Food and Chemical Toxicology 67 (2014) 123–130

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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

http://dx.doi.org/10.1016/j.fct.2014.02.0200278-6915/� 2014 Elsevier Ltd. All rights reserved.

Abbreviations: HSA, human serum albumin; PAC, paclobutrazol; PGRs, plant-growth 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: dshiney2004@yahoo.com.cn (S. Dong).

Sheying Dong a,b,⇑, Zhiqin Li a, Ling Shi a, Guiqi Huang a, Shuangli Chen a, Tinglin Huang b

a College of Sciences, Xi’an University of Architecture and Technology, Xi’an 710055, Chinab 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 a b s t r a c t

Article history:Received 28 September 2013Accepted 18 February 2014Available online 22 February 2014

Keywords:Plant-growth regulators (PGRs)Human serum albumin (HSA)Interaction molecular modelingSpectroscopic methods

The affinity between two plant-growth regulators (PGRs) and human serum albumin (HSA) was investi-gated by molecular modeling techniques and spectroscopic methods. The results of molecular modelingsimulations revealed that paclobutrazol (PAC) could bind on both site I and site II in HSA where the inter-action was easier, while uniconazole (UNI) could not bind with HSA. Furthermore, the results of fluores-cence 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 bind-ing affinity (Kb) and the amounts of binding sites (n) between PAC and HSA at 291 K were estimated as2.37 � 105 mol L�1 and 1, respectively, which confirm that PAC mainly binds on site II of HSA. An apparentdistance between the Trp214 and PAC was 4.41 nm. Additionally, the binding of PAC induced the confor-mational changes of disulfide bridges of HSA with the decrease of a-helix content. These studies providemore information on the potential toxicological effects and environmental risk assessment of PGRs.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Due to their low price and high effective ability to regulate plantgrowth and development, plant-growth regulators (PGRs) includ-ing synthetic compounds and natural plant hormones extractedfrom the organisms are generally applied for agricultural activitiesin China. They can inhibit gibberellin biosynthesis, slow down thecells extending, shorten internode, and maintain the cell numberand section number (Davis and Dernoeden, 1991). However, suchmaterials also promote toxic effects even at low concentrations.What is more, long-term heavy use of these substances results intheir residues in crop, soil and water bodies, and endangers thesafety of the entire ecosystem through the food chain (Valerónet al., 2009). Interaction between PGRs and proteins may interferewith the normal binding between proteins and other endogenoushormones in the body. Therefore, the affinity between PGRs andserum albumin was investigated for the potential toxicological ef-fects of PGRs. At present, paclobutrazol (PAC) and uniconazole

(UNI) are two kinds of important PGRs whose structures are shownin Fig. 1.

Human serum albumin (HSA), the most abundant carrier pro-tein in plasma, provides about 80% of the blood osmotic pressure(He and Carter, 1992). HSA molecule is a 585 amino acid residuemonomer of 66.500 g mol�1, which has sole tryptophan residue(Trp214). Heart-shaped three-dimensional structure of HSA mole-cule comprises of three structurally homologous domains that aredomain I (residues 1–195), II (196–383), III (384–585). And eachdomain is subdivided into two subdomains possessing commonstructural 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 ofbiological processes. It has been proved that the principle regions ofHSA to bind most of molecules are located within hydrophobic cav-ities in subdomains IIA and IIIA, which are corresponded to the Sud-low’s site I and site II (Roche et al., 2009; Sengupta and Hage, 1999).To characterize these interactions at the molecular level, opticaltechniques have become valuable tools because of their highsensitivity, rapidity and easy implementation. Particularly,three-dimensional fluorescence, synchronous fluorescence and cir-cular dichroism spectroscopy have been commonly used to studythe conformational changes of proteins (Hebert andMacManus-Spencer, 2010; Ren and Guo, 2012; Zhang and Ma,2013). In addition, molecular docking comprehensively considers

Fig. 1. Chemical structures of two PGRs.

