In vitro interaction of organophosphate metabolites with bovine serum albumin: A comparative 1H NMR, fluorescence and molecular docking analysis

Journal Pre-proof In vitro interaction of organophosphate metabolites with bovine serum albumin: A comparative 1H NMR, fluorescence and molecular docking analysis

Vandana Dahiya, Bibin G. Anand, Karunakar Kar, Samanwita Pal PII:

S0048-3575(19)30458-4

DOI:

https://doi.org/10.1016/j.pestbp.2019.10.004

Reference:

YPEST 4466

To appear in:

Pesticide Biochemistry and Physiology

Received date:

17 May 2019

Revised date:

15 October 2019

Accepted date:

15 October 2019

Please cite this article as: V. Dahiya, B.G. Anand, K. Kar, et al., In vitro interaction of organophosphate metabolites with bovine serum albumin: A comparative 1H NMR, fluorescence and molecular docking analysis, Pesticide Biochemistry and Physiology (2019), https://doi.org/10.1016/j.pestbp.2019.10.004

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© 2019 Published by Elsevier.

Journal Pre-proof

In Vitro Interaction of Organophos phate Metabolites with B ovi ne Serum Al bumin: A Comparati ve 1 H NMR, Fluorescence and Molecular Docking Analysis Vandana Dahiya a, Bibin G. Anand b , Karunakar Karc, Samanwita Pala,1 a

Department of Chemistry, Indian Institute of Technology Jodhpur, 342011, India.

b

c

Department of Bioscience and Bioengineering, Indian Institute of Technology Jodhpur, 342011, India.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India.

1

Corresponding author: Samanwita Pal

f

Email: [email protected]

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Tel: +91-0291-2801305 ORCID: Samanwita Pal: 0000-0003-3192-9237

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ABSTRACT – Since the exposure of organophosphate pesticides are known to cause severe health

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consequences, it is important to understand the mo lecular interaction of these pesticides metabolites with vital biomo lecules, especially with the proteins. Here, considering bovine serum albu min (BSA) as a model protein,

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we have examined its interaction with t wo selected organophosphate metabolites, 3,5,6-trich loro-2-pyridinol (TCPy) and parao xon methyl (PM). TCPy and PM are resultant metabolites of two most widely used

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organophosphate pesticides chlorpyrifos and parathion respectively. 1 H NM R line broadening, selective spin-

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lattice relaxat ion rate measurements, saturation transfer difference (STD) NM R of both TCPy and PM were carried out in the presence and absence of BSA. The obtained values of the affin ity index (A), bind ing constants

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(Ka) and thermodynamic parameters indicated strong organophosphates-BSA interaction. Further, fluorescence quenching data on TCPy-BSA and PM-BSA interactions strongly supported the NMR results, besides providing the stoichiometry of these comp lexes. Molecular docking analysis unraveled viab le, strong hydrogen bonds and electrostatic interactions in TCPy-BSA and PM -BSA co mp lexes. This study also revealed substantial timedependent changes in the 1 H NM R intensity of PM in the presence of BSA, wh ich suggests faster degradation of PM with increasing protein concentration during protein-metabolite interactions. The hydrolys is is attributed to the esterase-like action of BSA. The result provides key insights into the direct interaction of the organophosphate metabolites with a biologically important carrier protein, serum albumin. KEYWORDS- NM R spin-lattice relaxat ion, affinity index, saturation transfer difference, fluorescence quenching, molecular docking, hydrolysis ABBREVIATIONS

Journal Pre-proof Nuclear Magnetic Resonance (NMR), 3,5,6-trich loro-2-pyridinol (TCPy), Parao xon methyl (PM ), bovine seru m albumin (BSA), selective relaxation (R1 SE ), non-selective relaxat ion (R1 NS), saturation transfer difference (STD) quenching constant (Kq ), Stern-Volmer constant (Ksv ). 1. Introducti on Organophosphate (OP) pesticides are one of the most widely used pesticides [1,2]. Due to their specified mo lecular structural properties viz. presence of phosphorous and carbonyl mo ieties [3], the OP co mpounds can be easily absorbed through lungs, skin and gastrointestinal tract which in turn creates severe health complications to younger animals and hu man [4]. OP imparts their to xicity by attaching to red blood cells

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(RBC) that allows them to interact with acetylcholinesterase (AChE). Th is interaction causes inhibition of AChE activit ies at synaptic junctions which further results in accumu lation of A Ch E at nerve endings, leading to

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hyper activation of receptors [5]. Consequently, the impact of o rganophosphate pesticides on living systems has

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become a major issue these days [6].

OP pesticides are capable of creat ing acute health hazards due to their presence in soil, air, surface water as

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well as in groundwater in the form o f the parent molecule or the hydrolysis products long after their applicat ion [7,8]. The hydrolysis of organophosphate pesticides yields a dialkyl phosphate and a leaving group [9].

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Although it might be expected that this react ion results in a decreased to xicity, as the leaving group and dialkyl

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phosphate do not inhibit cholinesterase enzymes, there are several reports that confirm the potential to xic effects of these metabolites due to their greater water solubility and mobility [10–12]. Hence, besides the

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Organophosphate pesticides, their degradation products also play crucial roles in polluting the non -target environment [10]. Therefore , it will be equally significant to study the long term impact of these metabolites in tissues of humans and animals [13].

