Chemosphere 108 (2014) 26–32

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Study on the interaction of catalase with pesticides by flow injection chemiluminescence and molecular docking Xijuan Tan a, Zhuming Wang b, Donghua Chen a, Kai Luo a, Xunyu Xiong a, Zhenghua Song a,⇑ a Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry & Material Science, Northwest University, 229 North Taibai Road, Xi’an 710069, China b Key Laboratory of Western Mineral Resources and Geological Engineering of Ministry of Education, College of Earth Sciences and Land Resources, Chang’an University, 126 Yanta Road, Xi’an 710054, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The luminescence behaviors of

CAT–pesticides are studied by FI–CL & MD analysis.  The binding and thermodynamic parameters of pesticides to CAT are given.  The relationship of binding ability vs. structure of pesticide to CAT is analyzed.

a r t i c l e

i n f o

Article history: Received 4 December 2013 Accepted 22 February 2014

Handling Editor: S. Jobling Keywords: Catalase Pesticides Interaction mechanism Flow injection chemiluminescence Molecular docking

a b s t r a c t The interaction mechanisms of catalase (CAT) with pesticides (including organophosphates: disulfoton, isofenphos-methyl, malathion, isocarbophos, dimethoate, dipterex, methamidophos and acephate; carbamates: carbaryl and methomyl; pyrethroids: fenvalerate and deltamethrin) were first investigated by flow injection (FI) chemiluminescence (CL) analysis and molecular docking. By homemade FI–CL model of lg[(I0 I)/I] = lgK + nlg[D], it was found that the binding processes of pesticides to CAT were spontaneous with the apparent binding constants K of 103–105 L mol 1 and the numbers of binding sites about 1.0. The binding abilities of pesticides to CAT followed the order: fenvalerate > deltamethrin > disulfoton > isofenphos-methyl > carbaryl > malathion > isocarbophos > dimethoate > dipterex > acephate > methomyl > methamidophos, which was generally similar to the order of determination sensitivity of pesticides. The thermodynamic parameters revealed that CAT bound with hydrophobic pesticides by hydrophobic interaction force, and with hydrophilic pesticides by hydrogen bond and van der Waals force. The pesticides to CAT molecular docking study showed that pesticides could enter into the cavity locating among the four subdomains of CAT, giving the specific amino acid residues and hydrogen bonds involved in CAT–pesticides interaction. It was also found that the lgK values of pesticides to CAT increased regularly with increasing lgP, Mr, MR and MV, suggesting that the hydrophobicity and steric property of pesticide played essential roles in its binding to CAT. Ó 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +86 029 88303798; fax: +86 029 88302604. E-mail addresses: [email protected], [email protected] (Z. Song). http://dx.doi.org/10.1016/j.chemosphere.2014.02.075 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