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

the combined effect of ligands and receptors, which can avoidcircumstances for better local interaction and poor overall combina-tion as well as provide important information for molecular bindingmode and structural transformation of active small molecule. Thisaspect has attracted great interest among researchers in recentyears (Ding et al., 2011a; Wang et al., 2011a). Ding et al. (2011b) re-ported the binding of chlorantraniliprole with HSA by molecularspectroscopy and circular dichroism spectroscopy, and combinedwith molecular docking techniques to determine the bonding loca-tion. Han et al. (2012) investigated chlorpyrifos binding with HSAand bovine serum albumin (BSA) employing molecular spectros-copy, electrochemistry and molecular docking methods. Saquibet al. (2011) studied in detail the interaction between phorate andHSA. However, to the best of our knowledge, none of the publishedstudies were reported on the interaction of PGRs with HSA. On theother hand, there still exist some problems such as less type of pes-ticides, less technologies integration and systems analysis.

In this work, the binding of HSA with two kinds of PGRs (PACand UNI) was predicted by molecular modeling techniques. Subse-quently, the binding interactions under simulative physiologicalconditions were studied by UV–Vis absorption spectrometry, fluo-rescence spectroscopy, three-dimensional (3D) fluorescence spec-troscopy and circular dichroism spectroscopy (CD spectroscopy).The quenching mechanism was discussed on the binding con-stants, number of the binding sites and basic thermodynamicparameters. Furthermore, the features of PAC leading to conforma-tional changes of HAS have been explored by conformational anal-ysis. When HAS binds with PAC (HSA–PAC), the normal bindingbetween HSA and other endogenous hormones in human bodymay be interfere. Therefore, we hope that this study will be helpfulfor understanding the impact of PGRs on HSA structure andfunction.

2. Materials and methods

2.1. Instruments and chemicals

UV–Vis absorption spectra were recorded on a Nicolet Evolution 300 UV–Visiblespectrophotometer with 1.0 cm quartz cells. All fluorescence spectra were per-formed on a FP-6500 fluorophotometer (Hitachi, Japan) equipped with a xenonlamp source and a 1.0 cm quartz cell. The CD spectra were carried out in a ChirascanCD spectrometer (Applied Photophyscics Limited, United Kingdom). All pH mea-surements were measured with IX-501A digital pH-meter (Department of Chemis-try, Peking University, China) combined with a glass-calomel electrode.

HSA was purchased from Sigma Chemical Co. Ltd and without further purifica-tion. All calculations reported for HSA were in terms of HSA with the molecularweight of 66,500. A 1.00 � 10�4 mol L�1 stock solution of HSA was prepared by di-rectly dissolving the proteins in 0.0667 mol L�1 phosphate buffer and stored at277 K. PAC and UNI were of analytical grade, and were purchased from Jinghongof Jiangsu Chemical Reagent Co. Ltd., China. The stock solutions of PAC and UNI withthe concentration of 1.00 � 10�2 mol L�1 were obtained by dissolving PAC and UNIin methanol, respectively. Other chemicals were all of analytical grade and doublydistilled water was used throughout the experiment.

2.2. Molecular modeling

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

2.3. UV–Vis absorption spectrometry

Two separate experiments were carried out in the phosphate buffer.

Experiment 1: The concentration of PAC (5.0 � 10�5 mol L�1) or UNI(3.0 � 10�5 mol L�1) was kept constant, and different amounts of HSA were addedto the solution. The concentration range of HSA was 0–1.3 � 10�6 mol L�1, and 15different solutions were prepared.

Experiment 2: The concentration of HSA (5.0 � 10�7 mol L�1) was kept con-stant, and different amounts of PAC were added to the solution. The concentrationrange of PAC was 0–3.5 � 10�5 mol L�1.

The resulted solutions were allowed to stand for 15 min before analyzed on theUV–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 at5.0 � 10�7 mol L�1 while the concentration of the PAC was varied from 0 to3.2 � 10�5 mol L�1. After kept still for 15 min, the fluorescence spectra were re-corded in the range of 290–500 nm upon excitation wavelength at 280 nm, whichboth excitation and emission bandwidths were set at 5 nm.

2.5. Three-dimensional fluorescence spectra

The three-dimensional fluorescence spectra were recorded under the followingconditions: the emission spectra were recorded between 240 and 500 nm, the ini-tial excitation wavelength was set to 220 nm with increment of 5 nm, the numberof scanning curves was 37, and other scanning parameters were the same as thoseof fluorescence quenching studies.