Chlorpyrifos and parathion are the two most common ly used commercial pesticides which eventually degrade into 3,5,6-trichlo ro-2-pyrid inol (TCPy) [14] and parao xon methyl (PM) respectively [15– 17]. The molecu lar structure of these two metabolites is shown in Table S1. Effect of chlorpyrifos and parathion has been investigated thoroughly [18,19]; however, the effect of their metabolites viz., TCPy and PM have gained lesser attention. Nu merous studies suggest that chlorpyrifos and parathion are ext remely to xic to neuronal cells and tissues [20,21]. A quick review of the literature reveals that there are a number of instances where researchers have discussed both in vivo rat model-based experiments as well as in vitro spectroscopic methods to analyze OP pesticides toxicity in terms of the interaction of OP pesticides and metabolites with proteins [22–31].

Journal Pre-proof To the best of our knowledge, a few reports are available to comment on the interaction of OP with proteins. Among various possible pesticide-protein interactions, it is important to study pesticides-serum albu min interaction that controls the free concentration of pesticide inside the body and therefore defines their effects [26,32– 34]. Further, the strong binding affinity of pesticides with plasma protein indicates their lesser diffusion to body tissues leading to lesser toxicity; however, a stronger complexation with plas ma protein may also hinder the excretion process of the metabolite. Therefore in this study, we attempted to analyze the interaction of TCPy and PM with serum albu min by in vitro 1 H NMR methods. We used ligand observed NMR experiments viz. chemical Shift change, NMR spin-

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lattice relaxation and saturation transfer difference (STD) to predict the d istribution and fate of these two OP metabolites with plasma protein. Fluorescence quenching and molecular docking analysis revealed that these

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metabolite-protein co mplexes not only reinforced the NM R results but also offered a better understanding of the complexation in terms of stoichio metry and various mo lecular interactions. BSA is common ly used for

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investigating biological interaction studies due to its commercial availability and homology with hu man serum

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albumin [35,36]. BSA is a mu ltifunctional protein that possesses catalytic properties against a variety of xenobiotic substrate; a property of BSA formally known as an en zy me-like or a pseudo-enzymatic activity [37]. Therefore, hydrolysis study of OP in the presence of BSA is able to provide useful informat ion regarding

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catalytic degradation of these molecules [38]. 1 H NM R besides determining the structure of these harmful

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pesticides under various environmental conditions [39] is also capable of analyzing molecular dynamics.

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Although TCPy and PM interaction with seru m albumin may not directly indicate their to xicity towards body tissues but on the other hand, this can help us to predict their behavior with other proteins. 2. Materials and Methods 2.1. Materials

Bovine seru m albu min (98% purity, nuclease and protease free) was purchased fro m Hi-Media and d issolved in phosphate buffer (40 mM, pH ±7.4). TCPy and PM were purchased from Sig ma -Aldrich. Warfarin and ibuprofen were obtained fro m Sig ma-A ldrich. A ll the substances were used without further purification. D 2 O and DMSO at 99.9% purity were procured from Sigma-Aldrich. 2.2. NMR Measurements BSA was dissolved in deuterated PB (40 mM ) at p H 7.4 whereas TCPy and PM stock solutio ns in deuterated PB were prepared with a solvent combination o f 3:2 D2 O:DMSO [40–43] for NM R measurements. The solvent combination was used due to the very low solubility of PM in water. The reaction mixture was then transferred

Journal Pre-proof to an NMR samp le tube and a series of 1 H NM R spectra were acquired under standard observation conditions as a function of time. 2.2.1. NMR Analysis Change in chemical shift, proton spin-lattice relaxat ion rates, saturation transfer difference (STD) have been monitored to characterize the interaction of OP metabolites and BSA. All the NM R spectra were recorded on a Bru ker Ascend 500 MHz WB NMR spectrometer equipped with a BBFO probehead. Chemical shifts were referenced to the residual solvent signal of HDO at 4.69 pp m. The spin -lattice relaxation rates were measured using the inversion recovery (180°-τ m-90°-acquisition)n pulse sequence with the in itial 180° pulse being either

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non-selective or selective. The sequences employed were preceded by solvent presaturation to suppress residual water signal. All the experiments were carried out at two different temperatures viz., 300 K and 310 K over a

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spectral width of 9.00 pp m for various recovery periods ranging fro m 0-30 s. 32 K data points were collected during the acquisition period with a repetition t ime of 40 s. A total of 8 scans were collected for each

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experiment. The selective inversion was achieved by emp loying a Gauss1.1000 shaped 180° pulse. Relaxat ion

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times were extracted by plotting the experimental signal intensities against the recovery period (τ m). A nonlinear least-square fitting procedure based on the Levenberg-Marquardt algorithm [44] was used to extract the relevant relaxation rates.

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The maximu m experimental error in the relaxat ion rate measurements was 5% throughout the experiments.

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The linear regression analysis of the experimental data was used for affin ity index calculat ion. For affin ity index

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calculation, in all experiments the ligand concentration was 1 mM and the protein concent ration was varied fro m 1- 5 μM in steps of 1 μM whereas the protein concentration was fixed to 5 μM in binding constant calculation with TCPy and PM concentration changed from 1 mM-4 mM. The affin ity index was calculated according to a method proposed by Martini et al. 2010[42]. For STD NMR, the concentration of BSA was kept 10 μM while metabolite’s concentrations were maintained as 400 μM. For the STD NMR studies, the standard Bruker NM R pulse program STDDIFFESGP.3 was imp lemented at 300 K with 2048 scans. The selective saturation of BSA protein peak region was done by using cascades of 50 ms Gaussian pulses. The on-resonance irradiation was performed at 3 ppm; the off-resonance frequency was set at 30 ppm. The protein signal suppression was done using 30 ms spin lock filter. 2.2.2. NMR relaxation for ligand-protein interaction Nuclear Magnetic Resonance (NMR) has emerged as one of the most powerful tools to understand structural and dynamical in formation of molecu le at ato mic level. NM R o ffers both protein -based and ligand-based