X. Tan et al. / Chemosphere 108 (2014) 26–32

1. Introduction The massive usage of pesticides in agriculture leads to the widely spread of pesticides into the environment ranging from soil to foodstuff, resulting in continued wildlife and human exposure (Köck-Schulmeyer et al., 2012; Rutsaert et al., 2013; Abrantes et al., 2010). Organophosphate (OP), carbamates (CM) and pyrethroid (PY) are primarily utilized pesticides to protect crops or gardens from insects (Gupta and Milatovic, 2012; Schleier and Peterson, 2011). OP and CM, which are commonly known as anticholinesterase agents, are being phased out of use gradually due to biomagnification or high non-target toxicity, and PY as neurotoxic agents have been widely used nowadays. Because the long-term pesticide exposure might pose the potential risks of health effects on no-targets mainly via the interactions of proteins with pesticides, it is of great importance to investigate the interaction behavior between protein and pesticide at molecular level. The protein–small molecule interaction has become a hot spot in the fields of biology (Azami-Movahed et al., 2013; Agostino et al., 2013), medicine (Khan et al., 2012; Yoshimura et al., 2013; Tan and Song, 2014), environment (Xie et al., 2010; Akiyoshi et al., 2012) and chemistry (Dobretsov et al., 2013; Saha et al., 2013; Wang et al., 2013) in recent decades. Catalase (CAT, MW 240 kDa) presents in the perixisomes of nearly all aerobic cells and serves to protect tissues against damage from hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water without the production of free radicals (Schroeder et al., 1982). It is also one of the first enzymes proposed to be an effective marker of oxidative stress (Livingstone et al., 1993). CAT exists as a dumbbell-shaped tetramer of four identical subunits, each subunit formed by a single polypeptide chain with a heme as a prosthetic group (Reid et al., 1981). The interaction of CAT with small molecule has been studied in vitro by approaches including spectrometry (Zhao et al., 2007; Li et al., 2008; Zhang and Jin, 2008), calorimetry (Zhao et al., 2007) and equilibrium dialysis (Ruso et al., 2001), etc. Yet, no report on the interactions of CAT with pesticides using chemiluminescence (CL) method combined with flow injection analysis (FIA) has been described. In this current work, it was first found that pesticides (including disulfoton, isofenphos-methyl, malathion, isocarbophos, dimethoate, dipterex, methamidophos, acephate, carbaryl, methomyl, fenvalerate and deltamethrin, Scheme S1) obviously quenched the CL intensity from luminol–CAT system and the CL intensity decrements were proportional to the logarithm of pesticides’ concentrations within ranges from 0.3 to 30 nmol L 1. The binding parameters of CAT with pesticides were obtained using the FI–CL model of protein–small molecule interaction, lg[(I0 I)/I] = lgK + nlg[D] (Wang and Song, 2010), giving the binding ability of pesticides to CAT, and the major interaction force was speculated by the thermodynamic parameters of CAT–pesticides association process. It is well known that molecular docking (MD) is a method to predict and understand molecular recognition, find the predominant binding mode and binding affinity between the protein and ligand (Brink and Exner, 2009; Lie et al., 2011), and give a threedimensional structural explanation of the protein–ligand interaction (Gumede et al., 2012; Hou et al., 2013). In this paper, by MD the specific binding sites of pesticides on CAT and the binding mode were obtained, which was a beneficial complementary explanation to the CL results for understanding the interaction mechanism of CAT with pesticides.

2. Material and methods 2.1. Reagents All reagents were of analytical pure grade, and the deionized water used in this work was passed through a Milli-Q system

27

(Millipore, Bedford, MA, USA, 18.2 MX cm). Luminol (Fluka, Biochemika, Switzerland) and CAT from bovine liver (C40, Sigma–Aldrich, St. Louis, MO, USA) were used as received without further purification. Pesticides (OP: disulfoton, isofenphos-methyl, malathion, isocarbophos, dimethoate, dipterex, acephate and methamidophos; CM: carbaryl and methomyl; PY: fenvalerate and deltamethrin) with a concentration of 5 mg mL 1 (ethanol as solvent) were supplied by Material Evidence Identifying Center of Xi’an Public Security Bureau, China. Luminol stock solution of 2.5  10 2 mol L 1 was prepared by dissolving 0.44 g luminol in 100 mL of 0.1 mol L 1 NaOH solution in a brown calibrated flask. The stock solution of 5.0  10 6 mol L 1 CAT was prepared by dissolving 30.0 mg lyophilized powder in 25.00 mL deionized water. All stock solutions of pesticides with the concentration of 1.0  10 4 mol L 1 were prepared in deionized water. Working standard solutions of pesticides were prepared daily from the above stock solutions by appropriate dilution as required. All of the stock solutions were stored at 4 °C. 2.2. Apparatus The FI mode was shown in Fig. S1. The FI–CL apparatus (Xi’an Remex Analysis Instrument Co. Ltd., Xi’an, China) consisted of the sampling system (IFFM-E), the photomultiplier tube (PMT), and the PC with an IFFM-E client system (Remax, Xi’an, China). Poly Tetra Fluoro Ethylene (PTFE) tubing (1.0 mm i.d.) was used to carry and deliver the solutions. A six-way valve with a loop of 100 lL was used for quantitatively injecting luminol into carrier stream. The CL detector contained a flow cell made by coiling 15 cm of colorless glass tube (1.0 mm i.d.) into a spiral disk shape with a diameter of 2.0 cm and placed close to the PMT, and it is important to ensure that the sample compartment and PMT were light tight. The CL signal produced in flow cell was detected by the PMT without wavelength discrimination, with output recorded by computer. The temperature of the solutions was controlled in a water bath (T ± 0.1 °C). The F-4500 fluorophotometer (Hitachi, Kyoto, Japan) was applied to fluorescence measurements of CAT with pesticides (Supplementary Material). 2.3. General procedures Each solution was placed in a water bath to control the temperature. Before the running step was started, the whole flow system was washed using deionized water until a stable baseline was recorded. With a flow rate of 2.0 mL min 1 on each flow line, 100 lL luminol was quantitatively injected into the carrier stream by the six-way valve and then merged with the mixed solution of CAT and pesticide. The whole mixture was thereafter delivered into the flow cell in an alkaline medium to produce CL emission. The CL signal was detected by the PMT with the negative voltage of 700 V. The concentration of pesticide was quantified by the decrement of CL intensity. 2.4. The optimization of CL experimental conditions The effect of luminol concentration on the CL intensity was tested over the ranges of 5.0  10 7 to 5.0  10 4 mol L 1, it was found that the CL signal increased steadily with increasing luminol concentration until 2.5  10 5 mol L 1, and tended to be stable thereafter. Therefore, 2.5  10 5 mol L 1 luminol was chosen as the optimum concentration. Due to the alkaline mediumdependent nature of the luminol CL reaction, NaOH was introduced into the luminol solution to enhance the sensitivity of the system. A series of NaOH solutions over the ranges of 5.0  10 3 to 0.2 mol L 1 was examined, and the concentration of