2.6. CD spectra

The CD spectra were measured from 190 to 260 nm with 1.0 mm path lengthoptical circular quartz cells. Each result was the average of the three scans. The scanslit width of 1 nm, response time of 1 s and scan rate of 100 nm min�1 were con-stantly maintained throughout all experiments. The concentration of HSA was1.00 � 10�6 mol L�1 at pH 7.4 under constant nitrogen flush. The data were ex-pressed in terms of mean residue ellipticity (abbreviated as MRE), using the meanresidue weights of 114.3 (66500/582) for the intact molecular weight of the HSAand the cell path length. Blank phosphate buffer solution was used as a spectral ref-erence and subtracted from the sample spectra.

3. Results and discussion

3.1. Molecular modeling

To obtain insight into interactions of UNI/PAC with HSA, molec-ular modeling simulations were applied to examine the binding ofUNI/PAC at the active site of HSA. Ehits molecular docking programconsidered 32 conformations of compound by using a systematicsearch algorithm and scoring function based on protein family.The results indicated that for the binding of UNI at the active siteof HAS, any conformation and scoring did not provided in visuali-zation software CheVi. Although the structure of UNI was similar tothat of PAC, carbon–carbon double bond was connected with thebenzene ring and is near to the benzene ring for UNI. Thus, thenon-normal O–H� � �Ph hydrogen bond was formed, which re-stricted the formation of hydrogen bond between OH group inUNI and HAS because of its saturation. In order to confirm theviewpoint, the optimized configurations of PAC and UNI are carriedout at B3LYP/6-31G level. It can be found that the hydroxyl wasnearer to the benzene ring in UNI. An apparent distance betweenthe hydroxyl and the benzene ring is 1.91583 Å. Meantime, therigidity in UNI leads to the increase of the steric resistance.

S. Dong et al. / Food and Chemical Toxicology 67 (2014) 123–130 125

Consequently, a conclusion may be safely drawn that UNI would bedifficult to interact with HSA.

The docking results showed PAC could bind to the two sites ofHSA. The docking score was �5.56 for site I, and �7.26 for site IIwhere the interaction between PAC and HAS was easier owe tomore negative scoring. The optimum binding mode between PACand 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 pocketof HSA. Furthermore, as shown in Fig. 2B, there were hydrophobicinteractions of the hydrophobic group of PAC with the residuesAla291, Leu219, Leu264, Leu260, Leu238, Ile290, Trp214, Trp150,Lys199, and Arg218 of HSA. Therefore, the hydrophobic interactionwas extremely significant for the stability of the PAC and HSAcomplexes.

Fig. 2C and D showed the optimum binding mode between PACand HSA at site II. It was found in Fig. 2C that PAC was locatedwithin a hydrophobic pocket of HSA. There were hydrophobicinteractions of the hydrophobic group of PAC with the residuesVal433, Leu43, Leu453, Leu487, Leu407, Phe403, Asn391, Gly431,Ile388, Cys492, and Cys438 of HSA (Fig. 2D). It was important tonote that hydrogen bonding of hydroxyl with amino hydrogen ofArg410 in HSA was generated. The formation of hydrogen bond de-creased the hydrophilicity, which resulted in the increasing ofhydrophobicity to stabilize the PAC–HSA system (Wang et al.,2011b).

Therefore, the results obtained from molecular modeling sug-gest that PAC bind both on both site I and site II of HSA wherethe interaction between PAC and HAS was more easier, and theinteraction between PAC and HSA is dominated by hydrophobicforce and hydrogen bonds.

Fig. 2. Docking of PAC into site I (A and B) and site II (C and D) of HSA. The surface of tpresented using line in B and D. The ligand structure is represented using ball and stick mline.

3.2. UV–Vis absorption spectra

The interaction between PAC or UNI and HSA was confirmed byUV–Vis absorption spectra. Fig. 3A and B displayed the UV–Visabsorption spectra of PAC and UNI with different concentrationsof HSA, respectively. From Fig. 3A, it can be seen that with the addi-tion of the concentration of HSA, the absorption peak at about206 nm appeared, which was related to the a-helix structure ofHSA (Chang et al., 2005). Although the absorption intensity gradu-ally increased with the bathochromic shift of its absorption band,the linear relationship between DA of the PAC–HSA system andconcentration of HSA was poor. Meanwhile, with the increase ofHSA, the absorption intensity of the peaks at 221 nm increasedgradually, but changed slightly at 270 nm. These results impliedthat PAC interacted with HSA (Liu et al., 2007). In Fig. 3B, however,with the addition of the concentration of HSA, the absorptionintensity near 206 nm gradually increased. Further, a linear plot(Fig. 3B inset) for DA of the UNI-HSA system versus the concentra-tion of HSA was obtained. The changes in absorption intensity areonly caused by the concentration of HSA according to the Lambert–Beer’s law, suggesting UNI does not interact with HSA. The conse-quence of UV–Vis spectra is in agreement well with that obtainedfrom molecular modeling.

