Food and Chemical Toxicology 67 (2014) 123–130

Contents lists available at ScienceDirect

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

The interaction of plant-growth regulators with serum albumin: Molecular modeling and spectroscopic methods Sheying Dong a,b,⇑, Zhiqin Li a, Ling Shi a, Guiqi Huang a, Shuangli Chen a, Tinglin Huang b a b

College of Sciences, Xi’an University of Architecture and Technology, Xi’an 710055, China School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China

a r t i c l e

i n f o

Article history: Received 28 September 2013 Accepted 18 February 2014 Available online 22 February 2014 Keywords: Plant-growth regulators (PGRs) Human serum albumin (HSA) Interaction molecular modeling Spectroscopic methods

a b s t r a c t The affinity between two plant-growth regulators (PGRs) and human serum albumin (HSA) was investigated by molecular modeling techniques and spectroscopic methods. The results of molecular modeling simulations revealed that paclobutrazol (PAC) could bind on both site I and site II in HSA where the interaction was easier, while uniconazole (UNI) could not bind with HSA. Furthermore, the results of fluorescence spectroscopy, three-dimensional (3D) fluorescence spectroscopy and circular dichroism (CD) spectroscopy suggested that PAC had a strong ability to quench the intrinsic fluorescence of HSA. The binding affinity (Kb) and the amounts of binding sites (n) between PAC and HSA at 291 K were estimated as 2.37  105 mol L1 and 1, respectively, which confirm that PAC mainly binds on site II of HSA. An apparent distance between the Trp214 and PAC was 4.41 nm. Additionally, the binding of PAC induced the conformational changes of disulfide bridges of HSA with the decrease of a-helix content. These studies provide more information on the potential toxicological effects and environmental risk assessment of PGRs. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Due to their low price and high effective ability to regulate plant growth and development, plant-growth regulators (PGRs) including synthetic compounds and natural plant hormones extracted from the organisms are generally applied for agricultural activities in China. They can inhibit gibberellin biosynthesis, slow down the cells extending, shorten internode, and maintain the cell number and section number (Davis and Dernoeden, 1991). However, such materials also promote toxic effects even at low concentrations. What is more, long-term heavy use of these substances results in their residues in crop, soil and water bodies, and endangers the safety of the entire ecosystem through the food chain (Valerón et al., 2009). Interaction between PGRs and proteins may interfere with the normal binding between proteins and other endogenous hormones in the body. Therefore, the affinity between PGRs and serum albumin was investigated for the potential toxicological effects of PGRs. At present, paclobutrazol (PAC) and uniconazole

Abbreviations: HSA, human serum albumin; PAC, paclobutrazol; PGRs, plantgrowth regulators; UNI, uniconazole. ⇑ Corresponding author at: College of Sciences, Xi’an University of Architecture and Technology, Xi’an 710055, China. Tel.: +86 29 82201203; fax: +86 29 82205332. E-mail address: [email protected] (S. Dong). http://dx.doi.org/10.1016/j.fct.2014.02.020 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.

(UNI) are two kinds of important PGRs whose structures are shown in Fig. 1. Human serum albumin (HSA), the most abundant carrier protein in plasma, provides about 80% of the blood osmotic pressure (He and Carter, 1992). HSA molecule is a 585 amino acid residue monomer of 66.500 g mol1, which has sole tryptophan residue (Trp214). Heart-shaped three-dimensional structure of HSA molecule comprises of three structurally homologous domains that are domain I (residues 1–195), II (196–383), III (384–585). And each domain is subdivided into two subdomains possessing common structural motifs (A and B), which are six-helix (A) and four-helix (B) subdomains, respectively (Sudlow et al., 1975). Protein–ligand interactions play an important role in a variety of biological processes. It has been proved that the principle regions of HSA to bind most of molecules are located within hydrophobic cavities in subdomains IIA and IIIA, which are corresponded to the Sudlow’s site I and site II (Roche et al., 2009; Sengupta and Hage, 1999). To characterize these interactions at the molecular level, optical techniques have become valuable tools because of their high sensitivity, rapidity and easy implementation. Particularly, three-dimensional fluorescence, synchronous fluorescence and circular dichroism spectroscopy have been commonly used to study the conformational changes of proteins (Hebert and MacManus-Spencer, 2010; Ren and Guo, 2012; Zhang and Ma, 2013). In addition, molecular docking comprehensively considers