Journal Pre-proof methods to analyze ligand-protein interaction [45,46]. NM R measurements based on chemical shift, relaxation, diffusion and magnetization transfer allow one to compare the free and the bound state of the molecule [47]. In the present study NMR non-selective (

) and selective (

) spin-lattice relaxation rates of the test pesticides

have been monitored to analy ze their possible interaction with bovine seru m albumin. Measurement of

and

is a ligand-based NMR approach that allows one to investigate ligand binding to protein by analyzing the change in R1 values due to molecular interaction resulting in change in mo lecular rotational correlation t ime ( c) [40]. The expressions of

and

have been given in Supporting Information fro m equations (1-8)

following the discussions available in various previous reports [48–50]. In all these reports, ligand binding to a

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protein has been modeled as an equilib riu m process as depicted in equation (1) exh ibit ing a fast chemical exchange between the bound and free state of the ligand

M L

ML

pr

(1)

[

with a thermodynamic equilibrium constant

] [ ][ ].

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is one of the most sensitive parameter that reflects ligand-receptor interaction since it is influenced by the

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change in NM R dynamics [51] brought out by such interactions. In case of fast exchange between protein and

and

respectively.

(2)

are experimentally measured selective relaxat ion rates in the bou nd and free state

and

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where

can be defined by equation (2):

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free ligand the

are fractions of the ligand in its free and bound state respectively. If [L]>>[M] then one

equation (3) [52]: [ ] where of

=

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may exp ress the change in selective spin-lattice relaxat ion rate due to ligand binding to protein as expressed in

[ ]

[ ] [ ]

and [L] and [M] are the concentration of the ligand and protein, respectively [40]. Plot

against [M] g ives straight line passing through origin with a slope known as affinity index [ ] which is

used to calculate the global bind ing affinity of the ligand with the protein [53]. The majo r advantage of using affinity index [ ] is that it reveals the strength of specific and non-specific interactions and also tells about the dynamics of the ligand-macro molecu le interaction process. Further, it does not depend on the intrinsic relaxation properties of any proton nuclei. Moreover, the calcu lation does not require any info rmation about the stoichiometry of the interaction [54]. The dimensions of [ ] are M −1 s −1 and the superscript T and subscript L defines temperature and ligand concentration.

Journal Pre-proof Equation (3) can be rewritten as: [ ] [ ] It is understood from equation (4) that a plot of 1/

versus the ligand concentration [L] should be linear.

The relaxation rate of the bound ligand can be evaluated from the slope of the plot while dissociation constant KD can be extracted fro m the intercept. At this juncture, it must be pointed out that differential motional dynamics of various parts of a ligand leads to mult iple correlat ion times. As a consequence of such segmental motions, the dipolar interaction among different portions of the molecule is modulated that can directly

f

influence the selective relaxat ion rate. In order to remove the effect of such differential dynamics of various needs to be normalized by the relaxat ion rate of the free

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parts of the molecule on selective relaxat ion rate,

ligand [54,55,56]. Similarly [ ] is then normalized to the relaxation rate of the free ligand and defined as the

[

pr

[ ]

[ ]

] (M −1 ) calculated by using equation (5): ] [ ]

Pr

Where

e-

‘normalized affinity index’ [

2.3. Thermodynamics of the binding interactions

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In general, the mo lecular forces contributing to the interactions of s mall molecu les with proteins include

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hydrogen bonding, van der Wall’s forces, electrostatic forces and the hydrophobic interactions [57,58]. The values of the thermodynamic parameters viz., change in Gibbs free energy (∆G), enthalpy change (∆H) and

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entropy change (∆S) can assist in defining the types of binding forces involved in the ligand -macro mo lecule interactions. These values were calculated for the test pesticides using equations (6-8)[58]  (  )

(6)

)



(8)

where K: thermodynamic equilibrium constant that is analogous to the binding constants at the temperature of measurements (300 K and 310 K); R: the universal gas constant. K1 and K2 are the binding constants at T1 and T2 respectively. 2.4. Fluorescence measurements BSA stock solution was prepared by dissolving in phosphate buffer (PB: 0.04 M; pH 7.4). The stock concentrations were determined to be 20 M for BSA. PM, TCPy, ibuprofen and warfarin stock solutions were

Journal Pre-proof prepared in DMSO. 10% DMSO was used in final solutions used for measurements. Fluorescence emission of BSA was measured by scanning the solutions using a Perkin Elmer, LS 55 fluorescence spectrophotometer in the range of 250-460 n m using a 1.0 cm quartz cuvette. The excitation and emission slit widths were set to 5 n m throughout the experiments with a scanning speed of 300 n m/ min. An appropriate volu me of metabolites solutions were added to BSA (1.0×10−6 mol·L−1 ) that ranged fro m 0-10×10−6 mol·L−1 and mixed at roo m temperature. The excitation wavelength was fixed at 280 n m. A ll solutions were kept in the refrigerator at 04°C. Analytical grade reagents were used for all experiments and double distilled water was used throughout the experiments.

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Site marker co mpetition experiments were conducted with the use of warfarin and ibuprofen, wh ich primarily binds to site I and site II of BSA respectively.

