Comparative Studies of the Binding of Six Phthalate Plasticizers to

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Comparative Studies of the Binding of Six Phthalate Plasticizers to Pepsin by Multi-spectroscopic Approach and Molecular Modeling Yanqing Wang, and Hongmei Zhang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Oct 2013 Downloaded from http://pubs.acs.org on October 31, 2013

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Journal of Agricultural and Food Chemistry

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Comparative Studies of the Binding of Six Phthalate

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Plasticizers to Pepsin by Multi-spectroscopic Approach and

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Molecular Modeling

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Yan-Qing Wang*, Hong-Mei Zhang

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Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and

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Environmental Protection Yancheng City, Jiangsu Province 224002, People’s

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Republic of China; Institute of Applied Chemistry and Environmental Engineering,

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Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People’s

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Republic of China

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*Corresponding author：：

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Yan-Qing Wang

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E–mail address: [email protected]

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Tel: +86 515 88233188

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Fax: +86 515 88233188

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Abstract

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To explore the binding mechanism of phthalate plasticizers with digestive proteases,

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their effects on conformation and activity of pepsin by multi-spectroscopic approach

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and molecular modeling were investigated. Fluorescence spectra combined with

19

UV-vis and circular dichroism (CD) spectra measurements indicated that the six

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phthalate plasticizers induced the changes of tertiary and secondary structure of

21

pepsin. The solvent polarity of environment around both Trp and Tyr residues on

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pepsin were affected by phthalate plasticizers. By analyzing the fluorescence 1

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quenching and theoretical calculation data, it was concluded that a binding site exists

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for each phthalate plasticizer in pepsin with different binding ability. The hydrophobic,

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hydrogen bonding and π-π sticking interactions were involved in the interactions

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between pepsin and phthalate plasticizers. Moreover, the activity assay indicated that

27

phthalate plasticizers were not powerfully inhibitors or activators for pepsin. These

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studies demonstrated that phthalate plasticizers could cause some negative effects on

29

pepsin. The present studies may provide a way to analyze the biological safety of

30

phthalate plasticizers on digestive proteases or other proteins.

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Keywords: phthalate plasticizers; pepsin; spectroscopy; binding mechanism;

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conformation

33 34 35 36 37 38 39 40 41 42 43 44 2


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INTRODUCTION

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As a class of industrial chemicals, phthalates plasticizers are widely used in

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consumer products such as building materials, food packaging, mouthing toys,

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children's articles, and medical devices which are made of polyvinyl chloride(PVC).1,2

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For example, diethylhexyl phthalate (DEHP) and diisonnonyl phthalate (DINP)

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constitute about 80% of phthalate production in plastic product especially including

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food and beverage packaging and all PVC medical and surgical products.3 Because

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phthalates are additives and not covalently bound to the plastic matrix of PVC, they

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are able to leach from PVC and enter into the environment.4,5

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Therefore, concerns have been raised over the effects of phthalate exposure from

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PVC materials. For example, Fierens et al have studied the effect of cooking at home

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on the levels of eight phthalates in foods. The results have shown that DEHP was the

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most abundant phthalate compound followed by diisobutyl phthalate (DiBP) and

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benzylbutyl phthalate (BBP).6 Staden et al have reviewed some effects of toxic

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phthalates used in plastic-packaged commercial herbal products.7 Currently there are

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limited studies that quantify the danger of phthalates to humans, but the binding

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interaction mechanism between phthalate and serum albumin have been studied in

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order to analysis the detailed physicochemical character of phthalate binding to serum

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albumin.8,9

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Nowadays, human are exposed to thousands of different chemicals each day.6 As

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endocrine-disrupting chemicals, partly phthalates migrate from food contact materials

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into foods during processing or bioaccumulation and transfer through the food 3



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chain.10, 11 When phthalates enter into human stomach, the digestive proteases may be

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the indirectly binding targets. However, little is known about the interaction of

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phthalates with digestive proteases and the effects of these phthalates on the activity

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of proteases. As one of digestive proteases, pepsin is released by the chief cells in the

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stomach to degrade food proteins into peptides by cleaving peptide bonds between

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hydrophobic and preferably aromatic amino acids.12 Because pepsin plays an essential

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role in digestion deconstruction of food, it is often used as an important model of the

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digestive proteases to investigate the interactions of small molecule with proteins.13-15

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In this report, we selected six phthalate plasticizers as the investigated models to study

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the interactions of them with pepsin. The molecular structure of dimethyl phthalate

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(DMP), dibutyl phosphate (DBP), dinoctyl phthalate (DOP), DINP, dicyclohexyl

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phthalate (DCHP) and DEHP were showed in Fig 1. The activity and conformation of

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pepsin and the binding mechanism of above six phthalate plasticizers with pepsin

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were investigated by means of spectroscopic techniques and molecular modeling

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method in order to elucidate the effect of phthalates on the pepsin activity and their

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relationships to human health. Figure 1.