Many researches have confirmed that HSA showed two featuredprotein absorption bands in the UV region around 206 nm and278 nm, respectively, while UV–Vis absorption spectrum of PAChad several peaks at 221, 266 and 274 nm. Clearly, the spectrumof mixture of the two substances would have a composite profileconsisting of strong overlapping spectra of the two separateanalytes. When the concentration of HSA was constant, with the

he HSA is displayed by QUICK method in A and C, and the residues of the HSA areodel. The interaction between the ligand and the protein is represented using dashed

Fig. 3. The absorption spectra of PAC (A) and UNI (B) in the presence of variousconcentrations of HSA. (A) cPAC = 5.0 � 10�5 mol L�1, cHSA from b to i: 0, 1.0, 3.0, 5.0,7.0, 9.0, 11.0 and 13.0 (� 10�7 mol L�1); a: 13.0 � 10�7 mol L�1 HSA. (B) cUNI =3.0 � 10�5 mol L�1, cHSA from a to g: 0, 1.0, 3.0, 5.0, 7.0, 9.0 and 11.0(� 10�7 mol L�1); Inset: plot of DA vs. HSA concentration in 206 nm.

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

addition of PAC, the absorption peak intensity of HSA at 206 nm in-creased without shift of its absorption band, and the absorbanceintensity at 278 nm changed slightly (Fig. 4). These phenomenaproved that the extent of peptide chain in HSA had changed afterthe addition of PAC, which induced bareness extent changes ofboth tryptophan residue and tyrosine residue (Li et al., 1998). Asshown in Fig. 4 inset, it is observed obviously that the difference

Fig. 4. The absorption spectra of HSA in the presence of various concentrations ofPAC. cHSA = 5.0 � 10�7 mol L�1, cPAC from a to g: 0, 0.5, 1.0, 1.5, 2.0, 3.0 and 3.5 (�10�5 mol�L�1); Inset: the absorption difference spectra of HSA, cPAC = 3.5 � 10�5

mol�L�1.

spectrum between PAC–HSA system and PAC is significantly differ-ent from the UV absorption spectrum of HSA, indicating thatground state molecules of PAC and HSA form complex.

The absorption relationship between PAC and HSA was ex-pressed by the Lineweaver–Burk equation (Xu et al., 2011):

ðA� A0Þ�1 ¼ A�10 þ K�1

A A�10 ½Q �

�1 ð1Þ

where KA is the binding constant, which can be calculated from theratio of the intercept on the slope, A0 and A are the absorbance ofPAC in the absence and presence of HSA respectively. From theabsorbance of PAC at 221 nm which changed regularly relatively,binding constants at different temperatures were calculated.

Essentially, the interaction forces between ligands and biologi-cal macromolecules may include hydrophobic force, multiplehydrogen bond, van der Waals force and electrostatic interactions(Liu et al., 2006). If the enthalpy change (DH) does not vary signif-icantly in the range of temperature studied, then the values of DHas well as entropy change (DS) can be evaluated from the van’tHoff equation (Xu et al., 2012):

lnK ¼ �DHRTþ DS

Rð2Þ

DG ¼ DH � TDS ð3Þ

where K is binding constant at the corresponding temperature andR is the gas constant. The values of DH and DS were obtained fromthe slope and intercept of the linear plot (Eq. (2)) based on lnK ver-sus 1/T. The free energy change (DG) was estimated from Eq. (3).The results in Table 1 declared that the reaction process was spon-taneous due to the negative value of DG. Ross and Subramanian(Ross and Subramanian, 1981) have characterized the sign andmagnitude of thermodynamic parameters associated with variouskinds of interaction that may take place in protein association pro-cess, as described below. For typical hydrophobic effect, both DHand DS are positive, while there are negative DH and DS for vander Waals force and hydrogen bond formation in a low dielectricmedium. Further, specific electrostatic interaction between ionicspecies in aqueous is expressed by a positive value of DS and a neg-ative DH (almost zero). In the present case of PAC–HSA complex,the positive DH and DS attested that hydrophobic effect played adominant role in the binding of PCA to HSA. According to the ther-modynamic data, the formation of the PAC–HSA complex is favoredentropy while it is disfavored enthalpy. The complex formation re-sults in a less ordered state, possibly due to activation of the mo-tional freedom of both the PAC and HSA molecules.

