Studies on conformational changes induced by binding of pendimethalin with human serum albumin

Chemosphere 243 (2020) 125270

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Studies on conformational changes induced by binding of pendimethalin with human serum albumin Md. Irshad Ahmad a, Angamba Meetei Potshangbam b, Mehjbeen Javed c, Masood Ahmad a, * a b c

Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, UP, 202002, India Manipur University, Department of Biotechnology, Canchipur, Imphal, Manipur, 795003, India Aquatic Toxicology Research Laboratory, Department of Zoology, Aligarh Muslim University, Aligarh, UP, 202002, India

h i g h l i g h t s
Pendimethalin (PND), widely used herbicide.
Its toxic residues persist in environment and can enter humans upon exposure.
HSA is a major transporter for endogenous & exogenous toxicants into man.
Hydrophobic interactions & H bond involved in the binding of PND with HSA.
PND induced functional perturbation in esterase-like activity of HSA.

a r t i c l e i n f o

a b s t r a c t s

Article history: Received 11 July 2019 Received in revised form 28 October 2019 Accepted 29 October 2019 Available online 5 November 2019

Pendimethalin (PND) is a widely used herbicide in modern means of agricultural practices. So, its toxic residues exist extensively in the environment and can enter human body. Therefore, the in vitro interaction of PND with human serum albumin (HSA) has been explored by employing various biophysical, molecular docking and dynamics simulation studies as well as enzyme kinetics to unravel its binding mechanism. The binding constant of the PND-HSA complex was about 104 M1 using Fluorescence quenching spectra. The negative value of Gibbs free energy change (DG0 ¼ 32.0 kJ mol1) indicates this interaction is a spontaneous process. A large negative DH0 and positive DS0 suggests that hydrophobic interactions and H-bonding are involved in the binding process of PND with HSA. The binding of PND can cause conformational and micro-environmental changes in HSA molecule, as shown by various biophysical and molecular dynamics simulation studies. The site marker competition and molecular docking and simulation experiments affirmed that the binding of PND to HSA occurs at or near site I. Esterase-like activity of HSA exhibited decline in the presence of PND revealed the direct involvement of Lys199 of subdomain IIA (Sudlow’s site I) in the binding process. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Circular dichroism Esterase activity Fluorescence Molecular docking Molecular dynamics simulations Pendimethalin

1. Introduction Herbicides are commonly used chemicals for killing or inhibiting the growth of unwanted plants in many agricultural areas (Bermudez-Couso et al., 2011). When these herbicides are applied on crops the major part of them is transported to various places and only a fraction of chemicals stays in the applied area (Salman and Hameed, 2010). Therefore, herbicides can contaminate air, water

* Corresponding author. E-mail address: [email protected] (M. Ahmad). https://doi.org/10.1016/j.chemosphere.2019.125270 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

and soil, which may lead to environmental pollution (Brkic et al., 2008). The human health can be affected by direct contact through air after aerosol application, accidental spills or indirect through consumption of contaminated food and water. When these chemicals are employed directly or indirectly by humans via air, food and drinking water, it may cause harm to human health (Wang and Li, 2011). The toxicokinetic behaviors of herbicides in human body mainly depend on the interactions of these chemicals with carrier plasma protein (Silva et al., 2010; Zhou et al., 2008). The compound pendimethalin (PND) belongs to dinitroaniline herbicide and, extensively used to check broadleaf weeds for lawn care maintenance. India ranks 2nd in Asia and 12th in the World in