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MATERIALS AND METHODS

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Materials

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Pepsin (from porcine stomach mucosa, Purified Enzymes, ≥3000 NFU/mg) and

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bovine hemoglobin (from bovine erythrocyte, SDS-PAGE purity, ≥95%) were

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purchased from Sangon Company (Shanghai, China) and used without further

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purification. The analysis standards of DMP, DBP, DOP, DINP, DCHP, and DEHP 4


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were obtained from Aladdin Industrial Corporation. The other chemical reagents such

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as citric acid, trichloroacetic acid, H3PO4, NaH2PO4, NaCl, etc. were all of analytical

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purity. The DMP (0.0125 M), DBP (0.0125 M), DOP (0.0125 M), DINP (0.0125 M),

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DCHP (0.0125 M), and DEHP (0.0125 M) solution were prepared in methanol.

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Pepsin solution (10 µM) was prepared in pH 2.00 citric acid buffer solution (0.025 M,

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0.1 M NaCl). The 0.5 wt% of bovine hemoglobin was prepared in pH 2.00 citric acid

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buffer solution. The muriatic acid (reagent grade, 37%) was used to adjust pH of

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buffer solution. The 10 % of trichloroacetic acid was prepared in water. During

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experiment, water was purified with a Millli-Q purification system.

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Spectroscopic Measurements

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UV-vis Absorption Spectroscopy

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The UV-vis spectra of pepsin (10.0 µM) in absence and presence of DMP, DBP,

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DOP, DINP, DCHP, or DEHP were recorded at 298 K on a SPECORD 50 (Jena,

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Germany) equipped with 1.0 cm quartz cells. The range of wavelength was from 250

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to 400 nm.

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Circular Dichroism Spectroscopy

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The CD spectra of pepsin (10.0 µM) in absence and presence of DMP, DBP, DOP,

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DINP, DCHP, or DEHP were made at 298 K on a Chirascan spectrometer (Applied

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Photophyysics Ltd., Leatherhead, Surrey, UK) equipped with a temperature control

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quantum. The cell path length of 1mm was used with the spectra range 190-250 nm.

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The scan speed was set at 20 nm/min with a bandwidth of 1 nm.

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Fluorescence Spectroscopy 5



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All fluorescence spectra of pepsin were recorded on LS–50B Spectrofluorimeter

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(Waltham, Massachusetts, USA) equipped with 1.0 cm quartz cells and a thermostat

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bath. Fluorescence emission spectra were recorded over a wavelength range of

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300-500 nm at an excitation wavelength of 280 nm, the scan speed was set at 500

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nm/min and the slit widths of emission and excitation were set 5.0 nm-5.0 nm. The

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wavelength interval ∆λ (λem-λex) were set at 15 nm and 60 nm to obtain the

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synchronous fluorescence of pepsin in the same experimental conditions as the

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fluorescence emission spectra.

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Molecular Modeling

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The binding models between phthalate plasticizers and pepsin were generated by

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the molecular docking program Autodock 4.2.3.16 The molecular structure of pepsin

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(PDB

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http://www.rcsb.org/pdb.17,18 All water molecules were removed and the polar

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hydrogen and the Gasteiger charges were added at the beginning of docking study.