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2.5. Molecular Docking

The molecular interaction between OP metabolites and BSA was studied by discovery studio 4.0. The

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structure of ligand TCPy and PM (CID23017 and 329757097) were obtained fro m pub -chem. The ligands were

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initially prepared for interaction studies using ‘prepare ligand wizard’ of Discovery studio 4.0 (DS4). X-ray crystal structure of BSA (PDB ID: 4F5S), was obtained fro m Protein Data Ban k (PDB). Prio r to docking , the protein molecu le was prepared through ‘prepare protein wizard’ of DS4. The structures were init ially p rocessed

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by removing water and hetero atoms leaving a nascent BSA mo lecule. The pre-p rocessing and protonation were

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carried out using CHA RM m force fields [59]. The ligands were then docked into a specific receptor site of BSA

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mo lecule as already reported in PDB. The docking steps were carried out for 10 different conformations with 10 different orientations by employing a refined C Docker protocol [60]. The simulations were executed in a Dell precision T5610 workstation [61]. 2.6. Hydrolysis study

The hydrolysis of PM and TCPy was analyzed by using NM R chemical shift. The fraction of hydroly zed PM was determined as a function of t ime by the integration of the p roton signals assigned to the two breakdown products: diethyl phosphate and 4-nitrophenol (4-NP) [62], relative to the corresponding signals from an intact PM molecu le. TCPy remained stable for the same t ime period used for PM hydrolysis study. During the study of metabolite-BSA interaction, it was observed that PM starts degrading after 8 h in the free form wh ile in the presence of BSA the rate of degradation is even faster [63]. A co mplete analysis of hydrolysis rate of both the metabolites has been pres ented in the Result section. Considering the effect of degradation, all the relaxat ion

Journal Pre-proof measurements were carried out within 6 hrs of samp le preparation in case of both free and complexed metabolite. 3. Results and Discussion 3.1. Quantification of ligand-protein interaction To analyze the ligand-protein interaction measurement of chemical shift (δ), line width, non -selective ( and selective (

)

) relaxat ion rates were carried out at different protein and ligand concent rations for both

TCPy and PM. Detailed 1 H chemical shift and line width values of TCPy and PM with and without BSA has been summarized in Table S2 in the Supporting Information.

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The chemical shift change is due to alteration in the chemical and electronic environment of TCPy and PM in the presence of BSA. The protein concentration was used in such a way so that the solution viscosity did not

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change with an increase in BSA concentration. To differentiate the genuine ligand-protein interaction over viscosity effect variable temperature selective and non-selective relaxat ion rate measurements were performed.

with the condition

<

remain ing true which confirms that the changes in ligand relaxat ion

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and

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It was found that with an increase in temperature the ligand in the presence of protein exhib its reduction in both

rate is due to the ligand-protein binding and not because of changes in viscosity of the solution [53]. Tab le S3 in and

decrease with increase in temperature wh ich confirms

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the supporting informat ion revealed that both

that the free metabolites experienced the fast motion condition even in the presence of BSA. The selective and

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non-selective proton relaxation rates with and without BSA provides informat ion about the interaction of these

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two OP metabolites. A representative set of experimental values of

and

of TCPy and Hb proton of PM

with varying BSA concentration has been presented in Table 1. Exactly similar values have been obtained for Ha proton while the methyl proton relaxat ion rates have not been considered as it is also influenced by other relaxation mechanis m besides dipolar interactions. In the absence of BSA, compared to

has a greater experimental value

indicating that both OP metabolites are in the fast re -orientational motion reg ime. On the other

hand, in the presence of protein

becomes larger co mpared to

due to slowing down of ligand’s

dynamics by complex format ion with albu min. These observations are evidence of the interaction of BSA and OP metabolites [64]. The rotational correlat ion times for both free and bound metabolite in case of metabolite BSA complex is given in Table 1 and is calculated by using Equation 1 and 2 given in supporting information. Table 1: K.

and

values for TCPy and PM (1mM ) in the absence and presence of variable concentration of BSA at 300

Journal Pre-proof TCPy

±0.002

0.199

0.208

1

0

±0.001

0.221

0.250

2 ±0.003

0.281

0.307

3 ±0.001

0.283

0.316

4 ±0.001

0.290

0.342

5

0.158 ±0.003

0

0

8.01×10-11

0.001

0.004

4.26×10-10

0.358

0.002

0.009

3.96×10-10

0.007

0.032

3.93×10-10

0.018

0.082

3.86×10-10

0.021

0.09

4.29×10-10

0.219

±0.005

±0.002

0.219

0.220

4.81×10-10

0.223

0.132 ±0.002

0.218±0.002

4.2×10-10

0.130

±0.002

0.123 ±0.001

0.88×10-11

0.220

0.066 ±0.002

(s 1 )

±0.003

0.024 ±0.003

(s 1 )

0.220 0

±0.005

(s 1 )

c # (s)



∆

0.668

0.717

0.859

4.56×10-10

4.72×10-10

f

0.184

(s)

±0.003

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0.197 0

(s 1 )

∆

0.225

±0.001

±0.002

0.225

0.236

pr

(s 1 )

R1 NS

e-

(s 1 )



∆

±0.003

±0.005

0.227

0.239

Pr

(µM)

c #

∆

BSA

PM

5.11×10-10

±0.004

±0.004

±0.001

al



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corresponds to the free ligand proton selective relaxation rate . * defines the observed relaxation rate of ligand protons in the presence of the protein . #Molecular correlation times (c) was calculated from and using eq. 1 and 2 given in supporting information. 3.2. Calculation of Normalized Affinity index [

] and binding constant (Ka )

To examine the strength of TCPy-BSA and PM-BSA interactions, the normalized affin ity index [

] of the

ligand-protein system was examined which symbolizes the strength of the interaction process. In Fig. 1, to extract the normalized affinity indices for both OP metabolite-protein systems, the experimental values of have been plotted against the concentration of BSA and fitted with equation (5). The normalized affinity indices for both the metabolites have been reported in Table 2. A close inspection of the values revealed that TCPy has ca. eleven times stronger affin ity towards BSA compared to PM. Th is indicates that the structure plays an important role in binding affin ity. Structural co mparison of TCPy and PM reveals that TCPy possesses three chlorine ato ms yielding a h igh possibility of halogen bonding with the protein [65,66]. The relevance of halogen bonding in biological systems is well known in the literature that documents halogen bonding between a halogenated ligand and protein backbone as well as side chain having Lewis base

