorescence of BSA. In order to study the quenching mechanism between maltol and BSA, the fluorescence quenching data were analysed using the Stern–Volmer equation (Shahabadi, Maghsudi, Kiani, & Pourfoulad, 2011):

F0 ¼ 1 þ K SV ½Q  ¼ 1 þ K q s0 ½Q F

ð1Þ

where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. KSV is the Stern–Volmer quenching constant, which was determined by linear regression of a plot of F0/F against [Q]. Kq is the quenching rate constant of biomolecule, s0 is the average lifetime of the fluorophore without quencher, the value of s0 of the biopolymer is 108 s (Lakowicz, 2006), and [Q] is the concentration of quencher. Fluorescence quenching could proceed via different mechanisms, usually classified as dynamic quenching and static quenching. Dynamic and static quenching can be distinguished by their

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number of binding sites pre albumin molecule n can be obtained from the double logarithm regression curve of log [(F0  F)/F] versus log [Q] based on the following equation:

log

Fig. 1. (A) Fluorescence spectra of BSA in the presence of maltol at different concentrations (pH 7.4, T = 298 K, kex = 280 nm). c(BSA) = 0.80 lM, and c(maltol) = 0, 1.59, 3.17, 4.74, 6.31, 7.87, 9.43, 10.98, 12.52, 14.06, and 15.59  105 M for curves a ? k, respectively; Curve m shows the emission spectrum of maltol only, c(maltol) = 0.80 lM. (inset) Molecular structure of maltol. (B) The Stern–Volmer plots for the fluorescence quenching of BSA by maltol at different temperatures.

different dependence on temperature and excited-state lifetime (Lakowicz, 2006). For the dynamic quenching, higher temperatures will result in faster diffusion and larger amounts of collisional quenching, hence the quenching constant values will increase with increasing temperature, but the reverse effect would be observed for static quenching. Fig. 1B shows the Stern–Volmer plots for the quenching of BSA by maltol at three different temperatures (298, 304, and 310 K). The curves were linear within the studied concentrations, which suggested that a single type of quenching existed in maltol–BSA interaction. The calculated KSV values at each temperature are shown in Table 1. The results showed that the values of KSV decreased with increasing temperature, and the Kq values obtained were (1.48 ± 0.01)  1012, (1.39 ± 0.01)  1012, (1.27 ± 0.01)  1012 L mol1 s1 at 298, 304 and 310 K, respectively, which were much greater than the maximum scatter collision quenching constant of various quenchers with biopolymers, 2.0  1010 L mol1 s1 (Lakowicz, 2006). All the evidences indicated that the possible quenching mechanism of maltol–BSA interaction was initiated by static quenching and the maltol–BSA complex was formed. 3.2. Binding constant and location of binding site Assuming small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant K and the