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The geometries of DMP, DBP, DOP, DINP, DCHP, and DEHP were optimized using

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DFT B3LYP/6-311++ G (d, p) by Gaussian 03.19 The lowest unoccupied molecular

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orbital's energy (ELUMO), the highest occupied molecular orbital's energy (EHOMO), the

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chemical potential (µ), the chemical harness (η), and the volume of molecule were

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obtained by analyzing the geometries data.20 During molecular calculation study, the

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grid box size of pepsin-phthalate plasticizers systems were 100 Å × 100 Å × 100 Å,

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with grid spacing of 0.375 Å. The GA population size was set at 150, the maximum

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number of energy evaluation was set at 2500000 and others used were default

ID

5PEP)

was

obtained

from

RCSB

6


Protein

Data

Bank,

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parameters. Molegro Molecular Viewer software (Molegro-a CLC bio company,

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Aarhus, Denmark) was selected to analysis the docking conformation with the lowest

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binding free energy.21

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Assay of Pepsin Activity

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Ason method was used to detect the pepsin activity.22 The 1 mL of pepsin (30.0 µM)

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was mixed with different volume of 0.008 M DMP, DBP, DOP, DINP, DCHP, or

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DEHP (0.00 mL, 0.05 mL, 0.10 mL, 0.15 mL, 0.20 mL) in 10 mL centrifuge tube.

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Then the different volume of buffer solution (2.00 mL, 1.95 mL, 1.90 mL, 1.85 mL,

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1.80 mL) were dropped into above solution and make a constant volume to 3 mL. The

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pepsin- phthalate systems were set at 37 ℃ for 20 min. Then 2 mL of bovine

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hemoglobin (0.5 wt %) solution was added in above solution. After 20 min, 2 mL of

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10 % trichloroacetic acid was added to terminate the above reaction. The supernatant

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was obtained by centrifuging at 12,000 rpm for 20 min twice and was measured via

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OD 275 nm. The each experiment was repeated three times in order to take the mean.

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The original activity of pepsin is 3000 NFU/mg, and the activity of pepsin in presence

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of DMP, DBP, DOP, DINP, DCHP, or DEHP was obtained by eq (1):

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Activity / (NFU/mg) =3000 ×

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RESULTS AND DISCUSSION

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The Molecular Properties of Phthalate Plasticizers

OD 275(pepsin − phthalate) OD 275(original pepsin)

(1)

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Theoretical calculation about quantitative structure–activity relationship can be

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applied for the prediction of biological activity from chemical structure or

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properties.20 For example, ELUMO represents the ability to obtain an electron and 7



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EHOMO represents the ability to donate an electron. The surface and contour of HOMO

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and LUMO molecular orbital plots of the optimized structures of various phthalates

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were shown in Figure 2. Table 1 listed the ELUMO, EHOMO, µ, η, and the volume of

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molecule for the series of selected phthalates. It can be seen that the charge density

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were mainly accumulated on the benzene ring, and -C=O in the HOMO and LUMO.

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However, difference about the distribution of electric charge between HOMO and

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LUMO existed. The ester groups of phthalates partly influenced the distribution of

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electric charge. Such as DMP and DCHP, the electric charge of them distributed

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evenly over the molecule. However, with the increasing length of carbon chain of

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ester groups, little electric charges distributed in ester groups. In addition, the

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molecular volume of phthalates became bigger with the increasing length of carbon

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chain in ester groups. If the phthalates bind with the amino acid residues of pepsin by

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charge transfer, the benzene ring and -C=O are the main binding groups. The binding

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forces may be π–π sticking or hydrogen bonding. The carbon chain of ester groups

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and the molecular volume of phthalates could affect the binding interactions of

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phthalates with pepsin for steric exclusion. In the light of the weak-interactions of

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phthalates with pepsin, the ester groups may be main contributor of hydrophobic

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binding force. The theoretical calculation data will be important for analyzing the

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following experimental results.

175

Figure 2.

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Table 1.

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Effect of Phthalate Plasticizers on Pepsin Conformation 8


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UV-vis Absorption Spectra

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Because pepsin has many aromatic amino acids including five tryptophan (Trp),

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sixteen tyrosine (Tyr) and fourteen phenylalanine (Phe), it gives an absorption peak at

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about 278 nm coming from the π-π* transition of aromatic amino acids.23 The change

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of this absorption peak is often used as a conformational probe to explore the structure

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changes of protein. As shown in Figure 3, the intensity of absorption peak at 278 nm

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increased with addition of phthalate plasticizer and the absorption maximum took a

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slight blue shift towards lower wavelength region (from 278 nm to 274 nm). These

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results implied that the micro-environmental hydrophobicity of the amino acid

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residues were changed by the binding interactions between pepsin and DMP, DBP,

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DOP, DINP, DCHP, or DEHP. The conformation of pepsin was also changed. Figure 3.