3.3. Analysis of fluorescence quenching of HSA by PAC

In general, fluorescence of HSA originates from tryptophan(Trp), tyrosine (Tyr) and phenylalanine (Phe) residues. Becausethe Phe residue has a very low quantum yield, and the fluorescenceof Tyr residue is almost totally quenched when it is ionized or closeto an amino group, a carboxyl group or a Trp. The intrinsic fluores-cence of HSA is mainly attributed to the Trp residue alone (Zhouet al., 2011), which is extremely sensitive to its environment. Ifemission peak exhibits blue shift, hydrophobicity around trypto-phan residues would increase and polarity would become smaller.In contrast, red shift indicated that the hydrophobic property de-creased and polarity increased (Hu et al., 2009). The fluorescenceemission spectrum of HSA quenched by PAC was shown in Fig. 5.It is obvious that HSA appears a strong fluorescence emission witha peak at 336 nm (kex = 280 nm), while the PAC possesses a veryweak emission under the present experimental conditions. Withthe increasing concentration of PAC, the fluorescence intensitiesof HSA decreased remarkably, and the emission peak shifted from336 nm to 331 nm. These phenomena implied that the interaction

Table 1Association constants Ka and relative thermodynamic parameters of PAC–HSA system at different temperature.

T (K) KA (104 L mol�1) R1 DHh (KJ mol�1) DGh (KJ mol�1) DSh (J mol�1 K�1) R2

288 5.541 0.9984 �26.24293 8.925 0.9990 61.40 �27.76 304.3 �0.9862298 15.487 0.9964 �29.28303 18.842 0.9966 �30.80

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

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 � 10�7

mol L�1, cPAC from a to h: 0, 8.0, 12.0, 16.0, 20.0, 28.0 and 32.0 (� 10�6 mol�L�1), (h)[PAC] = 8.0 � 10�6 mol L�1.

Fig. 6. Overlap plots of the fluorescence emission spectrum of HSA (a) with theabsorption spectrum of PAC (b). [HSA] = [PAC] = 5.0 � 10�7 mol L�1.

S. Dong et al. / Food and Chemical Toxicology 67 (2014) 123–130 127

between PAC and HSA occurred and altered the microenvironmentof tryptophan.

In protein–ligand binding studies, quenching can be induced bydifferent mechanisms. It is usually classified into dynamic quench-ing and static quenching, which are caused by collisional encoun-ters and ground-state complex formation between fluorophoreand quenchers, respectively (Skrt et al., 2012).

Dynamic quenching and static quenching can be described bySterne–Volmer (Chen et al., 2012) (Eq. (4)) and modified Sterne–Volmer equation (Peng et al., 2012) (Eq. (5)), respectively:

F0

F¼ 1þ KSV½Q � ¼ 1þ Kqs0½Q � ð4Þ

F0

F0 � F¼ 1

faKa½Q �þ 1

fað5Þ

Here F0 and F are the relative fluorescence intensities in the ab-sence and presence of the quencher, respectively. Kq is the quench-ing rate constant of the biomolecule. KSV is the Sterne–Volmerdynamic quenching constant. s0 is the average lifetime the biomol-ecule without quencher. The value of s0 of the biopolymer is10�8 s�1 (Lakowicz and Weber, 1973), and [Q] is the concentrationof quencher (PAC). Ka is the effective quenching constant for theaccessible fluorophores and fa is the fraction of accessiblefluorescence.

The possible quenching mechanism can be interpreted by fluo-rescence quenching spectra of HSA, and quenching data were alsoanalyzed 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 onquencher concentration [Q] and F0/(F0 � F) on the reciprocal valueof the quencher concentration [Q]�1 were linear with the slope. Thechange of Ka with temperature was consistent with KSV and KA ob-tained from absorption spectral data. Obviously, values of Kq weregreater than 2 � 1010 L mol�1 s�1, which proved that the binding ofPAC to HSA was a static quenching process. In addition, the differ-ence absorption spectrum between PAC–HSA and PAC at the sameconcentration was clearly distinct from that of lonely HSA in Fig. 4inset. In fact, the ground-state complex formation frequentlyresults in the perturbation of the absorption spectrum of thefluorophore, while the collisional quenching only affects the ex-cited state of fluorophores (Zhang et al., 2009). Consequently, theresult again confirmed that the quenching mechanism was a staticquenching initiated by the formation of the ground state PAC–HSAcomplex.