F0  F ¼ log K þ n log½Q  F

ð2Þ

From the intercept and slope of the regression curve based on Eq. (2), the values of K and n at three different temperatures were obtained (Table 1). The high linear correlation coefficient r indicated that assumptions underlying Eq. (2) were reasonable. The increasing trend of K with rising temperature indicated that the capacity of maltol binding to BSA was enhanced and the binding was an endothermic reaction (Zhang, Chen, Guo, & Wang, 2009). The values of n were approximately equal to 1, suggesting the existence of just a single binding site in BSA for maltol. The structure of BSA consists of three homologous domains (I– III), and each domain is composed of two subdomains (A and B). There are two major specific ligands-binding sites in BSA and the principal regions usually located in hydrophobic cavities in subdomains IIA and IIIA, which are called site I and site II, respectively (Jin, Zhu, Yao, & Wu, 2007). Sudlow, Birkett, and Wade (1976) have reported that warfarin and ibuprofen bind to site I and site II in serum albumin, respectively. The binding site of digitoxin was found to be independent of site I and site II (Sjoholm et al., 1979), which was defined as site III. In order to identify the maltol binding site on BSA, competitive experiments were carried out using warfarin, ibuprofen and digitoxin as site markers. Varied amounts of the maltol were added to a solution containing fixed concentrations of BSA and site probes, and then the fluorescence emission spectra were recorded with the excitation wavelength at 280 nm. The corresponding values of binding constant at 298 K were obtained to be (3.37 ± 0.07)  104 L mol1 for digitoxin–maltol–BSA system, (2. 47 ± 0.04)  104 L mol1 for ibuprofenmaltolBSA system and (0.95 ± 0.06)  104 L mol1 for warfarinmaltolBSA system, based on Eq. (2). Compared with the binding constant, (3.90 ± 0.08)  104 L mol1 of maltolBSA system, the value of K decreased remarkably upon the addition of warfarin, whereas ibuprofen or digitoxin had a little effect on the binding of maltol to BSA. The results meant that the binding site for maltol and warfarin was the same in BSA; that is to say, the maltol was most likely bound to the hydrophobic pocket located in subdomain IIA (Sudlow site I) near Trp-212 (Katrahalli, Jaldappagari, & Kalanur, 2010). 3.3. Binding mode Generally, there are four representative types of non-covalent interaction existing in small ligands binding to biological macromolecules, including hydrophobic interactions, hydrogen bonds, van der Waals force and electrostatic interactions. Ross and Subramanian (1981) have characterised the signs and magnitudes of the thermodynamic parameters associated with various kinds of interaction force that may happen during the process of ligand binding to protein. The thermodynamic parameters of binding reaction are the major evidence for confirming the intermolecular forces. If there is no significant change in temperature, enthalpy

Table 1 The quenching constants (KSV), binding constants (K), number of binding sites (n) and relative thermodynamic parameters for the interaction of maltol with BSA at different temperatures.

a b

T (K)

KSV ( 104 L mol1)

ra

K ( 105 L mol1)

n

rb

DH (kJ mol1)

DG (kJ mol1)

DS (J mol1 K1)

298 304 310

1.48 ± 0.01 1.39 ± 0.01 1.27 ± 0.01

0.9952 0.9953 0.9942

0.39 ± 0.08 2.52 ± 0.06 8.69 ± 0.10

1.12 ± 0.02 1.34 ± 0.01 1.49 ± 0.02

0.9989 0.9995 0.9992

119.2 ± 0.1

–26.41 ± 0.02 –30.95 ± 0.02 –35.49 ± 0.02

757.0 ± 0.4

r is the correlation coefficient for the KSV values. r is the correlation coefficient for the K values.

G. Zhang et al. / Food Chemistry 133 (2012) 264–270

change (DH) can be regarded as a constant, then its value and entropy change (DS) value can be calculated from the slope and intercept of the plot of logK versus 1/T based on the following Eq. (3). The value of free energy change (DG) can be obtained from Eq. (4):

DH DS þ 2:303RT 2:303R DG ¼ DH  T DS logK ¼ 

E¼

R60 R60

þ r6

¼1

F F0

267

ð5Þ

ð3Þ

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

ð4Þ

R60 ¼ 8:79  1025 K 2 N4 /J

In Eq. (3), R is the gas constant, and the temperatures used were 298, 304, and 310 K. The thermodynamic parameters for the interaction of maltol with BSA are listed in Table 1. The reaction between maltol and BSA is spontaneous with a negative value of DG. The values of DH and DS were 119.2 ± 0.1 kJ mol1 and 757.0 ± 0.4 J mol1 K1, respectively, which implied the binding process is mainly an endothermic and entropy-driven reaction. What is more, the positive DH and DS values indicated that hydrophobic interactions played a major role in the binding of maltol to BSA (Ross & Subramanian, 1981). 3.4. Energy transfer between BSA and maltol The fluorescence studies suggested the formation of maltol–BSA complex. The intrinsic fluorescence of BSA is mainly due to tryptophan. The non-radiative energy transfer occurs from the donor to acceptor when the emission spectrum of donor overlaps with the absorption spectrum of the acceptor. Therefore, the distance between BSA (donor) and bound maltol (acceptor) could be determined based on the Förster’s non-radiative energy transfer theory (Förster & Sinanoglu, 1996). There was a spectral overlap between the fluorescence emission spectrum of BSA and UV–Vis absorption spectrum of maltol (Fig. 2A). According to Förster’s theory, the energy transfer efficiency E is related to the distance R0 between donor and acceptor by the following equation:

Fig. 2. (A) The spectral overlaps of the fluorescence spectra of BSA (a) with the absorption spectra of maltol (b). c(BSA) = c(maltol) = 1.60 lM. (B) The UV–Vis spectra of the maltol–BSA system at pH 7.4. c(BSA) = 0.80 lM, and c(maltol) = 0, 1.59, 3.17, 4.74, 6.31, 7.87, 9.43  105 M for curves a ? g, respectively; Curve x shows the absorption spectrum of maltol only. c(maltol) = 0.159 lM. (inset) The UV spectra of the maltol–BSA system in the wavelength range of 240–300 nm.

ð6Þ

where K2 is the spatial orientation factor of the dipole for random orientations as in a fluid solution, N is the refractive index of the medium, U is the fluorescence quantum yield of the donor in the absence of the 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

ð7Þ

where F(k) is the fluorescence intensity of the donor at wavelength k, e(k) is the molar absorption coefficient of the acceptor at wavelength k. For ligand–BSA interaction, K2 = 2/3, N = 1.336 and U = 0.15 (Ding et al., 2009). According to Eqs. (5)–(7), the values of the parameters were J = 6.106  1015 cm3 L mol1, R0 = 2.11 nm, E = 0.106 and r = 3.01 nm. Obviously, the distance between maltol and BSA is less than 8 nm, and 0.5R0 < r < 1.5R0, implying that the energy transfer from BSA to maltol occurred with high probability (Hu, Liu, Zhang, Zhao, & Qu, 2005). The bigger r value compared with that of R0 again indicated the presence of static quenching mechanism in the binding of maltol to BSA (He et al., 2005). 3.5. Conformation investigation of BSA induced by maltol 3.5.1. UV–Vis absorption studies UV–Vis absorption spectroscopy is a simple but effective method in detecting the conformational changes of proteins and the complex formation. The difference absorption spectra of BSA with various amounts of maltol obtained by subtracting the corresponding spectrum of free maltol from those of maltol–BSA complex are shown in Fig. 2B. The strong absorption of BSA at 210 nm resulted from the p ? p⁄ transition of BSA’s characteristic polypeptide backbone structure C@O and was related to the changes in the conformation of peptide backbone associated with helix-coil transformation in the difference spectra of proteins (Polet & Steinhardt, 1968). The weak absorption peak at 280 nm was concerned with the polarity of the microenvironment around tyrosine and tryptophan residues of BSA (Li, Zhu, Xu, & Ji, 2011). From Fig. 2B, it can be observed that with increasing amounts of maltol added to the BSA solution, the intensity of the absorption peak of BSA at 210 nm obviously decreased, and the peak shifted slightly toward longer wavelength (from 210 to 213 nm), whereas the intensity of the peak of BSA at 280 nm increased, gradually accompanied by a slight blue shift at about 4 nm. These observations indicated that the interaction of maltol with BSA may cause the conformational changes in BSA and change the polarity of the microenvironment around tyrosine and tryptophan residues of BSA. Moreover, these results also confirmed that maltol interacted with BSA to form a ground-state complex and the fluorescence quenching was mainly a static quenching process (Liu et al., 2010). 3.5.2. Synchronous fluorescence spectroscopic studies Synchronous fluorescence spectroscopy has been widely used for characterising fluorescence properties of samples since the 1970s when it was introduced by Lloyd and Evett (Bani-Yaseen, 2011). It can provide information about the microenvironment of fluorophores, which are related to the conformational changes of