124

S. Dong et al. / Food and Chemical Toxicology 67 (2014) 123–130

the combined effect of ligands and receptors, which can avoid circumstances for better local interaction and poor overall combination as well as provide important information for molecular binding mode and structural transformation of active small molecule. This aspect has attracted great interest among researchers in recent years (Ding et al., 2011a; Wang et al., 2011a). Ding et al. (2011b) reported the binding of chlorantraniliprole with HSA by molecular spectroscopy and circular dichroism spectroscopy, and combined with molecular docking techniques to determine the bonding location. Han et al. (2012) investigated chlorpyrifos binding with HSA and bovine serum albumin (BSA) employing molecular spectroscopy, electrochemistry and molecular docking methods. Saquib et al. (2011) studied in detail the interaction between phorate and HSA. However, to the best of our knowledge, none of the published studies were reported on the interaction of PGRs with HSA. On the other hand, there still exist some problems such as less type of pesticides, less technologies integration and systems analysis. In this work, the binding of HSA with two kinds of PGRs (PAC and UNI) was predicted by molecular modeling techniques. Subsequently, the binding interactions under simulative physiological conditions were studied by UV–Vis absorption spectrometry, fluorescence spectroscopy, three-dimensional (3D) fluorescence spectroscopy and circular dichroism spectroscopy (CD spectroscopy). The quenching mechanism was discussed on the binding constants, number of the binding sites and basic thermodynamic parameters. Furthermore, the features of PAC leading to conformational changes of HAS have been explored by conformational analysis. When HAS binds with PAC (HSA–PAC), the normal binding between HSA and other endogenous hormones in human body may be interfere. Therefore, we hope that this study will be helpful for understanding the impact of PGRs on HSA structure and function.

Fig. 1. Chemical structures of two PGRs.

Experiment 1: The concentration of PAC (5.0  105 mol L1) or UNI (3.0  105 mol L1) was kept constant, and different amounts of HSA were added to the solution. The concentration range of HSA was 0–1.3  106 mol L1, and 15 different solutions were prepared. Experiment 2: The concentration of HSA (5.0  107 mol L1) was kept constant, and different amounts of PAC were added to the solution. The concentration range of PAC was 0–3.5  105 mol L1. The resulted solutions were allowed to stand for 15 min before analyzed on the UV–Visible spectrophotometer in the range of wavelength from 200 to 350 nm. 2.4. Steady state fluorescence measurements In the present investigation, the concentration of HSA was kept constant at 5.0  107 mol L1 while the concentration of the PAC was varied from 0 to 3.2  105 mol L1. After kept still for 15 min, the fluorescence spectra were recorded in the range of 290–500 nm upon excitation wavelength at 280 nm, which both excitation and emission bandwidths were set at 5 nm. 2.5. Three-dimensional fluorescence spectra The three-dimensional fluorescence spectra were recorded under the following conditions: the emission spectra were recorded between 240 and 500 nm, the initial excitation wavelength was set to 220 nm with increment of 5 nm, the number of scanning curves was 37, and other scanning parameters were the same as those of fluorescence quenching studies.

2. Materials and methods

2.6. CD spectra

2.1. Instruments and chemicals

The CD spectra were measured from 190 to 260 nm with 1.0 mm path length optical circular quartz cells. Each result was the average of the three scans. The scan slit width of 1 nm, response time of 1 s and scan rate of 100 nm min1 were constantly maintained throughout all experiments. The concentration of HSA was 1.00  106 mol L1 at pH 7.4 under constant nitrogen flush. The data were expressed in terms of mean residue ellipticity (abbreviated as MRE), using the mean residue weights of 114.3 (66500/582) for the intact molecular weight of the HSA and the cell path length. Blank phosphate buffer solution was used as a spectral reference and subtracted from the sample spectra.

UV–Vis absorption spectra were recorded on a Nicolet Evolution 300 UV–Visible spectrophotometer with 1.0 cm quartz cells. All fluorescence spectra were performed on a FP-6500 fluorophotometer (Hitachi, Japan) equipped with a xenon lamp source and a 1.0 cm quartz cell. The CD spectra were carried out in a Chirascan CD spectrometer (Applied Photophyscics Limited, United Kingdom). All pH measurements were measured with IX-501A digital pH-meter (Department of Chemistry, Peking University, China) combined with a glass-calomel electrode. HSA was purchased from Sigma Chemical Co. Ltd and without further purification. All calculations reported for HSA were in terms of HSA with the molecular weight of 66,500. A 1.00  104 mol L1 stock solution of HSA was prepared by directly dissolving the proteins in 0.0667 mol L1 phosphate buffer and stored at 277 K. PAC and UNI were of analytical grade, and were purchased from Jinghong of Jiangsu Chemical Reagent Co. Ltd., China. The stock solutions of PAC and UNI with the concentration of 1.00  102 mol L1 were obtained by dissolving PAC and UNI in methanol, respectively. Other chemicals were all of analytical grade and doubly distilled water was used throughout the experiment.

2.2. Molecular modeling The complex crystal structures based on the two major binding sites for site I and site II of HSA were selected for molecular docking, respectively. The crystal structures of HSA in complex with thyroxine and ibuprofen, 1HK1_THYROXINE (3,30 ,5,50 -tetraiodo-L-thyroxine)_site1.pdb and 2BXG_ IBUPROFEN_site2.pdb, were taken from the Brookhaven Protein Data Bank. The initial structures of PAC and UNI were generated by Chemdraw of molecular modeling software. The eHiTS program combined with visual chevi was used to discuss the interaction modes between PAC/UNI and HSA, respectively. Because eHiTS program used a systematic search algorithm and scoring function based on the protein family, the molecular docking considered 32 conformations of the compound and eventually got 32 kinds of docking models, which most negative scoring was best.