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terms of pesticides production (Borichaand and Fulekar, 2009; Government of India, 2002e2007). In U.S the evaluated annual consumption of PND for the year 2014 was ~10 million pounds (U.S. Geological Survey, 2014). PND has been identified as a pollutant in water sources in the Denmark, France, Spain and United States (Asman et al., 2005), (Barba-Brioso et al., 2010), (CORPEP, 2009), (Larson et al., 1999). According to U.S. (E.P.A) pendimethalin is categorized as a possible human carcinogen (Group C) (U.S. EPA, 1999). Earlier reports on agriculture health cohort have been noticed the correlation of PND exposure causes an increased incidence of cancer in animal and human particularly pancreatic, lung and rectal cancers (Alavanja et al. Blair, 2004), (Gabriella et al., 2009), (Hou et al., 2006). In a recent research, the cytotoxic potential of PND with special reference to genotoxicity on Channa punctatus and male rats was described by Ahmad and Ahmad (2015) and Ahmad et al. (2018). In another study the binding properties of PND with DNA have also been reported by Ahmad et al. (2016). In silico study also described the endocrine disrupting potential of pendimethalin which act as antiandrogen for binding in ligand pocket of androgen receptor (Ahmad et al., 2017). Human serum albumin (HSA), a highly abundant transport protein in blood plasma. Its physiological role is transportation of a variety of exogenous and endogenous ligands such as chemical contaminants and drugs (Simpson et al., 2010). Furthermore, these contaminants bind in the active sites of the protein resulting in structural and functional changes and may also cause toxic effects (Guo et al., 2010). Therefore, it is very important to study the mechanism of interaction and toxicity between the ligands and the major carrier protein like HSA. Lee et al. (2017) reported the strong binding of PND to HSA. The molecular mechanism and toxic effects of PND due to binding with HSA are noteworthy features of this protein that need assessment. Still, there is a paucity of articles in this area. Previous paper has practiced finite approaches to study the binding interaction of HSA with PND, and have ignored the molecular mechanism and its toxic effects due to the structural and functional changes in HSA. In the present work, we explored the in vitro mechanistic aspects of interaction between PND and HSA under physiological conditions by multi-spectroscopic, molecular modeling and dynamics simulation techniques. Detailed thermodynamic parameters of PND-HSA complex has been acquired by ITC. The PND induced functional perturbation in esterase-like activity of HSA was also estimated. Further, the effect of PND on conformations and the microenvironment of HSA were also discussed. The specific binding pocket of PND within the HSA molecule was explored using molecular dynamics simulation. This study provides a substantial amount of data to elucidate the transportation mechanisms of PND with HSA and is helpful for understanding the effect on protein function vis- a-vis PND toxicity in vivo.

2.2. UVeVis absorbance spectra measurement The UVevisible spectra of HSA alone and HSA-PND complex were obtained in the range 240e320 nm. The UV absorbance were performed by keeping the constant HSA (5 mM) concentration and titrating the PND (0e16 mM) concentrations at 298 K. 2.3. Steady state fluorescence spectra measurements Fluorescence spectra of HSA were recorded in the range of 300e425 nm after excitation of the Trp-214 at 295 nm. Both the emission and excitation slits were set at 5 nm. Titrations were performed by addition of varying concentrations of PND (0.66e6 mM) while keeping the concentration of HSA constant (4 mM) at these temperatures 298, 303, and 308 K. Respective blanks were subtracted.

2.4. Drug displacement measurements Different site specific markers were used for drug displacement experiment. Warfarin (WAR) is a specific marker for subdomain IIA (Sudlow’s site I) and diazepam (DIA) for subdomain IIIA (Sudlow’s site II). Further, a subordinate pocket has also been placed in between subdomain IIA and IIB for ibuprofen (IBU). The solutions containing HSA and the individual site markers in the ratio of 1:1 at 4 mM were used, and these complexes were titrated by increasing concentrations of PND ranging from 0.66 to 6 mM. The intrinsic fluorescence spectra of HSA were noted at a range of 300e425 nm and the fixed excitation wavelength was 295 nm. Both the emission and excitation slits were set at 5 nm.

2.5. Far-UV CD spectra measurements CD spectra of HSA and HSA-PND complex were obtained at 298 K. The molar ratios of HSA-PND are as follows: 1:0, 1:0.5 and 1:1 and blanks were subtracted. All spectra were noted in the range of 200e250 nm and the scan speed and path length were 100 nm min1 and 0.1 cm respectively. The changes in CD spectra of HSA-PND complex results were measured and analyzed. The acquired signals were expressed in mean residue ellipticity (MRE) in mdeg by using the following equation:

MRE ¼

qobs 10  n  C  l

(1)

where qobs is the observed ellipticity in millidegree (mdeg), c is the molar concentration of HSA, and the path length (l) of cell in cm.