Journal Pre-proof like character [67]. Halogen contains an anisotropic charge distribution with an equatorial negative charge on one region and a positive electrostatic potential on the outer region ( -hole) leading to halogen bonding. In literature, it is well reported that ligands with halogen atoms exh ibit stronger affin ity towards proteins via such halogen bonding [67–69]. Moreover, a co mparison of the normalized affin ity indices with that of vine pesticides-BSA interaction [42] unveiled that both TCPy and PM exhibit an extremely strong binding interaction. Consequently, the bio-availability and the distribution of the OP metabolites are going to be very different compared to that of the vine pesticides reported previo usly. 1.0

oo

0.8

f

PM TCPY

pr

0.4

e-

SE -1

R1N (S )

0.6

Pr

0.2

0.0 1

2

3

4

5

al

0

BSA Concentration (M)

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Fig. 1. Comparison of the linear regression analysis of TCPy and PM (H b) by selective relaxation rate enhancement of TCPy

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and PM against BSA concentration to calculate “normalized Affinity index” [

To analyze the stability of the complexes,

] .

for both TCPy-BSA and PM-BSA were measured at two

different temperatures with varying metabolite concentrations keeping the protein concentration fixed at 5 M. The experimental values of 1/

were then plotted against metabolite concentration as depicted in Fig. 2 (a)

and (b) for TCPy and PM respectively. The binding constant was calculated from the intercept while the relaxation rate of the bound ligand was extracted fro m the slope of the plot following equation (4). In the case of PM, with an increase in ligand concentration the change in 1/

value at 300 K and 310 K is almost

insignificant. One may reason out this observation by stating that with higher metabolite concentration , the rate of hydrolysis of PM increases that affects the relaxat ion rate wh ile higher temperature accentuates the hydrolysis process as well [51]. The parameters thus calculated have been tabulated in Table 2 reports the relaxat ion rates of the bound ligands (

) of the ligand-protein co mp lex at d ifferent temperatures. Moreover, the lower

values of PM at

Journal Pre-proof both the temperatures compared to TCPy once again confirm the weaker nature of the PM-BSA co mp lex. As expected the equilib riu m constants for the two metabolites also fo llo w the same t rend with TCPy showing a 3.5 times greater binding constant at 300 K while 1.4 times greater binding constant at 310 K compared to PM. Table 2: Normalized Affinity Index [

] ;

and equilibrium constant (Ka = 1/K D) for the interaction of BSA and TCPy

at different temperatures (300 K and 310 K).

[

] (300 K) s -1

Compounds

(310 K) s -1

(103) M-1

Ka (300K)

Ka(310K)

L mol -1

L mol -1

181.46±8.72

99.37±8.4

77.34±14.9

3456±27.38

4700±27.47

PM

16.04±2.23

42.15±3.7

59.64±5.7

965±21.74

3286±21.8

R2

0.986

0.989

0.988

0.989

f

TCPy

oo

0.988

pr

Table 3 presents all the relevant thermodynamic parameters for both TCPy -BSA and PM-BSA that were evaluated from the binding constants measured at two different temperatures. Analysis of Table 3 reveals that

e-

for both systems change in Gibb’s free energy shows negative value, whereas the change in enthalpy and the

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change in entropy show positive values. According to the views of Ross and Subraman ian (1981) [70], the negative ∆G values suggested that the binding interaction were spontaneous whereas the positive ∆H values

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showed that the format ion of comp lexes were endothermic. The positive values of ∆H and ∆S obtained for the interaction between metabolites and BSA revealed that hydrophobic interaction plays an important role in

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stabilizing the complexes [71]. Further, the magnitude of ∆G value determines the stability of ligand-protein

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complex o r b inding affin ity of the g iven ligand to the protein which confirms that the binding of TCPy is stronger than PM [72]. These results support that the affinity index analysis for TCPy indicating TCPy as a better binder than PM is correct. Extract ion of thermodynamic parameters has been carried out using two temperatures analysis since PM hydrolysis becomes faster at higher temperature causing experimental determination of relaxation rate ambiguous.

versus TCPy and (b) Plot of 1/∆

versus PM (H b) concentration both were ranging from 1 mM to

e-

Fig. 2. (a) Plot of 1/∆

pr

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Journal Pre-proof

4 mM with fixed protein concentration 5 M for binding constant (K a ) calculation.

Pr

Table 3: Thermodynamic parameters for TCPy -BSA and PM -BSA interaction. ∆G

∆H

∆S

(kJ mol-1)

(kJ mol-1)

(J mol-1K -1)

TCPy

-20.32

23.77

146.98

PM

-17.14

94.74

rn 372.93

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3.3. STD NMR Experiments

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Compounds

To further confirm the binding event between BSA -TCPy and BSA-PM, saturation transfer difference (STD) experiments were carried out. STD is a ligand-based 1 H NMR technique widely used for the detection of ligandprotein interactions [73,74]. This technique is based on the Nuclear Overhauser Effect (NOE) observed for the ligand peaks wh ile irrad iating the protein signals . In STD NM R, in the absence of any interaction between ligand and protein, no ligand signal appears in the STD difference spectrum suggesting non-existence of ligandprotein complex [52,75]. Further, analysis of ligand peak integrals in the STD difference spectrum with respect to STD reference spectrum enables determination of spatial p ro ximity of ligand protons to the protein-producing epitope maps. In the present case, based on the results sown in Fig. 3, we concluded that significant STD effect was observed in both the OP metabolites. However, epitope mapping was not advantageous in case of TCPy having a single proton. On the other hand, the relative STD values for PM protons were obtained on the basis of peak

Journal Pre-proof integral values which confirm that the Hb proton of PM was in close proximity with BSA than Ha and CH3 proton.

pr

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f

The STD data complemented the relaxation analysis present before.

