Potential Toxicity of Phthalic Acid Esters Plasticizer: Interaction of

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Potential toxicity of phthalic acid esters plasticizer: Interaction of dimethyl phthalate with trypsin in vitro Yaping Wang, Guowen Zhang, and Langhong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5046359 • Publication Date (Web): 11 Dec 2014 Downloaded from http://pubs.acs.org on December 22, 2014

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Journal of Agricultural and Food Chemistry

Potential toxicity of phthalic acid esters plasticizer:

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Interaction of dimethyl phthalate with trypsin in vitro

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Yaping Wang, Guowen Zhang*, Langhong Wang

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State Key Laboratory of Food Science and Technology, Nanchang University,

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Nanchang 330047, China

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________________________

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*

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Corresponding author. Professor Guowen Zhang, Ph.D, Tel.: +8679188305234; fax: +8679188304347. E–mail address: [email protected]

19 20 21 22 23 1

ACS Paragon Plus Environment


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ABSTRACT

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Dimethyl phthalate (DMP) is widely used as a plasticizer in industrial processes,

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and has been reported to possess potential toxicity to human body. In this study, the

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interaction between DMP and trypsin in vitro was investigated. The results of

28

fluorescence, UV–vis, circular dichroism and Fourier transform infrared spectra along

29

with cyclic voltammetric measurements indicated that the remarkable fluorescence

30

quenching and conformational changes of trypsin resulted from the formation of a

31

DMP–trypsin complex, which was driven mainly by hydrophobic interactions. The

32

molecular docking and trypsin activity assay showed that DMP primarily interacted with

33

the catalytic triad of trypsin and led to the inhibition of trypsin activity. The dimensions

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of the individual trypsin molecules were found to become larger after binding with DMP

35

by atomic force microscopy imaging. This study offers a comprehensive picture of

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DMP–trypsin interaction which is expected to provide insights into the toxicological

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effect of DMP.

38 39

KEYWORDS: Dimethyl phthalate; Trypsin; Interaction; Spectroscopy; Molecular

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docking; Atomic force microscopy

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INTRODUCTION

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Phthalic acid esters (PAEs), also known as dialkyl or alkyl aryl esters of

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1,2-benzenedi-carboxylic acid, are a class of refractory organic compounds frequently

51

added to plastics to enhance the flexibility of the materials. PAEs also serve as solvents

52

and emulsifiers used in pharmaceuticals, pesticides, health, beauty products and

53

children’s toys.1,2 Because of their wide applications and growing demand, PAEs have

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become one of the highest yielding chemicals in the world. Their worldwide production

55

are approximately amount to 6 million tones per year.3 Due to non-covalent bonds

56

existence between the phthalate plasticizers and their parent materials, PAEs can be

57

significantly leached into the environment and lead to environmental contamination

58

which may cause common exposures in the general population.4 As a kind of

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environmental endocrine disrupting chemicals, PAEs show estrogen effects and a variety

60

of biological toxicity. Investigations have shown that PAEs can reduce fertility and

61

pregnancy rates in humans and animal models, and can increase miscarriages and other

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gestational complications.5 Epidemiologic studies have also found that early phthalate

63

exposure can induce neurodevelopmental damage.6 Moreover, they are suspected to

64

cause liver, kidney and thyroid gland tissue damage.7 Some PAEs, such as dimethyl

65

phthalate, diethyl phthalate, dibutyl phthalate, benzylbutyl phthalate and di-(2-ethylhexyl)

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phthalate have been classiﬁed as priority environmental pollutants by the U.S.

67

Environmental Protection Agency, European Union and the China National

68

Environmental Monitoring Center.8 In recent years, excess of some PAEs has been

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detected sometimes in liquor and beverage in China, their safety has attracted much

70

attention, thus most of PAEs have been listed as kinds of illegal food additives by the

71

Ministry of Health, China.

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Dimethyl phthalate (DMP, structure shown in Figure 1A) is one of the most frequently

3



73

used PAEs. The impact of this chemical on the environment and its toxicity to living

74

organisms is of great concern today. DMP and its intermediates are suspected to be

75

responsible for functional disturbances in the nervous systems and liver in animals.9

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Known for endocrine-disrupting and oestrogenic activity, it may also promote

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chromosome injuries in human leucocytes and can interfere with the reproductive system

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and normal development of animals and humans.10 Dillingham et al. have reported that

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DMP exhibited teratogenicity, mutagenicity and cellular toxicity.11 Additionally, repeated

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inhalation of the vapor of DMP will cause the irritation of the nasal mucous membrane

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and lead to noticeable kidney damage.12

82

Proteinases are a kind of important proteins which can cleave the covalent peptide

83

bond between amino acids and play an important role in many biologically relevant

84

processes.13 As a water-soluble globular protein, trypsin belongs to a serine protease that

85

cleaves peptide bonds at the carboxylic groups of lysine, arginine and ornithine. This

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enzyme is excreted by the pancreas into the small intestine and takes part in the digestion

87

of food proteins and other biological processes in vertebrates. Trypsin is a medium-sized

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protein owning 223 amino acid residues with a molecular weight of 23.5 KDa,14,15 and

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contains two domains of nearly equal size connected by six disulfide bonds. Every

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domain has six antiparallel β-foldings, and the active sites of trypsin are the catalytic

91

triad – His 57, Asp 102 and Ser 195 located between the two domains.16,17 Trypsin is

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often used as an important model of the digestive proteases to investigate the interactions

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of small molecules with proteins for it plays an essential role in digestion deconstruction

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of food. When DMP enters the human gastrointestinal tract, the digestive proteases may

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be the indirect binding targets and there might be interaction between DMP and trypsin.