For static quenching, the fluorescence intensity can also be usedto analyze the apparent binding constant (Kb) and the number ofbinding sites (n) using Eq. (6) when small molecules independentlybind to a set of equivalent sites on a macromolecule (Liu et al.,2012):

log½ðF0 � FÞ=F� ¼ log Kb þ n log½Q � ð6Þ

From Eq. (6), the values of Kb and n of PAC–HSA system at 291 Kwere obtained to be 2.37 � 105 mol L�1 and 1.357 which can berounded off to 1, respectively. These results indicate that there isa strong interaction between PAC and HSA, and there is one typeof binding site for PAC in HSA. A conclusion might be safely drawnthat PAC mainly bind on site II of HSA.

3.4. Energy transfer between PAC and human serum albumin

Generally, fluorescence resonance energy transfer (FRET) occurswhenever the emission spectrum of a fluorophore (donor) overlapswith the absorption spectrum of another molecule (acceptor).

Fig. 7. The three-dimensional fluorescence spectra and corresponding contour diagrams of HSA (A and C) and PAC–HSA (B and D). c(HSA)/(10�7 mol L�1) = 4.0, c(PAC)/(10�5 mol L�1) = 3.0.

Fig. 8. The CD spectra of the PAC–HSA system at pH = 7.4. cHSA = 1.0 � 10�6 mol L�1,c(PAC)/(1 � 10�6 mol L�1) = 0 (a), 1.0 (b), and 5.0 (c).

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

According to Förster’s non-radiative energy transfer theory(Förster, 1948), the efficiency of energy transfer depends mainlyon (i) the extent of overlap of emission spectrum of the donor(HSA) with absorption spectrum of the acceptor (PAC), (ii) therelative 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 fluores-cence emission spectrum of HSA and the UV absorption spectrumof PAC. The efficiency of energy transfer, E, can be calculated usingthe equation:

E ¼ 1� FF0¼ R6

0

R60 þ r6

ð7Þ

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

R60 ¼ 8:79� 10�25K2N�4/J ð8Þ

The term K2 is the relative orientation factor of the dipole, N is therefractive index of the medium, U is the fluorescence quantum yieldof the donor, and J is the overlap integral of the fluorescence emis-sion spectrum of the donor and the absorption spectrum of theacceptor (Fig. 6).

J ¼R1

0 FðkÞeðkÞk4dkR10 FðkÞdk

ð9Þ

where F(k) is the fluorescence intensity of the donor at wavelengthk, and e(k) is the molar absorption coefficient of the acceptor atwavelength k. In the present case, K2 = 2/3, N = 1.336 andU = 0.118 (Stryer, 1978). According to Eqs. (7)–(9), the values ofthe parameters were obtained to be J = 1.352 � 10�14 cm3 L mol�1,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 occurswith high possibility. Furthermore, the value of r was greater thanR0 in this study, suggesting that PAC could strongly quench theintrinsic fluorescence of HSA by a static quenching mechanism.

Table 2CD spectra results for PAC–HSA complex.

Protein HSA R1 k1max (nm) k2max (nm) MRE208 (deg cm2 dmol�1) a-Helix (%) Molar ellipticity h208 (deg cm2 dmol�1)

0 209.2 221.2 1.91 � 104 79.76 11.17 � 106

1.0 209.7 220.7 1.76 � 104 74.44 10.30 � 106

5.0 209.2 222.1 1.72 � 104 73.26 10.06 � 106

R1 = cPAC/cHSA.

S. Dong et al. / Food and Chemical Toxicology 67 (2014) 123–130 129

3.5. The effect of PAC on HSA conformation

3.5.1. Three-dimensional fluorescence spectraIn order to investigate the conformational changes of HSA, the

three-dimensional fluorescence spectroscopy was employed. Itcan extensively exhibit the fluorescence information of the sample,which makes the investigation of the characteristic conformationalchange of HSA more convenient and credible. Moreover, the con-tour spectra are also important. The three-dimensional fluores-cence spectra and contour ones for both HSA and PAC–HSA areshown in Fig. 7. By comparing the spectral changes of HSA in theabsence and presence of PAC, the conformational and micro-envi-ronmental changes in HSA can be obtained.