Fig.3. 1H and STD NMR spectra of TCPy (inset) and PM in the presence of BSA. NMR spectra were recorded at 300 K with 2.0 s

d6 at 500 M Hz.

3.4. Fluorescence Quenching Mechanism

Pr

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saturation time, 400 μM concentration of ligand and 1:40 receptor-to-ligand ratio in 40 mM PB (pH = 7.4±0.5), and 30% DM SO-

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Due to high sensitivity, selectiv ity and theoretical bases fluorescence spectroscopy is a popular method for studying ligand-protein interactions. As in Fig. 4 fluorescence intensity of BSA g radually decreased with

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metabolites interaction [14,76].

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increased concentration of both metabolites with a fixed concentration of BSA confirming the BSA -OP

Journal Pre-proof Fig. 4. Fluorescence spectra of BSA in the presence of various concentrations of (a) TCPY; (b) PM . BSA=1.0×10-6mol L-1, TCPY=PM =0-10-6mol L-1, ex=280 nm, pH=7.4, room temperature.

As described in the literature [77,78], the Stern-Volmer equation as given in equation 9 is used to examine the quenching mechanism by analyzing fluorescence data for both metabolites at room temperature.

F0 F  1  KSV Q  1  Kq 0 Q

(9)

where F0 and F are the fluorescence intensities of fluorophore in the absence and in presence of quencher, respectively; [Q] is the concentration of quencher, Ksv is the Stern-Volmer constant; Kq is the quenching rate

f

constant for bimo lecular quenching;  0 : the average fluorescence lifetime of the fluorophore without the

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quencher. Table 4 su mmarized all the relevant data. A closer inspection of the quenching constants clearly indicates that both the metabolites exh ibit static quenching and forms ground state complex with BSA. The

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Stern-Vo lmer constant confirms that TCPY shows high affinity to BSA than PM confirming the NMR analysis

3.4.1. Site marker competitive experiments

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reported in the previous section [79].

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Further to understand the binding site of these OP metabolites, a set of fluorescence quenching experiments in the presence of BSA site markers viz., warfarin for the site I and ibuprofen for site II were performed (Fig. 5). It

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of BSA.

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was concluded fro m the fluorescence site marker co mpetition experiment that both TCPY and PM bind to site I

Fig. 5. Competition of (a) TCPY and (b) PM with warfarin and ibuprofen with BSA fluorescence quenching. BSA=Warfarin=Ibuprofen=1.0×10-6 mol L-1, TCPY=PM =0-10-6 mol L-1, ex=280 nm, pH=7.4, room temperature.

Journal Pre-proof The quenching constant in the presence of site marker was determined for both metabolites. The results illustrated in Table 4 shows that in the presence of warfarin, significant changes observed for both metabolites , whereas in the presence of ibuprofen changes were trivial that confirms both the metabolites bind to Site I. Table 4: Stern-Volmer constant and the quenching constant of BSA with TCPY and PM in the absence and presence of site

S ite-Marker

Ksv(L mol -1)

Kq(L mol -1 s-1)

R2

BSA-TCPY

Blank

2.1×105±0.035

2.1×1013±0.035

0.998

Warfarin

5.7×104±0.019

5.7×1012±0.019

0.991

Ibuprofen

1.5×105±0.016

1.5×1013±0.016

0.995

Blank

4.09×104±0.029

4.09×1012±0.029

0.989

warfarin

2.7×104±0.015

2.7×1012±0.015

0.983

Ibuprofen

3.3×104±0.026

3.3×1012±0.026

0.992

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BSA-PM

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S ystem

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markers

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3.5. Molecular Docking

Insilco docking studies were further carried out for a better understanding of the in vitro interaction of these

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pesticide molecules with BSA besides validating the experimentally obtained NMR and fluorescence data. The mo lecular docking data clearly revealed the strong interaction of TCPY and PM with BSA. It was well known

al

that the BSA mono meric structure has two binding sites Site I and Site II and three hydrophobic subdomain I,

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IIA and IIIA respectively[80]. The observed results shown in Fig. 6 h ighlighted the binding microenviron ment of TCPy. The molecu le was surrounded by two hydrophobic residues PRO117, LYS114. It forms two

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electrostatic bonds with amino acid residues LYS114, A RG185 and one hydrogen bond with LYS114 residue with Cdocker interaction energy of -17.99 kcal/ mo le in the subdomain site I and IIA of BSA. These docking results stated that the hydrophobic and electrostatic interactions with hydrogen bond promote TCPy -BSA interaction. Similarly, the docked co mplex of PM with BSA shown in Fig. 7 actively interacts through hydrogen bonding with three amino acid residues ARG185, LYS114, THR518. Moreover, LYS116, PRO516, LYS114, ARG185 were involved in hydrophobic interaction andARG185, GLU182 in electrostatic interactions int o subdomain IIA and IIIA with Cdocker interaction energy of -31.89 kcal/mole.

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Journal Pre-proof

Fig.6. Interaction of TCPY with the BSA (PBD ID 4F5S): (a) The green colored highlighted region is the Site of Interaction

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(b) displays a snapshot of BSA-TCPY complex with its respective interacting residues.