2.3. UV–Vis absorption spectrometry Two separate experiments were carried out in the phosphate buffer.

3. Results and discussion 3.1. Molecular modeling To obtain insight into interactions of UNI/PAC with HSA, molecular modeling simulations were applied to examine the binding of UNI/PAC at the active site of HSA. Ehits molecular docking program considered 32 conformations of compound by using a systematic search algorithm and scoring function based on protein family. The results indicated that for the binding of UNI at the active site of HAS, any conformation and scoring did not provided in visualization software CheVi. Although the structure of UNI was similar to that of PAC, carbon–carbon double bond was connected with the benzene ring and is near to the benzene ring for UNI. Thus, the non-normal O–H  Ph hydrogen bond was formed, which restricted the formation of hydrogen bond between OH group in UNI and HAS because of its saturation. In order to confirm the viewpoint, the optimized configurations of PAC and UNI are carried out at B3LYP/6-31G level. It can be found that the hydroxyl was nearer to the benzene ring in UNI. An apparent distance between the hydroxyl and the benzene ring is 1.91583 Å. Meantime, the rigidity in UNI leads to the increase of the steric resistance.

S. Dong et al. / Food and Chemical Toxicology 67 (2014) 123–130

Consequently, a conclusion may be safely drawn that UNI would be difficult to interact with HSA. The docking results showed PAC could bind to the two sites of HSA. The docking score was 5.56 for site I, and 7.26 for site II where the interaction between PAC and HAS was easier owe to more negative scoring. The optimum binding mode between PAC and HSA at the site I was shown in Fig. 2A and B. From Fig. 2A, we observed that PAC might entirely bind to a hydrophobic pocket of HSA. Furthermore, as shown in Fig. 2B, there were hydrophobic interactions of the hydrophobic group of PAC with the residues Ala291, Leu219, Leu264, Leu260, Leu238, Ile290, Trp214, Trp150, Lys199, and Arg218 of HSA. Therefore, the hydrophobic interaction was extremely significant for the stability of the PAC and HSA complexes. Fig. 2C and D showed the optimum binding mode between PAC and HSA at site II. It was found in Fig. 2C that PAC was located within a hydrophobic pocket of HSA. There were hydrophobic interactions of the hydrophobic group of PAC with the residues Val433, Leu43, Leu453, Leu487, Leu407, Phe403, Asn391, Gly431, Ile388, Cys492, and Cys438 of HSA (Fig. 2D). It was important to note that hydrogen bonding of hydroxyl with amino hydrogen of Arg410 in HSA was generated. The formation of hydrogen bond decreased the hydrophilicity, which resulted in the increasing of hydrophobicity to stabilize the PAC–HSA system (Wang et al., 2011b). Therefore, the results obtained from molecular modeling suggest that PAC bind both on both site I and site II of HSA where the interaction between PAC and HAS was more easier, and the interaction between PAC and HSA is dominated by hydrophobic force and hydrogen bonds.

125

3.2. UV–Vis absorption spectra The interaction between PAC or UNI and HSA was confirmed by UV–Vis absorption spectra. Fig. 3A and B displayed the UV–Vis absorption spectra of PAC and UNI with different concentrations of HSA, respectively. From Fig. 3A, it can be seen that with the addition of the concentration of HSA, the absorption peak at about 206 nm appeared, which was related to the a-helix structure of HSA (Chang et al., 2005). Although the absorption intensity gradually increased with the bathochromic shift of its absorption band, the linear relationship between DA of the PAC–HSA system and concentration of HSA was poor. Meanwhile, with the increase of HSA, the absorption intensity of the peaks at 221 nm increased gradually, but changed slightly at 270 nm. These results implied that PAC interacted with HSA (Liu et al., 2007). In Fig. 3B, however, with the addition of the concentration of HSA, the absorption intensity near 206 nm gradually increased. Further, a linear plot (Fig. 3B inset) for DA of the UNI-HSA system versus the concentration of HSA was obtained. The changes in absorption intensity are only caused by the concentration of HSA according to the Lambert– Beer’s law, suggesting UNI does not interact with HSA. The consequence of UV–Vis spectra is in agreement well with that obtained from molecular modeling. Many researches have confirmed that HSA showed two featured protein absorption bands in the UV region around 206 nm and 278 nm, respectively, while UV–Vis absorption spectrum of PAC had several peaks at 221, 266 and 274 nm. Clearly, the spectrum of mixture of the two substances would have a composite profile consisting of strong overlapping spectra of the two separate analytes. When the concentration of HSA was constant, with the

Fig. 2. Docking of PAC into site I (A and B) and site II (C and D) of HSA. The surface of the HSA is displayed by QUICK method in A and C, and the residues of the HSA are presented using line in B and D. The ligand structure is represented using ball and stick model. The interaction between the ligand and the protein is represented using dashed line.