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G. Zhang et al. / Food Chemistry 133 (2012) 264–270

proteins. This technique has been applied to investigate the microenvironment of amino acid residues based on measuring the possible shift in maximum emission wavelength, the shift in position at emission maximum corresponding to the changes of the polarity around the chromophore molecule. Synchronous fluorescence spectra were obtained by simultaneously scanning excitation and emission monochromators. These spectra afford the characters of tyrosine and tryptophan residues in BSA, when the Dk between excitation wavelength and emission wavelength are set at 15 and 60 nm (Ding et al., 2009). The synchronous fluorescence spectra of BSA upon addition of maltol at Dk = 15 and 60 nm are displayed in Fig. 3A and B. It was apparent from Fig. 3A and B that the maximum emission wavelength of tyrosine residue had an obvious red shift (from 295 to 300 nm), and the fluorescence band of tryptophan residues red shifted slightly from 292 to 294 nm, suggesting that the polarity around the tyrosine and tryptophan residue increased and the hydrophobicity decreased (Zhang, Liu, Chi, & Gao, 2011).

ratio of maltol to BSA of 10:1. This result indicated that maltol bound with the amino acid residues of main polypeptide chain of the protein and destroyed their hydrogen bonding networks, resulting in some unfolding of the polypeptides of BSA (Zhang, Dai, Zhang, Yang, & Liu, 2008). Furthermore, the CD spectra of BSA in the absence and presence of maltol were observed to be similar in shape, implying the structure of the protein was also predominantly a-helix even after binding to maltol (Katrahalli et al., 2010).

3.5.3. Circular dichroism studies Further evidence for conformational changes in BSA was obtained by circular dichroism (CD), which is a sensitive technique to monitor the conformational changes in the protein. The CD spectra of BSA in the absence and presence of maltol are shown in Fig. 3C. The spectra of BSA exhibit two negative bands in the UV region at 209 and 221 nm, which are characteristic of a-helix of protein and both contributed to n-k⁄ transfer for the peptide bond of a-helical structure (He et al., 2006). As shown in Fig. 3C, the CD intensity of BSA decreased (shifting to zero levels) without any significant shift of the peaks with increasing molar ratio of maltol to BSA, which suggested that the a-helical content decreased. The contents of different secondary structures of BSA were calculated by the online Dichroweb software and presented in Table 2. Compared with the free BSA, it was obvious that the content of a-helix was reduced from 59.9% to 53.2%, the contents of b-sheet, b-turn and random coil structures were increased from 5.6% to 8.4%, from 12.4% to 13.5%, and from 22.1% to 24.9%, respectively, at a molar

3.5.4. FT-IR studies Infrared spectroscopy is a high-resolution technique in protein studies in a wide variety of environments. The intramolecular forces which are responsible for maintaining the secondary and tertiary structures of BSA will be altered after maltol binding with BSA, resulting in conformational changes of the protein (Neault, Benkirane, Malonga, & Tajmir-Riahi, 2001). In order to obtain detailed information on the binding of maltol to BSA, the FT-IR experiments were carried out in Tris–HCl buffer solution at pH 7.4. Infrared spectra of proteins exhibit a number of amide bands, which represent different vibrations of the peptide moiety. Among these amide bands of the protein, the amide I band (1700– 1600 cm1, mainly C@O stretch) and amide II band (1600– 1500 cm1, C–N stretch coupled with N–H bending mode) both have a relationship with the secondary structure of protein, and amide I band is more sensitive to the change of protein secondary than amide II band (He et al., 2006). Fig. 4A shows the FT-IR spectra of free BSA and the difference spectra after binding with maltol. As

Table 2 Secondary structure of free BSA and maltol–BSA systems (CD spectra) at pH 7.4. Molar ratio [maltol]:[BSA]

a-Helix (%)

b-Sheet (%)

b-Turn (%)

Random coil (%)

0:1 5:1 10:1

59.9 54.3 53.2

5.6 8.1 8.4

12.4 13.3 13.5

22.1 24.3 24.9

Fig. 3. The synchronous fluorescence spectra of BSA in the presence of maltol. (A) Dk = 15 nm, (B) Dk = 60 nm. c(BSA) = 8.00  107 mol L1, and c(maltol) = 0, 1.59, 3.17, 4.74, 6.31, 7.87, 9.43, 10.98, 12.52, 14.06, and 15.59  105 M for curves a ? k, respectively. (C) The CD spectra of BSA in the presence of increasing amounts of maltol. c(BSA) = 0.80 lM, the molar ratios of maltol to BSA were 0:1 (a), 5:1 (b) and 10:1 (c), respectively.