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Fro m this data it’s evident that hydrogen bonding, hydrophobic interaction and electrostatic interaction play a major ro le in triggering active and strong interaction between BSA and metabolite comp lexes as given in Table

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rn

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5 and 6 [81]. These results are in favor with findings from the thermodynamic study.

Fig. 7. Interaction of PM with the BSA (PBD ID 4F5S): (a) The red colored highlighted region is the Site of Interaction (b) displays a snapshot of BSA-PM complex with its respective interacting residues. Table 5: Summary of interactions between TCPY-BSA Interacting Residues ARG185: N5

Bond Length (Å) 4.5

Interaction Type Electrostatic

Journal Pre-proof PRO117: Cl2

5.0

Hydrophobic

LYS114: 04

5.1

Hydrophobic

LYS114:N5

5.0

Electrostatic

LYS114:O4

1.8

Hydrogen Bond

Table 6: Summary of interactions between PM -BSA Bond Length (Å)

Interaction Type

2.9

Hydrogen Bond

LYS116- C15

4.6

Hydrophobic

LYS114:O4

1.8

Hydrogen Bond

PRO516: C16

4.6

Hydrophobic

THR518: O5

2.5

Hydrogen Bond

LYS114: C16

4.1

Hydrophobic

ARG185:O6

5.5

Electrostatic

GLU182:N8

5.3

Electrostatic

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ARG185: O6

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Interacting Residues

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3.6. Hydrolysis of TCPy and PM

An interesting observation of spectral changes in terms of appearance and disappearance of 1 H NM R peaks of

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OP metabolites was made as a function of time during the relaxat ion studies that triggered our interest to

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understand the fate of these OP metabolites in solution in presence and absence of serum albumin. OP metabolites are generally fo rmed via natural degradation of OP pesticides due to various processes that are

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either hydrolytic or o xidative or chemical o r photolytic in nature [82]. Fig. 8 exh ibits the generation and further degradation of the OP metabolites TCPy and PM into several small products [83,84]. The degradation of TCPy and PM via hydrolysis can be conveniently fo llo wed by mon itoring changes in 1 H NMR peak intensities that in addition also provides valuable informat ion on the possible effect o f seru m albu min on the degradation process. A preliminary measurement of 1 H NM R spectra of TCPy and PM in different solvent systems over time revealed a number of stimulat ing facts related to hydrolysis of these molecules in an aqueous mediu m. Due to sparing solubility of PM in water, the stock solution of PM was prepared in dimethyl sulfo xide (DMSO) while that of TCPy was prepared both in water and in DMSO. To quantify hydrolysis of TCPy and PM in the presence and in the absence of protein, three different sets of solutions were used. In the first case , the metabolites were dissolved in 100% DM SO wh ile in the second and third cases 3:2 ratio of DMSO:D 2 O solutions were used as the solvent. A maximu m of 5 M of serum albumin was added to the third solution.

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(B)

(A)

f

Journal Pre-proof

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Fig. 8. The generation and degradation of the OP metabolites TCPy (A) and PM (B) in to several small products.

Finally, the 1 H NM R spectra with water presaturation over a total time period of 120 hrs were recorded for all

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the three solutions to compare the hydrolysis in different solvent conditions for both the metabolites. The

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comparison of 1 H NM R spectra of the pesticides dissolved in three different solvents revealed that PM was comparably stable in 100% DMSO solvent for a period of 120 h rs while the degradation process was prominent in DMSO:D2 O solvent mixture and became even mo re pronounced in presence of BSA after an init ial period of

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ca. 6 hrs as shown in supporting information (Fig. S1 and S2). On the other hand, TCPy remained stable over

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the complete 120 h rs time period in all the three solutions. A representative stack plot of 1 H NM R spectra of PM

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in the absence and in the presence of BSA (1 M) at different time intervals have been given in Fig. 9. Similar spectra for TCPy have been provided in the Supporting Information ( Fig. S3). The hydrolysis products of PM generated due to P–O bond hydrolysis were identified as p-nitrophenol (NP) and dimethyl phosphoric acid by monitoring the disappearance of the aro matic resonances of PM (8.722 pp m and 7.867 ppm) and the appearance of the aromat ic resonances at 8.281 pp m and 6.678 ppm for NP in the 1 H NMR spectrum. In order to evaluate the hydrolysis rate constant as well as the half-life of the process, 1 H NM R peaks were integrated to calculate the percentage of PM remaining at different time intervals of the measurement.

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pr

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f

Journal Pre-proof

Fig. 9. 1H NM R stack plot of the breakdown of PM in to p-nitrophenol (NP) at 7.4 pH (A) The hydrolysis of PM in absence

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of BSA and (B) The hydrolysis of PM in presence of BSA (1 M ) at different time intervals from 0 hrs, 12 hrs, 24 hrs, 36 hrs, 48 hrs, 72 hrs, 84 hrs, 96 hrs and 5 days at temperature 300 K. t = 0 h, stands for the spectra recorded immediately after

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dissolving PM . * indicates the peak corresponding to the hydrolysis product with significant intensity.