126

S. Dong et al. / Food and Chemical Toxicology 67 (2014) 123–130

spectrum between PAC–HSA system and PAC is significantly different from the UV absorption spectrum of HSA, indicating that ground state molecules of PAC and HSA form complex. The absorption relationship between PAC and HSA was expressed by the Lineweaver–Burk equation (Xu et al., 2011): 1 1 1 ðA  A0 Þ1 ¼ A1 0 þ K A A0 ½Q 

ð1Þ

where KA is the binding constant, which can be calculated from the ratio of the intercept on the slope, A0 and A are the absorbance of PAC in the absence and presence of HSA respectively. From the absorbance of PAC at 221 nm which changed regularly relatively, binding constants at different temperatures were calculated. Essentially, the interaction forces between ligands and biological macromolecules may include hydrophobic force, multiple hydrogen bond, van der Waals force and electrostatic interactions (Liu et al., 2006). If the enthalpy change (DH) does not vary significantly in the range of temperature studied, then the values of DH as well as entropy change (DS) can be evaluated from the van’t Hoff equation (Xu et al., 2012):

lnK ¼ 

DH DS þ RT R

DG ¼ DH  T DS

Fig. 3. The absorption spectra of PAC (A) and UNI (B) in the presence of various concentrations of HSA. (A) cPAC = 5.0  105 mol L1, cHSA from b to i: 0, 1.0, 3.0, 5.0, 7.0, 9.0, 11.0 and 13.0 ( 107 mol L1); a: 13.0  107 mol L1 HSA. (B) cUNI = 3.0  105 mol L1, cHSA from a to g: 0, 1.0, 3.0, 5.0, 7.0, 9.0 and 11.0 ( 107 mol L1); Inset: plot of DA vs. HSA concentration in 206 nm.

addition of PAC, the absorption peak intensity of HSA at 206 nm increased without shift of its absorption band, and the absorbance intensity at 278 nm changed slightly (Fig. 4). These phenomena proved that the extent of peptide chain in HSA had changed after the addition of PAC, which induced bareness extent changes of both tryptophan residue and tyrosine residue (Li et al., 1998). As shown in Fig. 4 inset, it is observed obviously that the difference

ð2Þ ð3Þ

where K is binding constant at the corresponding temperature and R is the gas constant. The values of DH and DS were obtained from the slope and intercept of the linear plot (Eq. (2)) based on lnK versus 1/T. The free energy change (DG) was estimated from Eq. (3). The results in Table 1 declared that the reaction process was spontaneous due to the negative value of DG. Ross and Subramanian (Ross and Subramanian, 1981) have characterized the sign and magnitude of thermodynamic parameters associated with various kinds of interaction that may take place in protein association process, as described below. For typical hydrophobic effect, both DH and DS are positive, while there are negative DH and DS for van der Waals force and hydrogen bond formation in a low dielectric medium. Further, specific electrostatic interaction between ionic species in aqueous is expressed by a positive value of DS and a negative DH (almost zero). In the present case of PAC–HSA complex, the positive DH and DS attested that hydrophobic effect played a dominant role in the binding of PCA to HSA. According to the thermodynamic data, the formation of the PAC–HSA complex is favored entropy while it is disfavored enthalpy. The complex formation results in a less ordered state, possibly due to activation of the motional freedom of both the PAC and HSA molecules. 3.3. Analysis of fluorescence quenching of HSA by PAC

Fig. 4. The absorption spectra of HSA in the presence of various concentrations of PAC. cHSA = 5.0  107 mol L1, cPAC from a to g: 0, 0.5, 1.0, 1.5, 2.0, 3.0 and 3.5 ( 105 molL1); Inset: the absorption difference spectra of HSA, cPAC = 3.5  105 molL1.

In general, fluorescence of HSA originates from tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) residues. Because the Phe residue has a very low quantum yield, and the fluorescence of Tyr residue is almost totally quenched when it is ionized or close to an amino group, a carboxyl group or a Trp. The intrinsic fluorescence of HSA is mainly attributed to the Trp residue alone (Zhou et al., 2011), which is extremely sensitive to its environment. If emission peak exhibits blue shift, hydrophobicity around tryptophan residues would increase and polarity would become smaller. In contrast, red shift indicated that the hydrophobic property decreased and polarity increased (Hu et al., 2009). The fluorescence emission spectrum of HSA quenched by PAC was shown in Fig. 5. It is obvious that HSA appears a strong fluorescence emission with a peak at 336 nm (kex = 280 nm), while the PAC possesses a very weak emission under the present experimental conditions. With the increasing concentration of PAC, the fluorescence intensities of HSA decreased remarkably, and the emission peak shifted from 336 nm to 331 nm. These phenomena implied that the interaction

127

S. Dong et al. / Food and Chemical Toxicology 67 (2014) 123–130 Table 1 Association constants Ka and relative thermodynamic parameters of PAC–HSA system at different temperature. T (K)

KA (104 L mol1)

R1

288 293 298 303

5.541 8.925 15.487 18.842

0.9984 0.9990 0.9964 0.9966

DHh (KJ mol1)

DGh (KJ mol1) 26.24 27.76 29.28 30.80

61.40

DSh (J mol1 K1)

R2

304.3

0.9862

R1 is the correlation coefficient for Lineweaver–Burk plots. R2 is the correlation coefficient for van’t Hoff plots.

Fig. 6. Overlap plots of the fluorescence emission spectrum of HSA (a) with the absorption spectrum of PAC (b). [HSA] = [PAC] = 5.0  107 mol L1.