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The binding of some exogenous environmental pollutants with trypsin can affect the

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conformation as well as the activity of trypsin and then cause the pathological changes in

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human body. Wang et al. reported that the in vitro interaction of a widely used dye acid

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yellow 23 with trypsin induced the conformational changes of trypsin and led to the

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inhibition of enzyme activity.18 To the best of our knowledge, no investigation on the

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interaction of DMP with trypsin has been previously reported, thus exploring the

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DMP–trypsin interaction is significantly important because of the contribution to the

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understanding the toxicity mechanism of DMP at the molecular level.

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The present study was focused on the binding interaction between DMP and trypsin in

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physiological buffer (pH 7.4) with the use of a combination of spectroscopic, atomic

106

force microscopy (AFM), cyclic voltammetric (CV) and molecular modeling approaches.

107

The binding mode, binding site and thermodynamic parameters of DMP with trypsin

108

were characterized and the conformational changes of trypsin induced by DMP were

109

determined.

110

MATERIALS AND METHODS

111

Materials. Trypsin from bovine pancreas was purchased from Sigma Chemical Co.,

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(St. Louis, USA), its stock solution (1.0 × 10−4 mol L−1) was prepared by dissolving its

113

crystals (11.8 mg) in 5 mL of Tris−HCl buffer (0.05 mol L−1 Tris base, 0.05 mol L−1 HCl

114

and 0.1 mol L−1 NaCl, pH 7.4) and then diluted to the required concentrations with the

115

buffer. DMP (purity ≥ 99.5%) obtained from Aladdin Industrial Co., was prepared as a

116

1.0 × 10−2 mol L−1 solution in anhydrous methanol. N-α-benzoyl-L-arginine ethyl ester

117

(BAEE, from Aladdin Industrial Co., Shanghai, China) was dissolved in pH 7.4 Tris−HCl

118

buffer to form a 6.0 × 10−3 mol L−1 solution. All other chemicals were of analytical

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reagent grade, and used without further purification. Ultrapure water was used

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throughout the experiment.

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Fluorescence Measurements. Fluorescence measurements were performed on a

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Hitachi spectrofluorimeter (Model F-7000, Hitachi, Japan) using a 1.0 cm path length

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quartz cuvette placed in a thermostat bath. A 3.0 mL solution containing 1.5 × 10−6 mol

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L−1 trypsin was titrated by successive additions of 1.0 × 10−2 mol L−1 DMP stock solution.

125

After the solutions were mixed thoroughly and kept for 5 min, the fluorescence spectra of

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the solutions were measured in the wavelength range of 300−500 nm upon exciting the

127

samples at 280 nm using 2.5/2.5 nm slit widths. The corresponding emission spectra of

128

free DMP were measured as blanks (i.e., successive addition of DMP solutions into 3.0

129

mL Tris−HCl buffer) to eliminate background signal. Additionally, fluorescence

130

intensities were corrected for the inner-filter effect before analysis of the binding and

131

quenching data using the relationship:19

132

Fc = Fm e ( A1 + A2 ) / 2

133

where A1 and A2 are the absorbance of DMP at excitation and emission wavelengths, Fc

134

and Fm represent the corrected and measured fluorescence, respectively. The intensity of

135

fluorescence used in this paper was the corrected fluorescence intensity.

(1)

136

Synchronous fluorescence spectra of trypsin in the absence and presence of DMP were

137

measured by setting the excitation and emission wavelength interval (∆λ) at 15 and 60

138

nm. The appropriate blank corresponding to the free DMP and buffer was subtracted to

139

correct for background fluorescence.

140

UV–vis Absorption Measurements. The UV–vis absorbance spectra of trypsin, DMP

141

and DMP–trypsin mixtures were recorded at room temperature on a Shimadzu UV–2450

142

spectrophotometer (Shimadzu, Japan) at wavelengths between 200 and 320 nm.

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The enzyme activity was measured spectrophotometrically by continuously measuring

144

N-α-benzoyl-L-arginine (BA) formation at wavelength of 253 nm with BAEE as the

145

substrate.20 A series of assay solutions dissolved in 0.05 mol L−1 Tris−HCl buffer (pH 7.4)

146

containing different concentrations of DMP and a fixed concentration of trypsin (1.5 × 6


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10−6 mol L−1) were prepared and incubated for 3 h at 37 ºC (the concentration of

148

methanol was less than 0.03% in the total reaction volume). The assay was initiated by

149

adding the DMP–trypsin complex solution to the substrate (BAEE, 7.0 × 10−4 mol L−1)

150

and the absorbance of the mixture at 253 nm was then determined every 20 s at room

151

temperature. The enzymatic activity without DMP was defined as 100%. Relative

152

enzymatic activity (%) = (slope of reaction kinetics equation obtained by reaction with

153

inhibitor)/(slope of reaction kinetics equation obtained by reaction without inhibitor) ×

154

100%.21

155

Molecular Simulation. Molecular modeling of the DMP−trypsin complexation was

156

performed using AutoDock 4.2 and AutoDockTools 1.5.6 (ADT). The crystal structure of

157

trypsin

158

(http://www.rcsb.org/pdb).22 Water molecules were removed, polar hydrogens were

159

added and Kollman united atom partial charges were assigned during protein preparation.