It can be seen that the Rayleigh scattering peak (kex = kem) andthe second-ordered scattering peak (kem = 2kem) displayed tomatch with the chine-like pattern in Fig. 7A and B, as well as thepencil-like pattern in Fig. 7C and D. At the same time, there aretwo hump-like peaks in the three-dimensional fluorescence spec-tra 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 thespectral behavior of Trp and Tyr residues on HSA, and the maxi-mum emission wavelength and the fluorescence intensity of theresidues are sensitively related to the polarity of their microenvi-ronment. Peak b (F = 297.7, kex = 230.0 nm, kem = 336.0 nm) showsthe behavior and intensity of polypeptide backbone structures inHSA 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 965.1and 261.3 decreased, indicating that the binding of PAC with HSAdecreased the polarity of tryptophan and tyrosine residues, buriedmore amino acids in the hydrophobic pocket. These changes re-sulted 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 thatthe binding of HSA with PAC enhanced hydrophobicity of hydro-phobic cavity micro-environment for amino acid (Zhang et al.,2012). All these phenomena and analysis revealed that the bindingof PAC with HSA induced some micro-environmental and confor-mational changes in HSA.

3.5.2. CD spectraCD spectroscopy is a quantitative technique to investigate the

conformation of HSA in aqueous solution. CD spectra of HSA withvarious concentrations of PAC at pH 7.40 were shown in Fig. 8.As the experimental data indicated, the CD spectrum of HSA aloneexhibits negative ellipticity below 260 nm and gives no signalabove this wavelength. The free and asymmetric HSA displaystwo 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 pep-tide bonds in the a-helix, and the peak at 209 nm is contributed top ? p⁄ transfer for the peptide interlinkage of a-helix (Yang andGao, 2002). From this study, we observed that the magnitude ofthese induced CD bands continued to decrease monotonically uponaddition of PAC. Nonetheless, the shape of CD spectra and the

position of two negative bands were almost no change (the specificvalues are shown in Table 2).

CD spectrum is usually presented in molar ellipticity [h(k)] unit(deg cm2 dmol�1), for proteins, the mean residue ellipticity (MRE)can be calculated as follows (Teng et al., 2011):

MRE ¼ hobs

10� n� l� cpð10Þ

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

a-helix ¼ MRE208nm � 400033;000� 4000

� �� 100% ð11Þ

Here 4000 is the gross MRE of the b-form and random coil confor-mation at 208 nm, and 33,000 is the gross MRE of a pure a-helixof 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 contentof HSA from 79.76% (in free HSA) to 73.26% (in bound form) at amolar ratio of PAC to HSA of 0:1, 1:1 and 5:1, respectively, indicat-ing that the PAC combined with the amino acid residues of themain polypeptide chain of protein, and perturb interior electro-static networks. Hence, the calculated results in Table 2 suggestthat the binding of PAC with HSA causes extensive conformationalchanges in predominantly of a-helix structure of HSA.

4. Conclusions

In this article, the interactions of two kinds of PGRs includingPAC and UNI with HSA were investigated by the combination ofmolecular docking and spectroscopic methods for the first time.The results indicated PAC mainly bind on site II in HSA, and the ma-jor interaction forces were hydrophobic and hydrogen bond inter-actions, while UNI could not bind with HSA. The analysis offluorescence illustrated that the binding of PAC to HSA was a staticquenching process, and binding affinity (Kb) and the amounts ofbinding sites (n) between PAC and HSA at 291 K were estimatedas 2.37 � 105 mol L�1 and 1, respectively. Meanwhile, PAC boundto the Trp214 of HSA with a distance of 4.41 nm. Furthermore, re-sults of 3D fluorescence and CD revealed that the binding of PACinduced the conformational changes of disulfide bridges of HSAwith the decrease of a-helix content. This new assay would providenoteworthy insight into the toxicology and environmental riskassessment 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 befound 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 NaturalScience Foundation of China (No. 50830303), overall InnovationProject of Science & Technology in Shaanxi Province (No.2011KTCG03-07), the National Natural Science Foundation of Chi-na (No. 51008242), and the Program for Science and TechnologyResearch of Shaanxi Province (No. 2012k08-12).

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