Fig. 5. Emission spectra of HSA in the presence of various concentrations of PAC (T = 298 K, pH = 7.4, kex = 280 nm), the Sterne–Volmer (A) and modified Sterne– Volmer (B) plots of PAC–HSA system at different temperatures. cHSA = 5.00  107 mol L1, cPAC from a to h: 0, 8.0, 12.0, 16.0, 20.0, 28.0 and 32.0 ( 106 molL1), (h) [PAC] = 8.0  106 mol L1.

between PAC and HSA occurred and altered the microenvironment of tryptophan. In protein–ligand binding studies, quenching can be induced by different mechanisms. It is usually classified into dynamic quenching and static quenching, which are caused by collisional encounters and ground-state complex formation between fluorophore and quenchers, respectively (Skrt et al., 2012). Dynamic quenching and static quenching can be described by Sterne–Volmer (Chen et al., 2012) (Eq. (4)) and modified Sterne– Volmer equation (Peng et al., 2012) (Eq. (5)), respectively:

F0 ¼ 1 þ K SV ½Q  ¼ 1 þ K q s0 ½Q F

ð4Þ

F0 1 1 ¼ þ F 0  F fa K a ½Q fa

ð5Þ

Here F0 and F are the relative fluorescence intensities in the absence and presence of the quencher, respectively. Kq is the quenching rate constant of the biomolecule. KSV is the Sterne–Volmer dynamic quenching constant. s0 is the average lifetime the biomolecule without quencher. The value of s0 of the biopolymer is 108 s1 (Lakowicz and Weber, 1973), and [Q] is the concentration of quencher (PAC). Ka is the effective quenching constant for the accessible fluorophores and fa is the fraction of accessible fluorescence. The possible quenching mechanism can be interpreted by fluorescence quenching spectra of HSA, and quenching data were also analyzed using the Stern–Volmer (A) and modified Stern–Volmer (B) plots of PAC–HSA system in Fig. 5.

It can be seen from Fig. 5 that the dependences of F0/F on quencher concentration [Q] and F0/(F0  F) on the reciprocal value of the quencher concentration [Q]1 were linear with the slope. The change of Ka with temperature was consistent with KSV and KA obtained from absorption spectral data. Obviously, values of Kq were greater than 2  1010 L mol1 s1, which proved that the binding of PAC to HSA was a static quenching process. In addition, the difference absorption spectrum between PAC–HSA and PAC at the same concentration was clearly distinct from that of lonely HSA in Fig. 4 inset. In fact, the ground-state complex formation frequently results in the perturbation of the absorption spectrum of the fluorophore, while the collisional quenching only affects the excited state of fluorophores (Zhang et al., 2009). Consequently, the result again confirmed that the quenching mechanism was a static quenching initiated by the formation of the ground state PAC–HSA complex. For static quenching, the fluorescence intensity can also be used to analyze the apparent binding constant (Kb) and the number of binding sites (n) using Eq. (6) when small molecules independently bind to a set of equivalent sites on a macromolecule (Liu et al., 2012):

log½ðF 0  FÞ=F ¼ log K b þ n log½Q 

ð6Þ

From Eq. (6), the values of Kb and n of PAC–HSA system at 291 K were obtained to be 2.37  105 mol L1 and 1.357 which can be rounded off to 1, respectively. These results indicate that there is a strong interaction between PAC and HSA, and there is one type of binding site for PAC in HSA. A conclusion might be safely drawn that PAC mainly bind on site II of HSA. 3.4. Energy transfer between PAC and human serum albumin Generally, fluorescence resonance energy transfer (FRET) occurs whenever the emission spectrum of a fluorophore (donor) overlaps with the absorption spectrum of another molecule (acceptor).

128

S. Dong et al. / Food and Chemical Toxicology 67 (2014) 123–130

Fig. 7. The three-dimensional fluorescence spectra and corresponding contour diagrams of HSA (A and C) and PAC–HSA (B and D). c(HSA)/(107 mol L1) = 4.0, c(PAC)/ (105 mol L1) = 3.0.

E¼1

F R6 ¼ 6 0 F 0 R0 þ r 6

ð7Þ

Here F and F0 are the fluorescence intensities of HSA in the presence and absence of PAC respectively, r is the distance between the acceptor and the donor, and R0 is the critical distance when the transfer efficiency is 50%.

R60 ¼ 8:79  1025 K 2 N4 /J

ð8Þ

The term K2 is the relative 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 (Fig. 6). Fig. 8. The CD spectra of the PAC–HSA system at pH = 7.4. cHSA = 1.0  106 mol L1, c(PAC)/(1  106 mol L1) = 0 (a), 1.0 (b), and 5.0 (c).