160

The 3D structure of DMP was constructed in Sybyl × 1.1 (Tripos Inc., St. Louis, U.S.),

161

and its energy-minimized conformation was obtained with the help of the MMFF94 force

162

field using MMFF94 charges. During the docking study, a grid box defined to enclose the

163

entire binding site of trypsin with dimensions of 126 Å × 126 Å × 126 Å and a grid

164

spacing of 0.375 Å was used. The Lamarckian genetic algorithm (LGA) was applied to

165

search binding conformations and a total of 100 runs were carried out during docking

166

simulation.

(PDB

code

2ZQ1)

was

taken

from

the

Protein

Data

Bank

167

Atomic Force Microscopy (AFM) Measurements. AFM measurements were carried

168

out in tapping mode using an Asylum Research atomic force microscope (MFP-3D SA)

169

at room temperature. Silicon nitride probes from Olympus (USA) with spring constants

170

of 0.3–4.8 N m−1 were used. The tip radius of the probes was 9+/-2 nm and rectangular

171

cantilevers with a length of 240 µm was used. The scanning rate was set at 1 Hz. Samples

7



172

imaged in air were prepared by drop 5 µL of the trypsin solution in the absence and

173

presence of DMP on freshly cleaved mica at the required concentrations and dried

174

overnight. Images were processed with the Igor Pro 6.3 software package and were

175

presented unﬁltered.

176

Cyclic voltammetric (CV) Experiments. CV experiments were carried out on a

177

CHI-660B electro-chemical workstation (Chenhua Instrumental Company, Shanghai)

178

with a three-electrode system. The working electrode was a glassy carbon disk electrode,

179

an Ag/AgCl electrode was selected as the reference electrode with a Pt wire as the

180

counter electrode. The concentration of DMP was kept at 1.0 × 10−5 mol L−1 and the

181

trypsin solution was added at different concentrations in the range of 0–1.5 × 10−6 mol

182

L−1. All test solutions were thoroughly deoxygenated by bubbling high-purity nitrogen

183

for 10 min. A stream of nitrogen was blown gently across the surface of the solution in

184

order to maintain the solution anaerobic throughout all the experiments.

185

Circular Dichroism (CD) Spectra. CD spectra in the far-UV region (190–250 nm)

186

were recorded at room temperature on a Bio-Logic MOS 450 CD spectrometer

187

(Bio-Logic, Claix, France) using a quartz cell with 1 mm path length in pH 7.4 Tris–HCl

188

buffer under constant nitrogen flush. The concentration of trypsin was kept at 1.5 × 10−5

189

mol L−1 while varying the DMP concentration by keeping the molar ratios of DMP to

190

trypsin as 0:1, 2:1 and 5:1. All the obtained CD spectra were corrected for buffer signal.

191

The online SELCON3 program (http://dichroweb.cryst.bbk.ac.uk/ html/home.shtml) was

192

used to analyze the CD spectroscopic data to obtain the contents of different secondary

193

structures of trypsin.

194

Fourier Transform Infrared (FT−IR) Measurements. FT–IR spectra were

195

performed on a Thermo Nicolet-5700 FT–IR spectrometer (Thermo Nicolet Co., USA)

196

equipped with a germanium attenuated total reflection (ATR) accessory and a DTGS KBr

8


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detector. All spectra were taken via the ATR method with resolution of 4 cm–1 and 60

198

scans. The FT–IR spectra of free trypsin (1.5 × 10−5 mol L−1) and DMP–trypsin complex

199

(the molar ratio of DMP to trypsin was kept at 5:1) were recorded in the range of

200

1800–1400 cm−1 at room temperature. The corresponding absorbance contributions of

201

buffer and free DMP solutions were recorded and substracted with the same instrumental

202

parameters.

203

RESULTS AND DISCUSSION

204

Fluorescence Quenching of Trypsin by DMP. Fluorescence quenching of protein

205

usually attributed to a variety of molecular interactions including excited-state reactions,

206

energy transfer, ground-state complex formation and collisional quenching.23 As shown

207

in Figure 1A, trypsin displayed a strong emission peak at 347 nm on excitation at 280 nm,

208

and the peak intensity of trypsin decreased gradually with increasing the amounts of

209

DMP. When the added concentration of DMP reached 1.64 × 10−4 mol L−1, nearly 51.7%

210

decrease in the fluorescence intensity of the peak accompanied by a 4 nm red shift was

211

observed, suggesting that DMP interacted with trypsin and the microenvironment of the

212

fluorescence chromophore of trypsin was altered upon binding with DMP.