G. Zhang et al. / Food Chemistry 133 (2012) 264–270

seen in Fig. 4A, the peak position of amide I band shifted from 1653 to 1648 cm1 and the amide II band moved from 1547 to 1541 cm1. The changes of these peak positions indicated that maltol interacted with the C@O and C–N groups in the protein structural subunits, which resulted in the rearrangement of the

0.0025

(A)

1547

a

1653

0.0020

Absorbance

1541

b

0.0015

1648

0.0010

0.0005

269

polypeptide carbonyl hydrogen bonding pattern and ultimately changed the secondary structure of BSA. A quantitative analysis of the secondary structure of BSA before and after the interaction with maltol are given in Fig. 4B and C. According to the literature (Darwish et al., 2010), the band range 1640–1610 cm1 was generally assigned to b-sheet, 1650– 1640 cm1 to random coil, 1660–1650 cm1 to a-helix, 1680– 1660 cm1 to b-turn and 1700–1680 cm1 to b-antiparallel. The content of each secondary structure of BSA can be calculated based on the integrated areas of the component bands in amide I band. According to Fig. 4B, the free BSA contained major amounts of ahelix 58.8% (1653 cm1), random coil 22.2% (1643 cm1), b-turn 13.1% (1671 cm1), b-sheet 3.4% (1621 cm1) and b-antiparallel 2.5% (1689 cm1). Upon maltol–BSA complexation, a major decrease of a-helix occurred from 58.8% to 55.2% with an increase in random coil and b-sheet from 22.2% to 23.5% and from 3.4% to 4.3%, respectively, while b-turn and b-antiparallel increased slightly from 13.1% to 13.9% and from 2.5% to 3.1%. The decrease in a-helix and increase in random coil and b-sheet suggested a partial protein unfolding in the presence of maltol (Zhang, Ni, & Kokot, 2010). 4. Conclusions

0.0000

1800

1700

1600

1500

1400

-1

Wavenumbers(cm )

(B) 0.0020

1653

Absorbance

0.0015

0.0010

1643

0.0005

1671

1621

1689 0.0000

1700

1680

1660

1640

1620

1600

-1

Wavenumbers(cm )

(C) 0.0020 1651

Absorbance

0.0015

0.0010

Acknowledgements We gratefully acknowledge the financial support of the National Natural Science Foundation of China (Nos. 31060210, 21167013), the Natural Science Foundation of Jiangxi Province (20114BAB204019), and the Research Program of State Key Laboratory of Food Science and Technology of Nanchang University (SKLF–TS–200917, SKLF–MB–201002).

1640 1677

0.0005

1616

1693

References

0.0000

1700

1680

1660

Fluorescence, UV–Vis absorption, CD and FT–IR spectroscopic methods were applied to investigate the interaction between BSA and maltol under simulated physiological conditions. The results indicated that the fluorescence of BSA was quenched by maltol through a static quenching mechanism. The values of DH and DS were calculated to be 119.2 ± 0.1 kJ mol1 and 757.0 ± 0.4 J mol1 K1, respectively, suggesting that maltol could bind to BSA, mainly through hydrophobic interactions. There was a single class of binding site for maltol in BSA and maltol was located in subdomain IIA (site I) of BSA. The binding distance r was 3.01 nm based on Förster theory, which indicated that the energy transfer from BSA to maltol occurred with high possibility. Analysis of UV–Vis absorption, synchronous fluorescence, CD and FT-IR spectra showed that the binding of maltol to BSA induced changes in the secondary structure of the protein. Shahabadi et al. (2011) reported the interaction of 2-tertbutylhydroquinone (TBHQ), a food antioxidant, with BSA by multispectroscopic methods. Their results indicated that TBHQ bound to BSA with a high affinity and the quenching mechanism of BSA by TBHQ was a static procedure. The formation of TBHQ–BSA complex induced conformational changes of BSA. Our results obtained from this study showed that the binding of maltol to BSA is similar to that of TBHQ.