According to Förster’s non-radiative energy transfer theory (Förster, 1948), the efficiency of energy transfer depends mainly on (i) the extent of overlap of emission spectrum of the donor (HSA) with absorption spectrum of the acceptor (PAC), (ii) the relative orientation of the donor and acceptor dipoles and (iii) the distance between the donor and the acceptor. In this work, Fig. 6 showed the overlap between the fluorescence emission spectrum of HSA and the UV absorption spectrum of PAC. The efficiency of energy transfer, E, can be calculated using the equation:

R1 J¼

0

FðkÞeðkÞk4 dk R1 FðkÞdk 0

ð9Þ

where F(k) is the fluorescence intensity of the donor at wavelength k, and e(k) is the molar absorption coefficient of the acceptor at wavelength k. In the present case, K2 = 2/3, N = 1.336 and U = 0.118 (Stryer, 1978). According to Eqs. (7)–(9), the values of the parameters were obtained to be J = 1.352  1014 cm3 L mol1, R0 = 2.58 nm and r = 4.41 nm. The donor-to-acceptor distance, r < 7 nm, indicated that the energy transfer from HSA to PAC occurs with high possibility. Furthermore, the value of r was greater than R0 in this study, suggesting that PAC could strongly quench the intrinsic fluorescence of HSA by a static quenching mechanism.

129

S. Dong et al. / Food and Chemical Toxicology 67 (2014) 123–130 Table 2 CD spectra results for PAC–HSA complex. Protein HSA

R1 0 1.0 5.0

k1max (nm) 209.2 209.7 209.2

k2max (nm) 221.2 220.7 222.1

MRE208 (deg cm2 dmol1) 4

1.91  10 1.76  104 1.72  104

a-Helix (%)

Molar ellipticity h208 (deg cm2 dmol1)

79.76 74.44 73.26

11.17  106 10.30  106 10.06  106

R1 = cPAC/cHSA.

3.5. The effect of PAC on HSA conformation 3.5.1. Three-dimensional fluorescence spectra In order to investigate the conformational changes of HSA, the three-dimensional fluorescence spectroscopy was employed. It can extensively exhibit the fluorescence information of the sample, which makes the investigation of the characteristic conformational change of HSA more convenient and credible. Moreover, the contour spectra are also important. The three-dimensional fluorescence spectra and contour ones for both HSA and PAC–HSA are shown in Fig. 7. By comparing the spectral changes of HSA in the absence and presence of PAC, the conformational and micro-environmental changes in HSA can be obtained. It can be seen that the Rayleigh scattering peak (kex = kem) and the second-ordered scattering peak (kem = 2kem) displayed to match with the chine-like pattern in Fig. 7A and B, as well as the pencil-like pattern in Fig. 7C and D. At the same time, there are two hump-like peaks in the three-dimensional fluorescence spectra of HSA and PAC–HSA marked peak a and peak b. Peak a (F = 999.9, kex = 282.0 nm, kem = 336.0 nm) mainly reflects the spectral behavior of Trp and Tyr residues on HSA, and the maximum emission wavelength and the fluorescence intensity of the residues are sensitively related to the polarity of their microenvironment. Peak b (F = 297.7, kex = 230.0 nm, kem = 336.0 nm) shows the behavior and intensity of polypeptide backbone structures in HSA which are correlated with the secondary structures of protein (Sun et al., 2012). As showed in Fig. 7D, with the addition of PAC, the fluorescence intensities of peak a and peak b that were 965.1 and 261.3 decreased, indicating that the binding of PAC with HSA decreased the polarity of tryptophan and tyrosine residues, buried more amino acids in the hydrophobic pocket. These changes resulted in the slight folding of the polypeptide chain of the protein. The maximum emission wavelength of peak a (kex = 282.0 nm, kem = 335.0 nm) was almost no shift, and peak b (kex = 230.0 nm, kem = 324.0 nm) exhibited blue shift for 12 nm, suggesting that the binding of HSA with PAC enhanced hydrophobicity of hydrophobic cavity micro-environment for amino acid (Zhang et al., 2012). All these phenomena and analysis revealed that the binding of PAC with HSA induced some micro-environmental and conformational changes in HSA.

3.5.2. CD spectra CD spectroscopy is a quantitative technique to investigate the conformation of HSA in aqueous solution. CD spectra of HSA with various concentrations of PAC at pH 7.40 were shown in Fig. 8. As the experimental data indicated, the CD spectrum of HSA alone exhibits negative ellipticity below 260 nm and gives no signal above this wavelength. The free and asymmetric HSA displays two negative bands in the UV region at about 209 and 221 nm. The peak at 221 nm is contributed to the n ? p⁄ transition of peptide bonds in the a-helix, and the peak at 209 nm is contributed to p ? p⁄ transfer for the peptide interlinkage of a-helix (Yang and Gao, 2002). From this study, we observed that the magnitude of these induced CD bands continued to decrease monotonically upon addition of PAC. Nonetheless, the shape of CD spectra and the

position of two negative bands were almost no change (the specific values are shown in Table 2). CD spectrum is usually presented in molar ellipticity [h(k)] unit (deg cm2 dmol1), for proteins, the mean residue ellipticity (MRE) can be calculated as follows (Teng et al., 2011):