2.6. Calculations of esterase-like activity 2. Materials and methods 2.1. Chemicals and reagents HSA and PND (HPLC-98.8%) were bought from Sigma-chemical Co, USA. Ibuprofen and Warfarin were from Tokyo chemical industry Co, Japan. p-nitrophenyl acetate and Diazepam were purchased from Sisco research laboratory, India and Ranbaxy, India. Stock solution of PND (16 mM) was prepared by dissolving in ethanol. Working solutions of HSA and PND were obtained by diluting the stock solutions in sodium phosphate buffer (20 mM, pH 7.4).

The changes in esterase-like activity of HSA to p-NPA exposure were measured by spectrophotometric method. The constant concentrations of HSA (12 mM) were incubated for 4 h at various HSA/PND (1:0, 1:5, 1:10 and 1:15) molar ratios whereas, varying the substrate (p-NPA) concentrations from 0 to 700 mM. The absorbance of p-NPA products after hydrolysis was recorded at 405 nm for 60s. kcat and Km were calculated by fitting the data according to the following given Michaelis-Menten equations:

vo ¼

Vmax ½S Km þ ½S

(2)

Md.I. Ahmad et al. / Chemosphere 243 (2020) 125270

kcat ¼

Vmax ½E

(3)

where vo and vmax represents the initial and final velocities and kcat and Km are the kinetic-parameter. Whereas, [E] is concentration of enzyme and [S] is the substrate concentration (p-NPA). LineweaverBurk plot were plotted against 1/vo Vs 1/[S] at varying concentrations of p-NPA in the with and without PND.

Km 1 þ 1=vo ¼ Vmax Vmax þ ½S

(4)

2.7. Molecular docking setup Molecular docking experiments were performed to predict the preferred binding mode of PND with human serum albumin by using AutoDock 4.2 tools. The crystal structure of HSA (PDB ID: 1AO6) was derived from PDB. Pendimethalin structure was drawn by using CHEMSKETCH (D/Structure Elucidator, 2012) and mol file format was converted into pdb by OPEN BABEL (O’Boyle et al., 2011) software. The MMFF94 force field was implemented in Avogadro for energy minimization. The rotatable bonds were defined and Gasteiger charges were added to the ligand atoms. For docking calculations, the Lamarckian genetic algorithms (LGA) were implemented. The blind docking was carried out and center grid point set to 126  126  126 Å along X-, Y- and Z-axis with 0.375 Å grid spacing to recognize the binding sites in HSA. The population size was set to100. Each docking run were set to 250 000 for energy evaluations. The minimized and best docking poses were generated using PyMol software (The PyMol Molecular Graphics System, 2003). For the validation of docking protocol, HSA with original cocrystallized ligand (PDB ID: 2BXC) was extracted and re-docking at the same pocket. The low RMSD value of 1.262 Å of the original and re-dock ligand conformation suggest reliable binding prediction and hence docking protocol. The superimpose structure is shown in Fig. S2 (Supplementary material). The final docking figures were generated using PyMol software.