28

X. Tan et al. / Chemosphere 108 (2014) 26–32

2.5  10 2 mol L 1 NaOH was used in subsequent experiments. The effect of flow rate and the length of mixing tubes on the CL intensity of this FI–CL system were also tested. The flow rate of 2.0 mL min 1 on each flow line was chosen as optima, and the optimum lengths of mixing tubes were 10.0 and 15.0 cm for M1 and M2, respectively.

that the Imax for luminol–O2 system with pesticides has no obvious changes. By taking deltamethrin as a representative example, the Imax for luminol–deltamethrin system (curve 6, dash) only changes from 119 to 115 by 3.4% with the Tmax of 4.8 s.

2.5. MD study

As Fig. 1 shows, it is clear that the Imax for luminol–O2 system in the presence of CAT increases from 119 to 200 by a factor of 1.68, and the corresponding Tmax shortens from 4.8 to 3.8 s, indicating that the electron transfer rate of excited 3-aminophthalate could be accelerated by CAT. According to literature report (Chen et al., 2012), it is speculated that luminol could bind to the heme group of CAT with the electron transfer rate of luminol’s excited oxidation product accelerated by the Fe(III) in the active center of CAT, producing the CL intensity enhancement. It is also clear that the Imax for luminol–O2 system shows no obvious differences in the presence and absence of pesticides, suggesting that the interactions between luminol and pesticides could be ignored. Thus, the CL intensity quenching effect of pesticides on luminol–CAT system might be caused by the interactions of CAT with pesticides. The possible CL mechanism of luminol–CAT–pesticide system can be explained as follows: the acceleration of electron transfer rate of 3-aminophthalate by CAT leads to the complexation enhancement of CL (CEC), giving the enhanced CL intensity from luminol–O2 system; while the CAT–pesticides interactions cause the complexation enhancement of quenching (CEQ), showing the quenched CL intensity from luminol–CAT system.

The MD investigation was carried out by using AutoDock 4.2 suit of programs (http://autodock.scripps.edu). The structures of pesticides with minimum energy were generated by ChemBioOffice 2008. The crystal structure of CAT was taken from the RCSB Protein Data Bank with the entry code of 1TGU (http://www.rcsb.org/pdb/ explore/explore.do?structureId=1TGU). The numbers of grid points were set to 70 Å  70 Å  70 Å, with the grid box spacing of 0.375 Å. The grid center was set as (21.954 Å, 27.411 Å, 42.149 Å) (Teng et al., 2011; Qin and Liu, 2013). Semi-flexible docking simulations were performed using the Lamarckian Genetic Algorithm (LGA) to search for the optimum binding sites of pesticides to CAT. The population size was 150 and the maximum number of energy evaluations was 250,000. The conformations with the lowest binding free energy were used for further analysis by Pymol 1.6.0.0. 3. Results and discussion 3.1. The relative CL intensity–time profile of luminolCATpesticide system The relationship of the relative CL intensity of luminol–CAT– pesticide system vs. time in the FI mode is tested at a flow rate of 2.0 mL min 1 with results given in Fig. 1 (luminol: 2.5  10 5 mol L 1; CAT: 5.0  10 9 mol L 1; carbaryl, isocarbophos and deltamethrin: 5.0  10 10 mol L 1). It can be seen that the maximum CL intensity (Imax) for luminol–O2 system (curve 5, solid) is 119 at the time (Tmax) of 4.8 s; while in the presence of CAT (curve 1), the Imax is 200 with the Tmax of 3.8 s. It can also be seen that the Imax for luminol–CAT system with pesticides (curves 2–4) decreases from 200 to 168, 160 and 131 for carbaryl, isocarbophos and deltamethrin, respectively, with the same Tmax of 3.8 s. It is found