MRE ¼

hobs 10  n  l  cp

ð10Þ

where hobs is the CD in millidegree, n is the number of amino acid residues (585 for HSA), l is the path-length of the cell in cm, and cp is the mole concentration of HSA. The a-helix content of HSA was estimated from the values of MRE at 208 nm using the following equation (Naveenraj et al., 2012):

a-helix ¼

  MRE208nm  4000  100% 33; 000  4000

ð11Þ

Here 4000 is the gross MRE of the b-form and random coil conformation at 208 nm, and 33,000 is the gross MRE of a pure a-helix of HSA at 208 nm (Tabassum et al., 2012). The a-helical content of HSA was evaluated from Eqs. (10) and (11). The results (Table 2) revealed the decrease in a-helix content of HSA from 79.76% (in free HSA) to 73.26% (in bound form) at a molar ratio of PAC to HSA of 0:1, 1:1 and 5:1, respectively, indicating that the PAC combined with the amino acid residues of the main polypeptide chain of protein, and perturb interior electrostatic networks. Hence, the calculated results in Table 2 suggest that the binding of PAC with HSA causes extensive conformational changes in predominantly of a-helix structure of HSA. 4. Conclusions In this article, the interactions of two kinds of PGRs including PAC and UNI with HSA were investigated by the combination of molecular docking and spectroscopic methods for the first time. The results indicated PAC mainly bind on site II in HSA, and the major interaction forces were hydrophobic and hydrogen bond interactions, while UNI could not bind with HSA. The analysis of fluorescence illustrated that the binding of PAC to HSA was a static quenching process, and binding affinity (Kb) and the amounts of binding sites (n) between PAC and HSA at 291 K were estimated as 2.37  105 mol L1 and 1, respectively. Meanwhile, PAC bound to the Trp214 of HSA with a distance of 4.41 nm. Furthermore, results of 3D fluorescence and CD revealed that the binding of PAC induced the conformational changes of disulfide bridges of HSA with the decrease of a-helix content. This new assay would provide noteworthy insight into the toxicology and environmental risk assessment of PGRs. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

130

S. Dong et al. / Food and Chemical Toxicology 67 (2014) 123–130

Acknowledgments The authors appreciate the support from the National Natural Science Foundation of China (No. 50830303), overall Innovation Project of Science & Technology in Shaanxi Province (No. 2011KTCG03-07), the National Natural Science Foundation of China (No. 51008242), and the Program for Science and Technology Research of Shaanxi Province (No. 2012k08-12). References Chang, X.J., Huang, Y., He, Q., 2005. Study on the Interaction between Ir(IV) and gamma seroglobulinum humanum. Acta Chim. Sinica 63, 223–238. Chen, J.B., Zhou, X.F., Zhang, Y.L., Gao, H.P., 2012. Potential toxicity of sulfanilamide antibiotic: binding of sulfamethazine to human serum albumin. Sci. Total Environ. 432, 269–274. Davis, D.B., Dernoeden, P.H., 1991. Summer patch mad Kentucky blue-grass quality as Zumbado influenced by cultural practice. Agriculture 831, 670–677. Ding, F., Liu, W., Diao, J.X., Sun, Y., 2011a. Characterization of Alizarin Red S binding sites and structural changes on human serum albumin: a biophysical study. J. Hazard. Mater. 186, 352–359. Ding, F., Liu, W., Diao, J.X., Yin, B., Zhang, L., Sun, Y., 2011b. Complexation of insecticide chlorantraniliprole with human serum albumin: biophysical aspects. J. Lumin. 131, 1327–1335. Förster, T., 1948. Intermolecular energy migration and fluorescence. Ann. Phys. 2, 55–75. Han, X.L., Tian, F.F., Ge, Y.S., Jiang, F.L., Lai, L., Li, D.W., Yu, Q.L.Y., Wang, J., Lin, C., Liu, Y., 2012. Spectroscopic, structural and thermodynamic properties of chlorpyrifos bound to serum albumin: a comparative study between BSA and HSA. J. Photochem. Photobiol. B 109, 1–11. He, X.M., Carter, D.C., 1992. Atomic structure and chemistry of human serum albumin. Nature 358, 209–215. Hebert, P.C., MacManus-Spencer, L.A., 2010. Development of a fluorescence model for the binding of medium- to long-chain perfluoroalkyl acids to human serum albumin through a mechanistic evaluation of spectroscopic evidence. Anal. Chem. 82, 6463–6471. Hu, Y.J., Liu, Y., Xiao, X.H., 2009. Investigation of the interaction between berberine and human serum albumin. Biomacromolecules 10, 517–521. Lakowicz, J.R., Weber, G., 1973. Quenching of fluorescence by oxygen. A probe for structural fluctuations in macromolecules. J. Biochem. 12, 4161–4170. Li, X.J., Wang, Z.Q., Chen, J., Zhang, S.G., 1998. UV spectral study on the interaction of RE ions with BSA. Appl. Chem. 15, 5–8. Liu, Y.M., Li, G.Z., Song, W.K., Wang, J.J., 2006. Molecular simulation and interaction between human serum albumin and topotecan hydrochloride. Acta Phys. Chim. Sin. 22, 1456–1459. Liu, Q., Li, J., Tao, W.Y., Zhu, Y.D., Yao, S.Z., 2007. Comparative study on the interaction of DNA with three different kinds of surfactants and the formation of multilayer films. Bioelectrochemistry 70, 301–307. Liu, Y.H., Zhang, L.J., Liu, R.T., Zhang, P.J., 2012. Spectroscopic identification of interactions of Pb2+ with bovine serum albumin. J. Fluoresc. 22, 239–245. Naveenraj, S., Raj, M.R., Anandan, S., 2012. Binding interaction between serum albumins and perylene-3,4,9,10-tetracarboxylate – a spectroscopic investigation. Dyes Pig. 94, 330–337. Peng, X.L., Yu, J., Yu, Q., Bian, H.D., Huang, F.P., Liang, H., 2012. Binding of engeletin with bovine serum albumin: insights from spectroscopic investigations. J. Fluoresc. 22, 511–519. Ren, X.M., Guo, L.H., 2012. Assessment of the binding of hydroxylated polybrominated diphenyl ethers to thyroid hormone transport proteins using a site-specific fluorescence probe. Environ. Sci. Technol. 46, 4633–4640.