213

Quenching of protein fluorescence induced by a quencher can be classified as either

214

dynamic quenching or static quenching, and the quenching mechanism can be

215

distinguished based on their temperature dependence.24 In order to speculate the

216

fluorescence quenching mechanism, the fluorescence titration experiments were carried

217

out at 298, 304 and 310 K and the data were analyzed by the well-known Stern–Volmer

218

equation:

219

220

F0 = 1 + KSV [Q] = 1 + K qτ 0 [Q] F

(2)

where F0 and F represent the fluorescence intensities in the absence and presence of 9



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221

DMP, KSV is the Stern–Volmer dynamic quenching constant and its value can be

222

determined by linear regression of a plot of F0/F against [Q]. Kq is the quenching rate

223

constant of the biomolecule, Kq=KSV/τ0. τ0 is the average lifetime of the fluorophore

224

without quencher (τ0 = 2.80 × 10−9 s),25 and [Q] is the concentration of DMP.

225

The Stern–Volmer plots (Figure 1B) obtained at different temperatures showed a good

226

linear relationship, suggesting a single quenching procedure between DMP and trypsin

227

occurred, and the corresponding KSV values for the DMP–trypsin complex are listed in

228

Table 1. An inverse correlation between KSV and temperature (decrease tendency of KSV

229

with increasing temperatures) was observed, indicating that the probable fluorescence

230

quenching mechanism of trypsin by DMP was initiated by complex formation viz., static

231

quenching.26 Moreover, larger Kq values (1.45 × 1012, 1.39 × 1012 and 1.20 × 1012 L mol−1

232

s−1 at 298 K, 304 K and 310 K, respectively) for the DMP−tryspin complexation

233

compared to the reported maximum scatter collision quenching constant (2 × 1010 mol

234

L−1 s−1) also indicated that the quenching process was mainly governed by a static

235

quenching procedure.27

236

Binding Constants and Number of Binding Sites. To further characterize the binding

237

properties of DMP with trypsin, the binding constant K and the number of binding sites n

238

for the DMP–trypsin complex were determined using the following equation:28

log 239

F0 − F 1 = n log K − n log ( F − F )[Pt ] F [Q t ] − 0 F0

(3)

240

where F0 and F are the same as in Eq.(2). [Qt] and [Pt] stand for the total concentration of

241

the ligand and the protein, respectively. From the intercept and slope of the regression

242

curve of log (F0 – F)/F versus log1/([Qt] – (F0 – F)[Pt]/F0) (Figure 1C), the values of K

243

and n at the three temperatures were calculated (Table 1). Due to the n values

244

approximately equal to 1, it can be inferred that there was a single class of binding sites 10


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on trypsin for DMP. The increasing trend of K with rising temperature suggested that the

246

DMP−tryspin interaction was an endothermic reaction and the increasing temperature

247

was in favor of this binding.29 The K value at 298K was 3.92 × 103 L mol−1, which was

248

similar to that of previous report on DMP interaction with HSA.30

249

Prediction of Binding Site of DMP on Trypsin. As a serine protease, the enzymatic

250

mechanism of trypsin is similar to that of other serine proteases, with a catalytic triad

251

(His57, Asp102 and Ser195).31 Trypsin contains the primary substrate-binding pocket (S1

252

binding pocket) formed by amino acid residues 189–195, 214–220 and 225–228.32 To

253

predict the binding site of DMP on trypsin, the molecular modeling was employed to

254

simulate the binding mode between DMP and trypsin.

255

Cluster analysis of 100 docking runs showed a total of 9 multimember conformational

256

clusters at a root-mean-square deviation (rmsd) tolerance of 2.0 Ǻ (Figure 2A). The

257

cluster with the lowest binding energy (red histogram in Figure 2A) was found to contain

258

the highest number of the analyzed conformations (38 out of 100), which was the most

259

energetically favorable. Thus, the cluster possessed the lowest energy (−5.12 kcal mol−1)

260

and the most frequent locus was used for binding orientation analysis, and the obtained

261

energy information was given in Table 2. The calculated binding Gibbs free energy

262

(∆Gbinding = intermolecular energy + torsional energy) was –5.12 kcal mol–1, which was

263

equal to the predicted binding energy (–5.12 kcal mol–1), indicating that the docking

264

result was credible.

265

As shown in Figure 2B, DMP did not bind into the S1 binding pocket of trypsin, while

266

it had a high affinity very close to the active site pocket, especially adjacent to the

267

catalytic residues (His 57, Asp 102 and Ser 195). It was observed that DMP was

268

surrounded by the amino acid residues including Phe41, Cys42, Ala56, His57, Tyr59,

269

Asp102, Met104, Gly193 and Ser195. Moreover, two hydrogen bonds were formed

11



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between the oxygen atoms of DMP and the hydrogen atom on Ser195 and Gly193 of

271

trypsin, and their bond lengths were 1.712 and 2.187Å, respectively, which suggested the

272

involvement of hydrogen bonding in the DMP−tryspin interaction. The docking results

273

indicated that DMP actually interacted with the catalytic residues located in the region

274

close to the S1 binding pocket of trypsin, and the binding may affect the enzymatic

275

activity.