240 200

Relative CL intensity

1

160 6

80

Tmax = 3.8 s

40

Tmax = 4.8 s

0 5.0

10.0

3.3. The analytical performance for the determination of pesticides Under the optimal experimental conditions, a series of standard solutions of CAT was tested by luminol–O2 system, and a series of standard solutions of pesticides was determined by luminol–CAT system. It is found that the relative CL intensity from luminol–O2 system increases with increasing CAT concentration, and the CL intensity increment is proportional to CAT concentrations ranging from 0.3 to 30 nmol L 1, with the linear equation of DI = 13.9CCAT + 10.7 (R = 0.9966). In the presence of pesticide, the CL intensity decrement is proportional to the logarithm of pesticide concentration within ranges of 0.01–3.0 nmol L 1, following the general equation of DI = AlgC + B (R > 0.99, herein, the slope A represents the determination sensitivity of pesticides by luminol–CAT system). The linear equations, linear ranges and limit of detection (LOD, 3r) were listed in Table 1, showing that the determination sensitivity A of pesticides on luminol–CAT system ranks in order of fenvalerate > deltamethrin > disulfoton > isofenphos-methyl > malathion > isocarbophos > carbaryl > dimethoate > dipterex > methomyl > acephate > methamidophos. 3.4. The interaction parameters and binding modes of CAT with pesticides

120

0

3.2. The possible CL mechanism of luminol–CAT–pesticide system

15.0

20.0

25.0

30.0

Time/s Fig. 1. CL intensity–time profile of different CL systems. Curve 1: luminol–CAT system; curves 2–4: luminol–CAT system in the presence of carbaryl, isocarbophos and deltamethrin, respectively; curve 5: luminol system (solid curve); curve 6: luminol system in the presence of deltamethrin (dash curve). The concentrations of luminol and CAT were 2.5  10 5 and 5.0  10 9 mol L 1, and carbaryl, isocarbophos and deltamethrin were 5.0  10 10 mol L 1, respectively.

By FI–CL model of lg[(I0 I)/I] = lgK + nlg[D] (I and I0 refer to the CL intensity of luminol–CAT system with and without pesticides, respectively, and [D] refers to the pesticide concentration), the binding parameters of CAT with pesticides are listed in Table 2 and Table S1, with plots of lg[(I0 I)/I] vs. lg[D] for disulfoton, carbaryl and deltamethrin as examples shown in Fig. S2. It can be seen that the apparent binding constants K of CAT with OP and CM are at 103–104 L mol 1 level, and with PY are at 105 L mol 1 level, suggesting that the binding affinities of PY to CAT are higher than OP and CM to CAT. The numbers of binding sites n (0.82–0.97, approximately equal to 1.0) reveal that there is one dependant class of binding sites in CAT for pesticides. The binding abilities of pesticides to CAT follow the order: fenvalerate > deltamethrin >

X. Tan et al. / Chemosphere 108 (2014) 26–32 Table 1 Linear equation with of pesticide’s concentration range by luminol–CAT CL system.a Pesticides disulfoton isofenphos-methyl malathion isocarbophos dimethoate dipterex acephate methamidophos carbaryl methomyl fenvalerate deltamethrin a b