Roche, M., Dufour, C., Loonis, M., Reist, M., Carrupt, P.-A., Dangles, O., 2009. Olive phenols efficiently inhibit the oxidation of serum albumin-bound linoleic acid and butyrylcholine esterase. Biochim. Biophys. Acta 1790, 240–248. Ross, D.P., Subramanian, S., 1981. Thermodynamics of protein association reactions: forces contributing to stability. J. Biochem. 20, 3096–3102. 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. Sengupta, A., Hage, D.S., 1999. Characterization of minor site probes for human serum albumin by high-performance affinity chromatography. Anal. Chem. 71, 3821–3827. ˇ ., Ulrih, N.P., 2012. Interactions of different Skrt, M., Benedik, E., Podlipnik, C polyphenols with bovine serum albumin using fluorescence quenching and molecular docking. Food Chem. 135, 2418–2424. Stryer, L., 1978. Fluorescence energy transfers as a spectroscopic rular. Annu. Rev. Biochem. 47, 819–946. Sudlow, G., Birkett, D.J., Wade, D.N., 1975. The characterization of two specific drug binding sites on human serum albumin. Mol. Pharmacol. 11, 824–832. Sun, H.H., Zhang, J., Zhang, Y.Z., Yang, L.Y., Yuan, L.L., Liu, Y., 2012. Interaction of human serum albumin with 10-hydroxycamptothecin: spectroscopic and molecular modeling studies. Mol. Biol. Rep. 39, 5115–5123. Tabassum, S., Al-Asbahy, W.M., Afzal, M., Arjmand, F., 2012. Synthesis, characterization and interaction studies of copper based drug with Human Serum Albumin (HSA): spectroscopic and molecular docking investigations. J. Photochem. Photobiol. B 114, 132–139. Teng, Y., Liu, R.T., Li, C., Xia, Q., Zhang, P.J., 2011. The interaction between 4aminoantipyrine and bovine serum albumin: multiple spectroscopic and molecular docking investigations. J. Hazard. Mater. 190, 574–581. Valerón, P.F., Pestano, J.J., Luzardo, O.P., Zumbado, M.L., Almeida, M., Boada, L.D., 2009. Differential effects exerted on human mammary epithelial cells by environmentally relevant organochlorine pesticides either individually or in combination. Chem. Biol. Interact. 180, 485–491. Wang, G.K., Wang, D.C., Li, X., Lu, Y., 2011a. Exploring the binding mechanism of dihydropyrimidinones to human serum albumin: spectroscopic and molecular modeling techniques. Colloids Surf. B 84, 272–279. Wang, G.K., Li, X., Ding, X.L., Wang, D.C., Yan, C.L., Lu, Y., 2011b. Exploring the mechanism of interaction between 5-(ethoxycarbonyl)-6-methyl-4-(4methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one and human serum albumin: spectroscopic, calorimetric and molecular modeling studies. J. Pharm. Biomed. Anal. 55, 1223–1226. Xu, D.L., Wang, X.M., Ding, L.S., 2011. Spectroscopic studies on the interaction of ccyclodextrin-daunorubicin inclusion complex with herring sperm DNA. Carbohydr. Polym. 83, 1257–1262. 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. Yang, P., Gao, F., 2002. The Principle of Bioinorganic Chemistry. Science Press, Beijing. Zhang, G.W., Ma, Y.D., 2013. Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods. Food Chem. 136, 442–449. Zhang, H.X., Mei, P., Yang, X.X., 2009. Optical, structural and thermodynamic properties of the interaction between tradimefon and serum albumin. Spectrochim. Acta, Part A 72, 621–626. Zhang, J., Sun, H.H., Zhang, Y.Z., Yang, L.Y., Dai, J., Liu, Y., 2012. Interaction of human serum albumin with indomethacin: spectroscopic and molecular modeling studies. J. Solution Chem. 41, 422–435. Zhou, X.M., Yue, Y.Y., Yang, Q., Yan, N., Chen, X.G., 2011. Complexes between C.I. Acid Orange 6 and human serum albumin, a multi-spectroscopic approach to investigate the binding behavior. J. Lumin. 131, 1222–1228.