276

Thermodynamic Parameters and Binding Forces. A thermodynamic process was

277

considered to be responsible for the formation of a complex considering from the

278

dependence of binding constant on temperature.33 Therefore, the thermodynamic

279

parameters including enthalpy change (∆H°), entropy change (∆S°) and free energy

280

change (∆G°) of the interaction were analyzed to further characterize the bonding force

281

between DMP and trypsin using the following equations:

282

log K = −

283

∆G° = ∆H ° − T∆S °

284

where R is the gas constant (8.314 J mol−1 K−1), T is the experimental temperature (298,

285

304 and 310 K), and K is the binding constant at the corresponding temperature. The

286

calculated values of ∆H°, ∆S° and ∆G° are summarized in Table 1. The negative signal

287

for ∆G° showed that the interaction process was spontaneous. The positive ∆H° and ∆S°

288

values indicated that hydrophobic interactions played a major role in the binding of DMP

289

to trypsin based on the rule generalized by Ross and Subramanian.34 It was obvious that

290

the ∆G° value (–4.91 kcal mol–1) at 298 K was a little larger than the calculated ∆G°

291

value from molecular modeling. This difference could be explained by the fact that X-ray

292

structure of the protein from crystals was different from that of the aqueous system used

293

in this study.35

294

∆H ° ∆S ° + 2.303RT 2.303R

(4) (5)

Effect of DMP on Trypsin Activity. As shown in Figure 3, trypsin activity decreased 12


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295

with the incremental addition of DMP, and the enzymatic activity decreased to 62.3% of

296

the initial level when the concentration of DMP reached to 1.0 × 10−4 mol L−1, indicating

297

that the interaction of DMP with trypsin can inhibit the enzymatic activity. There are two

298

fundamental characteristics about the active site region of trypsin: (i) The catalytic triad

299

composed of amino acid residues His 57, Asp 102 and Ser 195; (ii) The S1 binding

300

pocket was near Ser 195 which can define the cut position of the peptidic bond.36 If a

301

ligand enters into the hydrophobic pocket or locates near this pocket, the enzymatic

302

activity could be affected.37 This experimental result further supported the molecular

303

docking studies.

304

Changes in Trypsin Topography. AFM is a very useful imaging technique in

305

visualizing the interaction between small molecules and protein owing to its high

306

resolution. To study the changes in trypsin topography following by the addition of DMP,

307

AFM was performed on the free trypsin and the DMP–trypsin complex in pH 7.4

308

Tris–HCl buffer.

309

As shown in Figure 4A, the trypsin was adsorbed evenly on the mica surfaces and its

310

topography image can be clearly observed. Cross-sectional images of two single trypsin

311

molecules are given. After averaging the width of 50 single trypsin molecules, 5.0 nm

312

was obtained for the mean width. The mean height of the individual trypsin molecules

313

adsorbed on mica was determined to be 0.8 nm. This average width was larger than the

314

reported data (3.8 nm) based on X-ray diffraction studies.38 Upon complexation with

315

DMP, trypsin molecules appeared to be more swollen on the mica substrate (Figure 4B),

316

and the mean width and height of the individual trypsin molecules reached 12.0 nm and

317

2.5 nm, respectively. The increase in the size indicated that DMP interacted with trypsin

318

and a DMP−trypsin complex was formed. After binding with DMP, the stable dispersion

319

of the protein aggregation became more evident in the AFM photograph, suggesting that

13



320

the formation of the DMP−trypsin complex may cause a major destabilization of the

321

native conformation of trypsin. From the three-dimensional graph of free trypsin and the

322

DMP−trypsin complex (Figure 4C and D), it was clearly observed that the interaction

323

between DMP and trypsin induced the topography image changes of trypsin and

324

aggregation, which may be due to the changes of microenvironment around the trypsin

325

molecule and the enzyme molecule exposing to a more hydrophobic environment after

326

interaction with DMP. To minimize the number of unfavorable factors to form a stable

327

structure, the trypsin molecule reduced its surface area in contact with water by

328

molecular aggregation. The results suggested that hydrophobic interactions existed

329

between trypsin and DMP, which was consistent with that of above thermodynamic

330

analysis.

331

Electrochemical Behavior of DMP in the Presence of Trypsin. The cyclic

332

voltammograms of DMP in the absence and presence of trypsin are shown in Figure 5.

333

DMP exhibited a deoxidization peak at −0.98 V in pH 7.4 Tris−HCl buffer which was the

334

characteristic of the electroactive moiety of DMP. When trypsin was added to the DMP

335

solution, a positive shift of the deoxidization peak along with a decrease in peak current

336

was found and no new peak was observed in the same scan potential range. These results

337

indicated that the binding interaction between DMP and trypsin occurred and the

338

electro-inactive complex was formed.39 It was difficult for DMP in the complex to make

339

contact with the electrode surface, and the free DMP concentration on the electrode

340

surface was reduced after bonding with trypsin, and thus the peak current was

341

decreased.40

342

Effect of DMP on the Conformation of Trypsin. UV–vis absorbance spectroscopy is

343

widely used to explore the conformational changes of protein and to investigate

344

ligand–protein complex formation. Figure 6 shows the difference absorption spectra of

14


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345

trypsin after interaction with DMP which were obtained by subtracting the corresponding

346

absorption spectra of free DMP from those of DMP–trypsin complex. Trypsin has two

347

main absorption peaks located at 205 and 280 nm, respectively. The stronger absorption

348

peak at 205 nm reflects the peptide bond absorption peak which is due to the π→π*

349

transition of trypsin’s characteristic polypeptide backbone structure C=O, and the weaker

350

one at 280 nm appears due to the absorption of aromatic amino acids.41 With the addition

351

of DMP to trypsin solution, the peak at 205 nm shifted obviously toward the longer

352

wavelength and the intensity decreased sharply, suggesting that the binding of DMP to

353

trypsin may cause the conformational change of trypsin. Moreover, decrease in the peak

354

intensity at 280 nm also suggested that the polarity of the microenvironment around Trp

355

and Tyr residues of trypsin was affected by the interaction with DMP.