Linear equations DI = AlgC + B

Rangesc (nmol L

DI = 34.1lgC + 371.4 DI = 29.9lgC + 324.8 DI = 20.0lgC + 213.3 DI = 17.1lgC + 189.9 DI = 15.2lgC + 187.3 DI = 13.2lgC + 140.4 DI = 11.5lgC + 132.4 DI = 11.1lgC + 124.1 DI = 15.6lgC + 170.5 DI = 11.6lgC + 141.3 DI = 37.3lgC + 406.8 DI = 36.2lgC + 404.7

0.03–3.0 0.03–3.0 0.03–3.0 0.01–1.0 0.03–3.0 0.01–3.0 0.03–3.0 0.03–3.0 0.01–3.0 0.01–3.0 0.03–1.0 0.03–1.0

1

)

LODs (nmol L

Rb 1

)

0.01 0.01 0.01 0.003 0.01 0.003 0.01 0.01 0.003 0.003 0.01 0.01

0.9975 0.9992 0.9968 0.9983 0.9961 0.9979 0.9980 0.9970 0.9957 0.9955 0.9971 0.9967

Each result is the average of five separate determinations. R: correlation coefficient.

Pesticides

K (L mol 1) FI–CL/FQ/MD

n FI–CL/FQ

disulfoton isofenphos-methyl malathion isocarbophos dimethoate dipterex acephate methamidophos carbaryl methomyl fenvalerate deltamethrin

6.58  104/6.66  104/6.54  104 5.95  104/6.07  104/6.05  104 3.41  104/3.53  104/3.38  104 2.54  104/2.69  104/2.35  104 8.81  103/8.92  103/8.77  103 7.99  103/8.11  103/8.01  103 6.31  103/6.18  103/5.90  103 4.71  103/4.65  103/4.53  103 3.73  104/3.84  104/3.53  104 4.84  103/4.81  103/5.33  103 9.11  105/9.19  105/9.35  105 5.35  105/5.56  105/6.80  105

0.92/0.95 0.92/0.94 0.91/0.92 0.90/0.91 0.88/0.90 0.87/0.89 0.84/0.83 0.82/0.81 0.91/0.93 0.82/0.82 0.97/0.98 0.95/0.96

The binding parameters were results at 298 K.

disulfoton > isofenphos-methyl > carbaryl > malathion > isocarbophos > dimethoate > dipterex > acephate > methomyl > methamidophos, which is generally similar to the order of A values, indicating that pesticide with a higher determination sensitivity may achieve a stronger binding ability to CAT. The enthalpy change (DH), entropy change (DS) and binding free energy change (DG) of CAT with pesticides are obtained using the Van’t Hoff equation of ln K = –DH/RT + DS/R (Lakowicz, 2006). The thermodynamic parameters of pesticides binding to CAT are listed in Table 3 and Table S2, with plots of ln K vs. 1/T of CAT with methamidophos, acephate, isocarbophos, carbaryl and deltamethrin as examples are shown in Fig. S3. It is shown that for the strong

Table 3 Thermodynamic parameters of CAT–pesticides by FI–CL/FQ/MD.a

a

Pesticides

DH (kJ mol 1) FI–CL/FQ

DS (J mol FI–CL/FQ

disulfoton isofenphos-methyl malathion isocarbophos dimethoate dipterex acephate methamidophos carbaryl methomyl fenvalerate deltamethrin

14.47/14.46 14.35/14.31 12.14/12.35 45.17/45.07 10.38/10.50 10.01/10.18 –28.35/–29.68 –25.99/–26.00 36.40/35.31 6.86/7.06 25.59/25.07 14.61/14.98

141.68/141.60 139.18/139.10 128.71/128.63 235.83/234.45 110.17/110.25 108.09/109.83 –22.50/–26.85 –17.46/–17.05 210.52/186.99 95.62/94.20 199.96/198.13 159.04/160.05

The shown DG values were results at 298 K.