1640

1620

1600

-1

Wavenumbers(cm ) Fig. 4. (A) The FT-IR spectra of free BSA (a) and difference spectra [(maltol– BSA)maltol solution] (b) in a pH 7.4 buffer solution in the region of 1800– 1400 cm1. c(BSA) = c(maltol) = 80 lM. The curve-fitted amide I region (1700– 1600 cm1) of free BSA (B) and its maltol complex (C).

Bani-Yaseen, A. D. (2011). Spectrofluorimetric study on the interaction between antimicrobial drug sulfamethazine and bovine serum albumin. Journal of Luminescence, 131, 1042–1047. Bjeldanes, L. F., & Chew, H. (1979). Mutagenicity of 1,2-dicarbonyl compounds: Maltol, kojie acid, diacetyl and related substances. Mutation Research, 67, 367–371. Darwish, S. M., Abu sharkh, S. E., Abu Teir, M. M., Makharza, S. A., & Abu-hadid, M. M. (2010). Spectroscopic investigations of pentobarbital interaction with human serum albumin. Journal of Molecular Structure, 963, 122–129.

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G. Zhang et al. / Food Chemistry 133 (2012) 264–270

Förster, T., & Sinanoglu, O. (Eds.). (1996). Modern quantum chemistry (vol. 3). New York: Academic Press, p. 93. Ding, F., Huang, J. L., Lin, J., Li, Z. Y., Liu, F., Jiang, Z. Q., et al. (2009). A study of binding of C.I. Mordant Red 3 with bovine serum albumin using fluorescence spectroscopy. Dyes Pigments, 82, 65–70. Gralla, E. J., Stebbins, R. B., Coleman, G. L., & Delahunt, C. S. (1969). Toxicity studies with ethyl maltol. Toxicology Applied Pharmacology, 15, 604–613. Hayashi, M., Kishi, M., Sofuni, T., & Ishidate, M. J. (1988). Micronucleus tests in mice on 39 food additives and eight miscellaneous chemicals. Food Chemical Toxicology, 26, 487–500. He, W. Y., Li, Y., Si, H. Z., Dong, Y. M., Sheng, F. L., Yao, X. J., et al. (2006a). Molecular modeling and spectroscopic studies on the biding of guaiacol to human serum albumin. Journal of Photochemistry Photobiology A, 182, 158–167. He, W. Y., Li, Y., Tang, J. H., Luan, F., Jin, J., & Hu, Z. D. (2006b). Comparison of the characterization on binding of alpinetin and cardamonin to lysozyme by spectroscopic methods. International Journal of Biological Macromolecules, 39, 165–173. He, W. Y., Li, Y., Xue, C. X., Hu, Z. D., Chen, X. G., & Sheng, F. L. (2005). Effect of Chinese medicine alpinetin on the structure of human serum albumin. Bioorganic and Medicinal Chemistry, 13, 1837–1845. Hu, Y. J., Liu, Y., Zhang, L. X., Zhao, R. M., & Qu, S. S. (2005). Studies of interaction between colchicine and bovine serum albumin by fluorescence quenching method. Journal of Molecular Structure, 750, 174–178. Jin, J., Zhu, J. F., Yao, X. J., & Wu, L. M. (2007). Study on the binding of farrerol to human serum albumin. Journal of Photochemistry Photobiology A, 191, 59–65. Katrahalli, U., Jaldappagari, S., & Kalanur, S. S. (2010). Probing the binding of fluoxetine hydrochloride to human serum albumin by multispectroscopic techniques. Spectrochimica Acta A, 75, 314–319. Lakowicz, J. R. (2006). Principles of Fluorescence Spectroscopy (3rd ed.). New York: Springer Publications. Leblanc, D. T., & Akers, H. A. (1989). Maltol and ethyl maltol: From the larch, tree to successful food additive. Food Technology, 43, 18–84. Li, D. J., Zhu, M., Xu, C., & Ji, B. M. (2011). Characterization of the baicalein-bovine serum albumin complex without or with Cu2+ or Fe3+ by spectroscopic approaches. European Journal of Medicinal Chemistry, 46, 588–599. Lin, S. Y., Li, M. J., & Wei, Y. S. (2004). Ethanol or/and captopril-induced precipitation and secondary conformational changes of human serum albumin. Spectrochimica Acta A, 60, 3107–3111.