356

Alteration in the microenvironment around the fluorophores (Tyr and Trp) of trypsin

357

was further explored by synchronous fluorescence spectra. Figure 7A and B displays the

358

effect of the increasing concentrations of DMP on the synchronous fluorescence spectra

359

of trypsin when the scanning interval ∆λ was fixed at 15 and 60 nm, respectively. Upon

360

the addition of DMP, the maximum emission wavelength of Tyr residue at ∆λ = 15 nm

361

showed inconspicuous shift (Figure 7A), indicating that DMP had little effect on the

362

microenvironment of Tyr residue, while the emission peak of Trp residue at ∆λ = 60 nm

363

displayed an obvious red shift (from 286 to 290 nm, shown in Figure 7B), suggesting that

364

the hydrophobicity decreased and the polarity increased around Trp residue.42

365

The ratios of synchronous fluorescence quenching (RSFQ) were calculated by using

366

the equation RSFQ = 1 − F/F0 (F0 and F are the fluorescence intensities of trypsin in the

367

absence and presence of DMP, respectively).29 As shown in Figure 7C, the data of RFSQ

368

at ∆λ = 60 nm were a little higher than the corresponding ones at ∆λ = 15 nm at the same

369

DMP concentrations, which may be due to more contribution of Trp residue to the

15



370

quenching of the intrinsic fluorescence and DMP was closer to Trp compared to Tyr

371

residue.

372

To detect the possible influence of DMP binding on the secondary structure of trypsin,

373

CD measurements were performed in the absence and presence of different amounts of

374

DMP (Figure 8). The CD spectrum of trypsin exhibited only one negative band at 200 nm.

375

Generally, the β-sheet shows a CD band at 210−220 nm, however, the irregular strands in

376

trypsin structure may cause the negative CD band shift from the ideal β-sheet position

377

toward the 200 nm region.43 With the addition of DMP, the intensity of the negative peak

378

increased with obvious blue shift, the similar findings have been reported for the

379

interactions of oxytetracycline and tetracycline with trypsin.14,44 Then, the CD spectra

380

data were analyzed by the online SELCON3 software, the contents of different secondary

381

structure of trypsin were obtained and listed in Table 3. The results showed that the

382

β-sheet structure was predominant with a high percentage in trypsin, which was identical

383

with the literature.45 When the molar ratios of DMP to trypsin increased, a decreasing

384

tendency of the β-sheet and β-turn contents were observed, while the α-helix and random

385

coil contents tended to increase, which indicated that DMP induced the secondary

386

structure changes of trypsin, and thus may influence the function of the enzyme.

387

Infrared spectroscopy is a powerful means for investigating the secondary structures of

388

proteins. The amide I band (1600 to 1700 cm−1, mainly C═O stretch) and amide II band

389

(1600 to 1500 cm−1, C–N stretch coupled with N–H bending mode) are main vibrational

390

bands of the peptide moiety associated with the secondary structure of proteins,46 and the

391

amide I band is more sensitive to the change of secondary structure of protein than the

392

amide II band.47 Upon DMP interaction, the FT–IR difference spectra of trypsin

393

[(DMP–trypsin solution)–DMP solution] were obtained to monitor the variation of these

394

vibrations (Figure 9A). It was apparent that the peak position of amide I of trypsin moved

16


Page 16 of 40

Page 17 of 40


395

from 1640 to 1646 cm−1 and the one of amide II shifted from 1550 to 1552 cm−1 after

396

addition of DMP, suggesting that DMP interacted with the C═O and C–N groups in the

397

protein polypeptides and caused the rearrangement of the polypeptide carbonyl hydrogen

398

bonding network.48

399

The curve-fitted spectra of trypsin infrared amide I bands before and after the binding

400

with DMP were analyzed to further characterize the secondary structure change of

401

trypsin according to the literature.49 As shown in Figure 9B, the free trypsin contained

402

β-sheet 37.4% (1632 cm−1), random coil 22.6% (1645 cm−1), α-helix 12.0% (1658 cm−1),

403

β-turn 20.5% (1675 cm−1) and β-antiparallel 7.5% (1692 cm−1). After interaction with

404

DMP ([DMP]:[trypsin] = 5:1), the contents of β-sheet, β-turn and β-antiparallel reduced

405

from 37.4% to 32.1%, from 20.5% to 19.4% and from 7.5% to 5.9%, while the α-helix

406

structure and random coil increased from 12.0% to 17.6% and from 22.6% to 25.0%,

407

respectively (Figure 9C). The results indicated that the binding of DMP to trypsin led to

408

the conformational changes of trypsin with an increase of α-helix and random coil

409

stability and loss of β-structure content.