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The experimentally measured integral values for Hb proton of PM were then plotted against time in Fig.10. The data were then fitted using equation (10) considering the hydrolysis process to be a pseudo-first-order reaction

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[85]:

(10)

Here t is the incubation time in minutes, C is the concentration of PM at t ime t, C 0 is the in itial concentration and kobsd in min -1 is the observed rate constant of the reaction. Table 7 summarizes observed rate constant and half-life in the absence and presence of BSA at pH 7.4 and 27 °C. A close inspection of Table 7 revealed that the hydrolysis rate and the half-life in the presence of protein was 12% faster compared to that of the pesticide without protein. Further, with an increase in protein concentration, the hydrolysis rate of PM increased. Fig. S4 in supporting information shows the dependence of the hydrolysis rate of PM on protein concentration measured for 12 hrs of sample preparat ion. A close inspection revealed that in the presence of protein with concentration ranging fro m 1-5 M the metabolite remained stable over an in itial period of 6 hrs. While above 5 M concentration the process of degradation in the presence of protein started even earlier. Therefore, the study clearly ind icates possible detoxificat ion of PM by serum albu min. On the other hand, TCPy does not undergo

Journal Pre-proof any degradation in the presence of protein. We further evaluated the PM hydrolysis rate with a higher protein concentration of 2 M . A representative stack plot of 1 H NM R spectra of PM in the presence of 2 M of protein and a plot of PM Hb proton intensity against time are provided in the Supporting Information by Fig. S5. An increase in hydrolysis rate was clearly observed with an increase in protein concentration signifying molecu lar interaction of PM with BSA with subsequent degradation. However, BSA did not exh ibit a similar effect in case

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Pr

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pr

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f

of TCPy as validated by the 1 H NMR spectra of TCPy recorded in the presence and absence of the protein.

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Fig. 10. Degradation of PM (H b) represented as a function of time in the absence of BSA ( ) and in the presence of 1 M BSA ( ) at pH 7.4 as determined by 1H NM R analysis.

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Table 7: Observed Pseudo-First-Order Disappearance Rate Constant (hydrolysis rate constant) for PM in absence and presence of BSA (1 M ) and half-lives for PM hydrolysis both for free PM and PM bound with BSA. Compounds

Hydrolysis Rate Constant

Half Life (t1/2)

R2

PM

(3.6±0.15)×10-4min1

(1925±81.8) min

0.926

PM +BSA

(4.1±0.17)×10-4min1

(1689±73.6) min

0.985

4. Conclusions Interaction of OP metabolites with proteins plays important ro le in understanding the effect of OP on biological systems. It is crucial to examine the binding ability of serum albumin towards OP since serum albumin possesses high availability in all vertebrates and may offer resistance to the adverse effects of OP. In vitro 1 H NMR relaxat ion measurements exemp lify a powerfu l tool to decipher such interac tion processes in solution state while time -dependent study of 1 H NM R peak intensity change in presence and absence of protein gives valuable information about the stability of OP metabolites. In the present case, 1 H NMR spectra of these

Journal Pre-proof metabolites recorded as a function of time reveal that TCPy remained stable in the solution in the presence of BSA while PM degradation was accelerated in presence of BSA. Our results in terms of the non -selective and selective spin-lattice relaxation rates point out that TCPy is a better binder to BSA, resulting in a more stable complex with the serum albu min with an affin ity constant c.a. eleven times greater than PM due to halogen bonding present in case of TCPy. Consequently, the BSA-TCPy co mplexation prevents the hydrolysis of TCPy and the complex behaves like a reservoir for TCPy in serum. On the other hand, PM with a much smaller affinity constant is not only prone to metabolis m due to its higher free concentration but also has a greater chance to diffuse to other body tissues [29]. Further, these results demonstrate that the structure of a compound

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f

plays major role in deciding the interaction with serum albumin. Acceleration of degradation rate of PM in the presence of BSA indicates a catalyzing effect of BSA that allows it to function as a degradation promoter.

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Hence, the protein can act as a scavenger for PM preventing other proteins fro m undergoing covalent alterations due to interaction with the said OP. Although compared to TCPy, PM engenders a greater risk to hu mans and

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other organisms [86,87] due to its higher free concentration in the serum; it has a greater chance to get

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metabolized by BSA in its free state. A complementary set of d ata consisting of NMR saturation transfer difference, fluorescence quenching and mo lecular docking enabled us to confirm the format ion of metabolite BSA co mplex and its binding interaction with certainty. The quenching data clearly indicated static binding of

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these metabolites with BSA Site I involving hydrogen bonding, electrostatic interaction and hydrophobic

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interaction as major interaction forces as revealed by molecu lar docking. To th e best of our knowledge, the

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present OP metabolites-BSA interaction is reported for the first time. Th is study will be of particular interest in ecotoxico logy of OP metabolites and environ mental risk assessment [88]. The enzy mat ic properties of BSA can be helpful in designing of new b iocatalysts which can operate in a waste-free and environ mentally acceptable way. The detailed informat ion regard ing degradation pathway of OP will provide therapeutic insights for designing new drugs against OP poisoning. Hence, current findings might be helpful in the understanding of the mechanism of toxicity of OP. Acknowl edg ments We th an k M HR D, Govt . o f Ind ia and IIT Jodh pur fo r p rov id ing student fello ws h ip and cont ing ency . The autho rs g rat efu lly ackno wledg e a spect ro met er grant to IIT Jod hpu r fro m DST, Ind ia an d a lso th an k Depart ment of Chemist ry; IIT Jodh pu r fo r ot her experimental facilit ies. W e also ackno wledg e Ms. Bhawna and Mr. Deepak for their constant support during the experiment al work. Notes

Journal Pre-proof This research did not receive any specific grant fro m funding agencies in the public, commercial, or not -forprofit sectors. The authors declare no competing financial interest. References [1]

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Journal Pre-proof Highlights: 

Affinity constant of BSA-TCPy is highly greater than BSA-PM.

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Thermodynamics parameters confirms electrostatic and hydrogen bonding between complexes. Fluorescence quenching data shows static quenching for both metabolites.

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Hydrolysis rate of PM was much faster than TCPy both in absence and presence of BSA.

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PM will greatly diffuse to body tissues with accelerated excretion compared to TCPy.

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