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1995), applied for high-range ionic interactions with grid spacing of 0.8 Å. Further, low-range ionic and Van der waals interactions were smoothly diminished at 0.0 Å. 3. Results and discussion 3.1. Physical changes in HSA upon PND interaction UV absorption spectroscopy is a simple technique which is widely used to evaluate the structural alterations in the proteins and protein-ligand complexes after binding with various ligands (Tunc et al., 2014). The UV spectra of PND-HSA complex and HSA alone are presented in Fig. S1 (a) (Supplementary material). The HSA (native) showed a prominent absorption peak at 278 nm, which is largely, ascribed due to the absorption of Tyr and Trp residues (Pace et al., 1995). The absorption maxima wavelength was increased with apparent blue shift upon subsequent inclusion of increasing concentrations of PND (Dangkoob et al., 2015). The experimental absorbance changes of HSA-PND complexes clearly suggest the possibility of strong binding between PND and HSA. Such a prominent blue shift in the absorption maxima peaks was possibly due to the changes in polarity, which would cause increased hydrophobicity around the tryptophan residue (Rabbani, 2017). 3.2. CD spectra changes in HSA upon PND binding Circular dichroism (CD) is a powerful and most sensitive technique used to investigate the secondary conformational alterations in HSA after binding with ligand. Secondary structure is allied with the biological function of proteins (Zhang et al., 2013). As shown in Fig. S1 (b) (supplementary material), the far-UV CD spectra of free HSA showed two negative bands at 208 and 222 nm, accorded to pp* and n-p* transition, which are the characteristic features of ahelix in proteins (Greenfield, 2006). The aehelical content of HSA increases slightly with the augmenting in the molar ratio of PND (from 1:0.5 to 1:1). These results are also in support of our previous studies which reported an elevation in aehelical content of HSA upon ligand binding (Greenfield, 2006; Rabbani, 2017; Zhang et al., 2012, 2013). The above findings, also suggests that in the presence of PND the secondary structure of HSA got disrupted.

2.8. Molecular dynamic simulation

3.3. Fluorescence quenching of HSA upon addition of PND

The molecular dynamics simulations of the Apo enzyme and HSA-PND complex were performed using Desmond from Schrodinger (Bowers et al., 2006) by implementing OPLS-AA 2005 force field (Kaminski et al., 2001; Jorgensen et al., 1996). The water model (TIP3P) was selected to solvate the Apo enzyme and HSA-PND complex. To generate the simulation systems an orthorhombic periodic boundary box was chosen. For maintaining the charge neutrality suitable of counter ions were added to the system. In order to avoid interactions with its own periodic image of protein complex the distance between the box and wall was kept greater than 10 Å. The Steepest descent method was considered to minimize the potential energies of the systems and so maximum steps set were 5000 and 25 kcal mol1/Å gradient threshold value. Further minimization was done until convergence a criteria of 1 kcal mol1/Å are arrived by Low-memory Broyden-FletcherGoldfarb-Shanno quasi-Newtonian (L-BFGS) method. Default parameters provided in Desmond was applied for equilibrations of pressure and temperature. Subsequent and final 25ns simulation run was carried out at constant temperature and pressure (300 k, 1 atm) with time step of 2fs on the equilibrated systems. The particle-mesh ewald method (PME) (Essmann et al.,

To determine the mechanism of interaction of drug molecules and other ligands with protein, the fluorescence quenching measurement has been used extensively (Lakowicz, 2006). The changes in fluorescence quenching were recorded, and the inner filter effects correction for fluorescence intensity in solution was calculated as follows:

Fcorr ¼ Fobs  eðAex þAem Þ=2

(5)

where corrected (Fcorr) and observed (Fobs) are fluorescence intensities, Aex and Aem are the excitation and emission fluorescence intensities, respectively. The HSA concentrations were maintained at 4 mM. The constant excitation was fixed at 295 nm and the emission and excitation slits were set at 5 nm. The characteristic spectra of HSA with or without PND revealed in Fig. 1 (a). A significant decline in the fluorescence intensity of HSA accompanied by slight blue shift was noticed upon subsequent addition of PND. Increased hydrophobicity and movements of charged groups as well as hydrophobic charges in the microenvironment around the fluorophore(s) are responsible for the blue shift and decrease in the emission maxima (Khanna et al., 1986). These changes observed in

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▪

Fig. 1. (a) Effect of PND on fluorescence spectra of HSA. (b) The SternVolmer plots for HSA fluorescence quenching by PND at ( ) 298, (C) 303 and (:) 308 K.

the fluorescence properties of HSA reflect conformational alterations in the three-dimensional structure of the protein following PND addition and further suggest the binding of PND to HSA. To investigate the mechanism of binding and thermodynamics of PND-HSA complex, quenching experiments were performed at following temperatures 298, 303 and 308 K. Fig. 1 (b) shows the F0/ F vs [Q] plots, and figures were calculated according to the SternVolmer equation 37:

Fo



F ¼ 1 þ Ksv ½Q  ¼ 1 þ Kq to ½Q 

formula:

log

ðFo  FÞ ¼ log Kb þ n log½Q  F

(7)

The results from Table 1 show that Kb values are in 4 orders of magnitude, representing a strong binding interaction between PND and HSA. Similar values of Kb ¼ ~  104 M1 have also been reported earlier for various ligands bound to HSA by means of fluorescence spectroscopy (Kalanur et al., 2010; Varlanand and Hillebrand, 2010; Zhang et al., 2011). The main driving forces involved in the PNDHSA interaction were identified with the help of van’t Hoff plot. While entropy (DS) and enthalpy (DH) change were determined from the intercept of van’t Hoff eq-3 and slope, the Gibbs free energy (DG) change of the phenomenon was governed by eq-4, as given below:

(6)

where F and Fo are the fluorescence intensities in the presence and absence of PND, [Q] is the molar concentration of quencher (PND), kq is the bimolecular quenching rate constant, Ksv is the SternVolmer quenching constant and to is the integral fluorescence lifetime of tryptophan (5.78  109 s). The nature and types of quenching can be monitored closely by assessing its temperature changes. At increasing temperatures will results in dissociation of weakly bound complexes and hence lead to decrease in static quenching whereas faster rate of diffusion causes increased in dynamic quenching (Yue et al., 2014). The results showed that the Ksv values were in the range 104 M1 with increasing temperatures (Fig. 1 and Table 1), implying that a significant interaction between PND and HSA (Rabbani, 2017). The kq values of HSA-PND composite shown in Table 1 fall within this range ~1013 M1 s1, which is considerably larger than the highest dynamic quenching constant (~1010 M1 s1) (Kang et al., 2004). Thus, with increasing temperature decrease in kq and Ksv figures also suggests that the genesis of HSA and PND complexes occurs through static quenching rather than by dynamic quenching of fluorescence.

logKb ¼

DS R



DH

(8)

RT

DG ¼ DH  TDS

(9)

where, R is the universal gas constant and T is temperature in Kelvin. These thermodynamic values have been illustrated in Table 1. The negative value of DG implies that the binding of PNDHSA complex was unprompted (29.81 ± 0.43, 30.04 ± 1.1, 30.27 ± 1.4 K J mol1 at 298, 303 and 308 K respectively). The positive value of DS advocates the involvement of both electrostatic and hydrophobic interactions in the binding reaction. A large negative DH reflects the role of Hbonding associated with the formation of HSA-PND complex. These values also rule out the possibility of electrostatic forces because these forces are usually accompanied by a very small DH value (Ross and Subramanian, 1981). From the above results we can conclude that the formation of PND-HSA complex was spontaneous, hydrophobic in nature wherein H-bonding played an important role for the stabilization of PND-HSA complex (Table 1).

3.4. Binding interaction and thermodynamics of HSA-PND complex Using the data generated as above, the binding constant (Kb) and the binding stoichiometry (n) of the HSA-PND complex were evaluated by help of the following modified SternVolmer

Table 1 Binding energy and thermodynamic parameters of PND and HSA obtained from fluorescence quenching experiments at three different temperatures. Temp (K)

Ksv (  104 M1)

Kq (  1013 M1s1)

Kb (  104 M1)

N

R2

DH

DS

TDS

DG

298 303 308

9.92 ± 0.39 8.35 ± 0.44 7.17 ± 0.29

1.72 ± 0.11 1.44 ± 0.13 1.24 ± 0.13

10.63 ± 1.1 9.85 ± 0.99 8.47 ± 0.67

1.01 1.0 1.02

0.997 0.998 0.996

16.17 ± 1.4

45.78 ± 2.2

13.64 ± 0.14 13.87 ± 0.15 14.10 ± 0.13

29.81 ± 0.43 30.04 ± 1.1 30.27 ± 1.4

DH, DS, TDS and DG are expressed in, KJ mol,1 J K1 mol,1 kJ mol,1 kJ mol1 respectively. The data are the means ± standard deviations of three independent trials.