410

In summary, nowadays human are exposed to thousands of different chemicals each

411

day, the chemicals may gather in organisms through food chain and induce acute and

412

chronic toxicity. The contaminants may show their toxicity by interacting with the

413

biological macromolecules and affecting the structure as well as function of the

414

macromolecules after entering organisms. Hence, exploring the toxic effects and

415

mechanism of contaminants at molecular level is helpful to perfect the toxicity evaluation

416

system of the contaminants and forecast the related effects on macro organisms or

417

populations. Also, it will be useful to provide reference and technical support for

418

formulating reasonable standard to protect human health. Investigation of DMP−trypsin

419

interaction by a combination of fluorescence, UV–vis absorption, circular dichroism,

17



420

Fourier transform infrared spectroscopic, atomic force microscopy, cyclic voltammetric

421

and molecular docking approaches revealed that DMP can bind with trypsin to form the

422

DMP–trypsin complex. DMP actually interacted with the catalytic residues (His 57, Asp

423

102 and Ser 195) located within the region close to the S1 binding pocket of trypsin and

424

resulted in the inhibition of the enzymatic activity. The binding process was primarily

425

driven by hydrophobic interactions, as the values of ∆H° and ∆S° were found to be 4.16

426

kJ mol−1 and 82.80 J mol−1 K−1, respectively. The binding of DMP to trypsin induced an

427

increase in α-helix and random coil contents and a decrease in β-sheet and β-turn

428

structure, and caused the change of surface morphology of trypsin and its molecular

429

aggregation. Additionally, the DMP binding to trypsin weakened the electrochemical

430

signal of DMP to some extent, which confirmed the formation of the DMP–trypsin

431

complex.

432

Park et al. have reported that exposure of C. riparius to a most commonly used

433

plasticizer Di-(2-ethylhexyl) phthalate (DEHP) induced the decrease of the serine-type

434

endopeptidase (SP) gene expression and the C. riparius SP was putatively classified as a

435

chymotrypsin-like protein which was primarily expressed in the gut where pollutants are

436

frequently encountered.50 As trypsin belongs to the chymotrypsin superfamily of serine

437

endopeptidases, the changes in gene expression might cause some adverse effects on

438

trypsin by DEHP. Our experimental results indicated that DMP, an analogue of DEHP,

439

can bind to trypsin and affect the structure and function of the enzyme under simulative

440

physiological conditions. Although in vitro results could not completely represent what

441

happens in vivo and the fully biochemical properties of DMP, this study will be helpful in

442

comprehensive understanding the mechanism of DMP affecting the conformation and

443

activity of trypsin in biological processes and its toxicity assessment in the environment.

444

ACKNOWLEDGEMENTS 18


Page 18 of 40

Page 19 of 40


445

The authors gratefully acknowledge the financial support of this study by the National

446

Natural Science Foundation of China (Nos. 31460422 and 21167013), the Natural

447

Science Foundation of Jiangxi Province (20143ACB20006 and 20142BAB204001), the

448

Joint Specialized Research Fund for the Doctoral Program of Higher Education

449

(20123601110005), the Program of Jiangxi Provincial Department of Science and

450

Technology (20141BBG70092), and the Research Program of State Key Laboratory of

451

Food Science and Technology of Nanchang University (SKLF–ZZB–201305,

452

SKLF–ZZA–201302 and SKLF–KF–201203).

453

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454

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loop–motion–controlled dynamic correlation as a determinant. Biophys. J. 2005, 89,

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(46) Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Determination of protein

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Biochemistry 1993, 32, 389–394.

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(47) Sandhya, B.; Hegde, A. H.; Kalanur, S. S.; Katrahalli, U.; Seetharamappa, J.

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Interaction of triprolidine hydrochloride with serum albumins: thermodynamic and

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binding characteristics, and influence of site probes. J. Pharm. Biomed. Anal. 2011, 54,

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(48) Li, Y.; Yao, X. J.; Jin, J.; Chen, X. G.; Hu, Z. D. Interaction of rhein with human

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(49) Zhang, G. W.; Wang, L.; Pan, J. H. Probing the binding of the flavonoid diosmetin

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to human serum albumin by multispectroscopic techniques. J. Agric. Food Chem. 2012,

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(50) Park, K.; Kwak, I. S. Expression of Chironomus riparius serine-type

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594

endopeptidase gene under di-(2-ethylhexyl)-phthalate (DEHP) exposure. Comp. Biochem.

595

Phys., Part B 2008, 151, 349–354.

596

597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

25



612

Figure captions

613

Figure1. (A) Effect of DMP on the fluorescence spectra of trypsin (pH 7.4, T = 298 K,

614

λex = 280 nm, λem = 347 nm). c(trypsin) = 1.5 × 10−6 mol L−1; c(DMP) = 0, 1.66, 3.32,

615

4.98, 6.62, 8.26, 9.90, 11.53, 13.16, 14.78 and 16.39 × 10−5 mol L−1 for curves a→k,

616

respectively; Curve x shows the emission spectrum of DMP only, c(DMP) = 1.66 × 10−5

617

mol L−1; Inset shows the molecular structure of DMP. (B) The Stern–Volmer plots for the

618

fluorescence quenching of trypsin by DMP at three different temperatures. (C) log

619

(F0–F)/F against log1/([Qt]–(F0–F)[Pt]/F0) plots of DMP−trypsin system.