189 190

CD Spectra

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As one of well-known biophysical techniques, CD spectroscopy is often used to

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elucidate the secondary structures of proteins in solution.24 Figure 4 showed the

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effects of six phthalate plasticizers on the far-UV CD spectra of pepsin. As can be

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seen from Figure 4, pepsin had a strong negative band at about 200 nm which

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indicated that pepsin has significant amount of β-sheet conformation and adopts

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predominantly disordered structure.24 With addition of phthalate plasticizers in pepsin

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solution, the shape of spectra did not change significantly, but the negative molar

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ellipticity showed decreases. The decreases in molar ellipticity at 200 nm suggested

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that phthalate plasticizers induced substantial secondary structure of pepsin. 9



200

Furthermore, the phthalate plasticizers with different ester groups caused varying

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degrees of molar ellipticity decreases. From DMP to DINP, the degrees of molar

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ellipticity decreases increased gradually. DEHP induced the strongest decrease

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compared to other five phthalate plasticizers; the main reason may be that DEHP has

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two chain branches in ester groups. The different structure of ester groups in phthalate

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plasticizers not only induce change in molecular volume, but also give them different

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hydrophobic properties. These different molecular structures could change the ability

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of inducing substantial secondary structure of pepsin. Figure 4.

208 209

Fluorescence Spectra

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If the binding interactions of small molecule with protein induced the changes

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around the aromatic amino acid residues in a protein, the fluorescence emission

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behavior of the protein are often affected.25 Since pepsin consists of 5 Trp and 16 Tyr

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residues which are the major contributions of intrinsic fluorescence of pepsin, the

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fluorescence emission intensity and spectra properties are used to analysis the tertiary

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structure of pepsin. It can be obviously seen that the fluorescence intensity of pepsin

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at 344 nm decreased with an increase in phthalate plasticizers concentration (Figure 5).

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Moreover, the occurrence of an isoactinic point at about 425 nm indicated the

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existence of bound and free phthalate plasticizers in the equilibrium of the binding

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system.26 In addition, the phthalate plasticizers with different ester groups caused

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varying degrees of fluorescence quenching.

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induced the smallest degree of fluorescence quenching of pepsin compared to other

Of the six phthalate plasticizers, DMP

10


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five phthalate plasticizers because DMP had smallest chemical potential and

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molecular volume. The fluorescence quenching data also indicated that there were

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binding interactions between pepsin and phthalate plasticizers. The binding ability of

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phthalate plasticizers with pepsin were different each other because of their different

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molecular structure.

227

Figure 5.

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Because the fluorescence emission of pepsin with an excitation wavelength of 280

229

nm does the combination of Trp and Tyr fluorescence, the synchronous fluorescence

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measurements are often used to separate the Trp and Tyr fluorescence and to analyze

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thoroughly the effects of phthalate plasticizers on the molecular microenvironment of

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pepsin. Figure 6 illustrated the synchronous fluorescence spectra of pepsin treated

233

with six phthalate plasticizers, respectively.

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141, 181, 190, 300), which are located in the β-sheet regions of the protein. As

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showed in Fig 6(A-2, B-2, C-2, D-2, E-2, F-2), the synchronous fluorescence intensity

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decreased obviously in presence of phthalate plasticizers. In addition, the λmax of Trp

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residues in pepsin did shift to longer wavelength (red shift), the observed red shift

238

suggested the possibility of conformational changes of pepsin which induced decrease

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in the hydrophobic environment around Trp residues. Except Trp residues, the λmax of

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Tyr residues in pepsin also shifted to longer wavelength in presence of phthalate

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plasticizers. These results indicated that the binding interactions of phthalate

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plasticizers with pepsin induced the changes in the solvent polarity of environment

243

around both Trp and Tyr residues.27 However, DMP and DBP induced the

Pepsin contains 5 Trp residues (Trp -39,

11



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fluorescence quenching of Tyr residues in pepsin, other four phthalate plasticizers

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induced the fluorescence increasing of Tyr residues in pepsin. Figure 6.