Liu, B. S., Xue, C. L., Wang, J., Yang, C., Zhao, F. L., & Lv, Y. K. (2010). Study on the competitive reaction between bovine serum albumin and neomycin with ponceau S as fluorescence probe. Journal of Luminescence, 130, 1999–2003. Neault, J. F., Benkirane, A., Malonga, H., & Tajmir-Riahi, H. A. (2001). Interaction of cisplatin drug with Na, K-ATPase: Drug binding mode and protein secondary structure. Journal of Inorganic Biochemistry, 86, 603–609. Polet, H., & Steinhardt, J. (1968). Binding-induced alterations in ultraviolet absorption of native serum albumin. Biochemistry, 7, 1348–1356. Ross, P. D., & Subramanian, S. (1981). Thermodynamics of protein association reactions: Forces contributing to stability. Biochemistry, 20, 3096–3102. Savory, J., Huang, Y., Herman, M. M., Reyes, M. R., & Wills, M. R. (1995). Tau immunoreactivity associated with aluminum maltolate-induced neurofibrillary degeneration in rabbits. Brain Research, 669, 325–329. Shahabadi, N., Maghsudi, M., Kiani, Z., & Pourfoulad, M. (2011). Multispectroscopic studies on the interaction of 2-tert-butylhydroquinone (TBHQ), a food additive, with bovine serum albumin. Food Chemistry, 124, 1063–1068. Sjoholm, I., Ekman, B., Kober, A., Pahlman, I. L., Seiving, B., & Sjodin, T. (1979). Binding of Drugs to Human Serum Albumin:XI The specificity of three binding sites as studied with albumin immobilized in microparticles. Molecular Pharmacology, 16, 767–777. Sudlow, G., Birkett, D. J., & Wade, D. N. (1976). Further characterization of specific drug binding sites on human serum albumin. Molecular Pharmacology, 12, 1052–1061. Sulkowska, A., Maciazek, M., Rownicka, J., Bojko, B., Pentak, D., & Sulkowski, W. W. (2007). Effect of temperature on the methotrexate-BSA interaction spectroscopic study. Journal of Molecular Structure, 834–836, 162–169. Zhang, G. W., Chen, X. X., Guo, J. B., & Wang, J. J. (2009). Spectroscopic investigation of the interaction between chrysin and bovine serum albumin. Journal of Molecular Structure, 921, 346–351. Zhang, Y. Z., Dai, J., Zhang, X. P., Yang, X., & Liu, Y. (2008). Studies of the interaction between Sudan I, and bovine serum albumin by spectroscopic methods. Journal of Molecular Structure, 888, 152–159. Zhang, H., Liu, R. T., Chi, Z. X., & Gao, C. Z. (2011). Toxic effects of different charged metal ions on the target-Bovine serum albumin. Spectrochimica Acta A, 78, 523–527. Zhang, Q. L., Ni, Y. N., & Kokot, S. (2010). Molecular spectroscopic studies on the interaction between Ractopamine and bovine serum albumin. Journal of Pharmaceutical Biomedical Analysis, 52, 280–288.