620

Figure 2. (A) Cluster analysis of the AutoDock docking runs of DMP with trypsin. (B)

621

The interaction site between DMP (showing stick representation) and trypsin (ribbon

622

form). The white dashed lines stand for hydrogen bonds.

623

Figure 3. Trypsin activity in the absence and presence of DMP at different

624

concentrations. c(trypsin) = 1.5 × 10−6 mol L−1, and c(BAEE) = 7.0 × 10−4 mol L−1.

625

Figure 4. AFM topography image of trypsin (A) and DMP–trypsin complex (B)

626

adsorbed onto mica with tapping mode in air, and the scan size of the image is 1.0 µm ×

627

1.0 µm. (C) and (D) are the three-dimensional graph for (A) and (B), respectively.

628

c(trypsin) = 1.5 × 10−9 mol L−1, c(DMP) = 1.5 × 10−8 mol L−1.

629

Figure 5. Cyclic voltammograms of DMP and DMP treated with different concentrations

630

of trypsin. c(DMP) = 1.0 × 10−5 mol L−1; c(trypsin) = 0, 5.0, 10.0 and 15.0 × 10−7 mol

631

L−1 for curves a→d, respectively.

632

Figure 6. The UV−vis absorption spectra of trypsin in the presence of DMP at different

633

concentrations. c(trypsin) = 1.5 × 10−6 mol L−1; c(DMP) = 0, 1.66, 3.32, 4.98, 6.62, 8.26

634

and 9.90 × 10−5 mol L−1 for curves a→g, respectively; Curve x shows the absorption

635

spectrum of DMP only. c(DMP) = 1.66 × 10−5 mol L−1. (inset) The UV spectra of the

636

DMP–trypsin system in the wavelength range of 260–300 nm.

26


Page 26 of 40

Page 27 of 40


637

Figure 7. Synchronous fluorescence spectra of trypsin with different concentrations of

638

DMP at ∆λ = 15 nm (A) and ∆λ = 60 nm (B). c(trypsin) = 1.5 × 10−6 mol L−1; c(DMP) =

639

0, 1.66, 3.32, 4.98, 6.62, 8.26, 9.90, 11.53, 13.16, 14.78 and 16.39 × 10−5 mol L−1 for

640

curves a→k, respectively. (C) Comparative evaluation of DMP effect on the RSFQ of

641

trypsin.

642

Figure 8. The CD spectra of trypsin in the presence of increasing the amounts of DMP.

643

c(trypsin) = 1.5 ×10−5 mol L−1, the molar ratios of DMP to trypsin were 0:1 (a), 2:1 (b),

644

and 5:1 (c), respectively.

645

Figure 9. (A) The FT–IR spectra of free trypsin (a) and difference spectra

646

[(DMP–trypsin solution)–DMP solution] (b) at pH 7.4 in the region of 1800–1400 cm−1.

647

c(trypsin) = 1.5 ×10−5 mol L−1, c(DMP) = 7.5 ×10−5 mol L−1. The curve-fitted amide I

648

region of free trypsin (B) and its DMP complex (C).

27



Page 28 of 40

Table 1. The quenching constants (KSV), binding constants (K) and relative thermodynamic parameters of the DMP–trypsin system. T (K)

KSV (×10 L mol−1)

Ra

K (×10 L mol−1)

n

Rb

298

4.07

0.9988

3.92

1.00

0.9986

304

3.90

0.9992

4.08

1.16

0.9996

310

3.37

0.9976

4.19

1.33

0.9969

3

3

a

R is the correlation coefficient for the KSV values.

b

R is the correlation coefficient for the K values.

∆H° (kJ mol−1)

28


∆G° (kJ mol−1)

∆S° (J mol−1 K−1)

−20.51 4.16

−21.01 −21.51

82.80

Page 29 of 40


Table 2. The binding free energies of DMP–trypsin interaction with the most favorable docking gesture. Complex DMP–trypsin

Intermol energy –1

Internal energy

Torsional energy

–1

–1

Binding energy

∆Gbinding

(kcal mol )

(kcal mol )

(kcal mol )

(kcal mol )

(kcal mol–1)

–6.31

–0.53

1.19

–5.12

–5.12

29


–1


Page 30 of 40

Table 3. Secondary structural analysis of free trypsin and DMP−trypsin systems from CD data. Molar ratio [DMP]:[trypsin]

α-Helix

β-Sheet

β-Turn

Random coil

(%)

(%)

(%)

(%)

0:1

13.8

43.8

20.6

21.8

2:1

15.9

40.9

20.1

23.1

5:1

17.1

39.1

19.8

24.0

30


Page 31 of 40


Figure 1 (A)

(B)

(C)

31



Figure 2 (A)

(B)

32


Page 32 of 40

Page 33 of 40


Figure 3

33



Figure 4 (A)

(B)

(C)

(D)

34


Page 34 of 40

Page 35 of 40


Figure 5

35



Figure 6

36


Page 36 of 40

Page 37 of 40


Figure 7 (A)

(B)

(C)

37



Figure 8

38


Page 38 of 40

Page 39 of 40


Figure 9 (A)

(B)

(C)

39



TOC Graphic

40


Page 40 of 40

Potential Toxicity of Phthalic Acid Esters Plasticizer: Interaction of

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