246 247

Binding Nature of Phthalate Plasticizers with Pepsin

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Binding Constants and Binding Sites

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By analyzing the UV-vis, CD and fluorescence data, a conclusion was draw that

250

there were existences of binding interactions of phthalate plasticizers with pepsin. In

251

order to analyze thoroughly the binding nature of them, binding constants and binding

252

sites were obtained by calculating the fluorescence quenching data (Figure 5) using

253

Eq(2). 28

254

log

  F0 -F 1 =nlogK A -nlog   F  [Q t ]-(F0 -F )[Pt ]/F0 

(2)

255

where F0 and F are the fluorescence intensities in the absence of phthalate plasticizer

256

and the corrected fluorescence intensity according to Ref [29] in the presence of

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phthalate plasticizer, respectively. [Qt] and [Pt] are the total phthalate plasticizer

258

concentration and the total pepsin concentration, respectively. The plot of log(F0-F)/F

259

versus log (1/ ([Qt] – (F0 – F) [Pt]/F0)) were showed in Figure 7. The binding constants

260

(KA) and binding sites (n) can be obtained from the slope and intercept of the plots in

261

Figure 7. The calculated results were showed in Table 2. It can be seen that the KA

262

values at 298 K increased in the order, KA (DMP) < KA (DCHP) < KA (DBP) < KA

263

(DOP) < KA (DINP) ≈KA (DEHP). In the light of structure-binding ability, the binding

264

ability of phthalate plasticizers with pepsin increased with the increase of molecular

265

volume of phthalate plasticizers. From DMP to DINP, the value of KA obviously 12


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increased with the growth of carbon chain of ester groups in phthalate plasticizers.

267

However, this difference did not affect the value of binding site (n). As also shown in

268

Table 2, the values of n indicated the existence of one binding site in pepsin for DMP,

269

DBP, DOP, DINP, DCHP or DEHP, respectively. Compared with the binding

270

interactions of phthalate plasticizers with serum albumin, the values of KA of pepsin

271

with phthalate plasticizers were lower than those of serum albumin with phthalate

272

plasticizers.8, 9 The difference of protein structures may result in the difference of

273

ligand binding abilities.

274

Figure 7.

275

Table 2.

276

The Binding Forces

277

The phthalate plasticizers consist of aromatic benzene ring, -C=O, and ester groups,

278

which can bind with the amino acid residues of pepsin by different molecular forces.

279

Such as the π-π stacking between benzene ring of phthalate plasticizers and aromatic

280

amino acid residues (Trp, Tyr or Phe), the hydrogen interactions between the -C=O of

281

phthalate plasticizers and pepsin, or the hydrophobic interactions may be involved in

282

the binding interactions of phthalate plasticizers with pepsin. In order to obtain the

283

thermodynamic parameters, eqs (3-5) were used to calculate the enthalpy change

284

(∆H○), the entropy (∆S○) and free-energy change (∆G○):30,31

285

ln

286

∆G Ο = − RT ln K A

( K A )2 ( K A )1

=

∆H Ο  1 1   −  R  T1 T2 

(3) (4)

13



∆H Ο − ∆G Ο T

287

∆S Ο =

288

The calculated results were also listed in Table 2. From the Table, according to the

289

point of view of Ross and Subramanian, there were many interaction forces involved

290

in the binding process.32,33 Firstly, the negative values of ∆G○ indicated that the

291

binding progress of phthalate plasticizers with pepsin were spontaneous. Secondly, the

292

positive values of ∆S○ were the main contribution of the source of ∆G○, which

293

indicated that the hydrophobic interaction was one of main interaction forces. Thirdly,

294

the negative ∆H○ values implied that there were hydrogen bonding in the interaction

295

between pepsin and DMP (DBP, DOP, DINP, or DEHP). The positive ∆S○ and ∆H○

296

values for the binding interaction of DCHP with pepsin indicated that the dinoctyl

297

enhanced the degree of hydrophobic interaction between DCHP and pepsin.

298

3.4. Theoretical calculation of the binding interactions of phthalate plasticizers with

299

pepsin

(5)

300

The best docked results of phthalate plasticizer-pepsin systems were showed in

301

Figure 8. Our results indicated that phthalate plasticizer can bind to pepsin with a

302

similar binding domain. These results implied that phthalate groups played an

303

important role in the binding interactions of phthalate plasticizers with pepsin. The

304

values of the calculated binding energy were -63.861 kJ/mol, -76.311 kJ/mol, -95.982

305

kJ/mol, -72.453 kJ/mol, -85.765 kJ/mol, and -68.280 kJ/mol for DMP, DBP, DOP,

306

DINP, DCHP and DEHP, respectively.

307

DEHP < DINP < DBP < DCHP < DOP. This order was in accordance with the number

308

of amino acid residues taking part in the binding interactions of phthalate plasticizers

The order of binding energy was DMP

Comparative Studies of the Binding of Six Phthalate Plasticizers to

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