1

K

polar methamidophos and acephate, the signs for DG, DH and DS are negative, suggesting that the binding process is spontaneous and exothermic, with hydrogen bond and van der Waals force as the main driving force according to Ross theory (Ross and Subramanian, 1981). For hydrophobic pesticides, the signs for DG and DH are negative and positive, respectively, indicating that the binding process is spontaneous and endothermic. The positive DH and DS reveals that the hydrophobic interaction force plays a major role in hydrophobic pesticides binding to CAT. Using the intrinsic fluorescence of CAT with kEm/kEx of 280 nm/ 340 nm, fluorescence quenching (FQ) method (Lakowicz, 2006; Manna and Chakravorti, 2013; Roy et al., 2013) is also employed to study the interactions of CAT (5.0 lmol L 1) with pesticides (from 1.0 to 70 lmol L 1). Results show that the K and n (Table 2 and Table S1), and DH, DS and DG (Table 3 and Table S2) are extremely close to those from FI–CL analysis. Apparently, the proposed FI–CL analysis shows higher sensitivity at least two orders of magnitudes than the FQ method. 3.5. CAT–pesticides binding investigation by MD

Table 2 Binding parameters of CAT–pesticides by FI–CL/FQ/MD.a

a

29

1

) DG (kJ mol 1) FI–CL/FQ/MD –27.49/–27.52/–27.47 –27.24/–27.29/–27.28 –25.86/–25.95/–25.83 –25.13/–25.27/–24.95 –22.50/–22.54/–22.52 –22.26/–22.30/–22.27 –21.68/–21.63/–21.52 –20.95/–20.92/–20.86 –26.08/–26.15/–25.50 –21.02/–21.01/–21.27 –34.00/–34.02/–34.12 –32.68/–32.78/–33.33

As we know, CAT consists of four identical subunits each with a heme situating in the active site which is accessible from the CAT surface through a main channel about 25–55 Å in length (Gouet et al., 1995; Bravo et al., 1999; Putnam et al., 2000; Diaz et al., 2004), and the main channel leads the ligand to the active site (Gouet et al., 1995). The MD results of pesticides to CAT are given in Tables 2 and 3 and Tables S3 and S4. It is found that all pesticides bind to the CAT cavity which locates among the four subdomains of CAT, with disulfoton, carbaryl and deltamethrin to CAT as examples shown in Fig. 2. For the hydrophobic pesticides to CAT, it is found that the binding cavity are mainly formed by hydrophobic residues ILE68, PRO69, PRO367, LEU365 and PRO390 (Table S3), suggesting that hydrophobic interaction is the dominant stable force for hydrophobic pesticides to CAT. It is also found that the numbers of hydrophobic residues involved in the interactions for hydrophobic OP to CAT are 13, 12, 10, 9, 8 and 6 for disulfoton, isofenphos-methyl, malathion, isocarbophos, dimethoate and dipterex, respectively (Table S3), following the same monotonic decreasing order of binding constants K. Similarly, the numbers of hydrophobic residues involved in CM and PY to CAT show the same trends as OP to CAT. Hydrogen bond (H-bond) is another binding force in the interactions of CAT with hydrophobic pesticides except deltamethrin (Table S4), with residues ARG65, ARG362 and HIS363 in different chains contributing to the H-bonds. For the hydrophilic acephate and methamidophos to CAT, docking results show H-bonds mainly contribute to the stabilities of CAT–acephate/methamidophos complexes. It is clear that the numbers of H-bonds are 3 and 2 for acephate and methamidophos to CAT, according well with the sequence of binding constants K from FI–CL analysis. The binding constants K (Table 2) and binding free energies DG (Table 3) for CAT pesticides interactions well agree with the results obtained by the proposed FI–CL analysis, following the same order of fenvalerate > deltamethrin > disulfoton > isofenphos-methyl > carbaryl > malathion > isocarbophos > dimethoate > dipterex > acephate > methomyl > methamidophos. 3.6. The relationship of structure–binding ability of pesticide to CAT It is interesting observed that the structural differences of pesticides show significant influence on their binding abilities to CAT. For hydrophobic OP pesticides, the dipterex with double bond between O and P has the lowest binding ability to CAT with the K of 7.99  103 L mol 1, and the disulfoton with double bond between S

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X. Tan et al. / Chemosphere 108 (2014) 26–32

disulfoton

A

a

b

B carbaryl

C

deltamethrin

c

Fig. 2. Docking modes of CAT binding to pesticides. For (a)–(c), the residues of CAT are represented using line model and the pesticides using stick model (Green, blue, yellow and red color refer to chain A–D of CAT). The pesticides in each figure are (a): disulfoton, (b): carbaryl, (c): deltamethrin, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

and P give the highest K of 6.58  104 L mol 1 to CAT. Comparing with the K of disulfoton to CAT, the two ester side chains at S connecting to the P through single bond in malathion decreases the K by 48.2%, and the additional amino bond in dimethoate decreases the K by 86.6%; while the substitution of N for O which connects to the P via single bond and the additional phenyl group in isofenphos-methyl decrease the K by 9.6%, and the isocarbophos without the two –CH3 moieties at N atom further decreases the K by 61.4%. For hydrophilic OP pesticides, it is found that the introduced acyl group in acephate causes an additional H-bond between acephate O 8 and A/HIS363 H of CAT with length of 1.9 Å, leading to the K 1.34-fold that of methamidophos. For CM pesticides, the hydrophobic naphthalene increases the K 7.7 times to that of methomyl. For PY pesticeds, the phenyl group in fenvalerate increases the K 1.7 times to that of deltamethrin.

Table 4 Physicochemical parameters of the studied pesticides.a Pesticides

lgP

Mr. (g mol

disulfoton isofenphos-methyl malathion isocarbophos dimethoate dipterex methamidophos acephate carbaryl methomyl fenvalerate deltamethrin

4.06 3.84 2.38 2.09 1.37 0.48 0.78 0.85 2.34 0.60 6.55 6.42

274.40 331.37 330.36 289.29 229.26 257.44 141.13 183.17 201.22 162.21 419.90 505.20

1

)

MR (cm3)

MV (cm3 mol

72.73 87.26 77.51 73.34 54.46 46.95 31.45 40.82 59.03 41.19 116.44 116.01

233.27 281.81 259.65 226.77 175.72 163.52 109.71 144.98 169.99 137.97 346.76 316.74

1

)

a The predicted data are from ChemSpider generated using the ACD/Labs’ ACD/ PhysChem Suite.

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X. Tan et al. / Chemosphere 108 (2014) 26–32

7

7

lgK = 0.0063 Mr + 2.72

lgK = 0.30 lgP + 3.77

6

6

R = 0.973

R = 0.900

5

lgK

lgK

5 4

4

3

3

2 -2

0

2

4

6

2

8

0

120

240

lgP

360

480

600

Mr

7

7 lgK = 0.026 MR + 2.70

6

lgK = 0.0094 MV + 2.47

6

R = 0.969

5

R = 0.936

lgK

lgK

5

4

4

3

3

2

0

30

60

90

120

150

2

0

80

MR

160

240

320

400

MV

Fig. 3. Correlations of lgK vs. physicochemical parameters of pesticides. For (a) to (d), the plots were lgK vs. lgP, Mr, MR and MV, respectively.

3.7. The correlations between lgK and the physicochemical parameters of pesticides The correlations of lgK values vs. the physicochemical parameters of pesticides, including the octanol/water partition coefficient (lgP), relative molecular mass (Mr), molar refractivity (MR) and molar volume (MV) with values listed in Table 4, are analyzed. As shown in Fig. 3, it is clear that the lgK values increase regularly with increasing lgP, Mr, MR and MV giving linear relationships with R >0.90, which suggests the hydrophobic, steric properties and molecular size of pesticides have great impacts on the binding abilities of pesticides to CAT. It is also clear that the slopes of lgK vs. lgP is far higher than lgK vs. Mr, MR and MV, demonstrating the hydrophobicity of pesticides is the very essential factor affecting their binding abilities to CAT, which offers a valuable insight into the toxic mechanisms of pesticides in vivo. 4. Conclusions The interaction of CAT with OP, CM and PY was studied by FI–CL analysis in combination with MD for the first time. The binding constants K of 103–105 L mol 1 and the numbers of binding sites about 1.0 were obtained, and the specific binding sites of pesticides on CAT were given by MD. According to the correlations between lgK and physicochemical parameters, it was suggested that the binding abilities of pesticides to CAT was closely related to their hydrophobic and steric properties, which offered the possibility for speculating pesticides’ action regularity in vivo. Acknowledgments This work was supported by the National Nature Science Foundation of China, China (No. 21275118), the Open Fund from Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, China, and the Northwest University

(NWU) Graduate Innovation and Creativity Funds (No. 10YZZ29), China.

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