Md.I. Ahmad et al. / Chemosphere 243 (2020) 125270

3.5. Thermodynamic parameters and binding mode by ITC ITC experiments were performed to determine the more accurate binding energy and thermodynamic parameters of PND-HSA complex. After heat correction of PND dilution effect, the ITC values were evaluated with single binding sites, and results are presented in Fig. 2 and Table 2. The titration of PND upon HSA results in negative heat deflection, representing that the binding is an exothermic process (Fig. 2). The result shows that PND had binding constant (Kb) 12.34 ± 0.13  104 M1. The negative values of Gibbs free energy change (DG0 ¼ 32.0 kJ mol1) suggested that the interaction process was spontaneous for complex formation. A large negative enthalpic change (DH0 ¼ 25.68 ± 1.4 kJ mol1) for the binding of PND to HSA revealed that the binding phenomenon was exothermic. Moreover, positive entropic change (TDS0 ¼ 6.32 kJ mol1) suggests that hydrophobic interactions and H-bonding were taking place in the binding process between PND and HSA. It should be pointed out that the thermodynamic parameters and binding energy values obtained by ITC (Table 2) differed quantitatively from fluorescence spectroscopy (Table 1). These changes can be explained on the basis of assumption in noncalorimetric approaches i.e. DH could not be based on temperature. Furthermore, the distinctness in the calculated values of

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thermodynamic parameters and binding energy achieved by fluorescence spectroscopy and ITC was due to the fact that the fluorescence spectroscopy compute only local changes around the Trp-214 whereas, global change in the property was measured by ITC (Watanabe et al., 2000; Zaidi et al., 2013a).

3.6. Identification of specific PND binding site on HSA Human serum albumin is a globular protein of 585 amino acids. This protein contains 3a-helical domains and all of which have two subdomains (A and B). The principal target regions of ligand binding sites are existed in hydrophobic cavities, deeply interior to the subdomains IIA and IIIA (Beauchemin et al., 2007). Many ligands bind specifically to HSA, for example warfarin (WAR) to subdomain IIA (Sudlow’s site I) whereas, diazepam (DIA) to subdomain IIIA (Sudlow’s site II). A secondary active site has been situated at the interface between subdomain IIA and IIB for ibuprofen (IBU) (Bian et al., 2006), (Ghuman et al., 2005), (Zaidi et al., 2013b). For recognizing the specific binding site of PND on HSA, drug displacement was carried out with distinct HSA-markers in a molar ratio (1:1) as a function of varying PND concentrations. As shown in Fig. 3 (a), addition of WAR into HSA solution resulted in the maximum emission intensity of HSA with the noticeable red shift, and the intensity maxima was lower as compare to HSA alone. The maximum fluorescence emission of the HSA decreased gradually, with slight red shift in the presence of PND. It is suggested that heightened polar region around the tryptophan site (Trp-214), indicating that PND-HSA complexes were hampered by adding WAR (Il’ichev et al., 2002). Fig. 3 (b & c) show the comparative fluorescence emission spectra of HSA-PND complex in the presence and absence of IBU or DIA. The discrepancy occurred in fluorescence intensity with IBU or DIA, of PND-HSA composite was nearly the same as it is without IBU or DIA, which suggested that IBU or DIA did not inhibit the linking of PND in its predictable binding location. Further, to understand the comparative influence of ibuprofen, warfarin or diazepam on the binding of PND to HSA, the quenching constant (Ksv) were investigated using the Stern-Volmer equation (Table 3). It is obvious from the results that the Ksv was significantly reduced with warfarin, whereas little change occurred with ibuprofen or diazepam as compared to isolated HSA. Hence, these results strongly pointing to the fact that major binding site of PND is found within subdomain IIA (Sudlow’s site I) of HSA.

3.7. Synchronous fluorescence

Fig. 2. Isothermal titration calorimetry profile of HSAPND interaction for calculating the binding energy and thermodynamic parameters. Upper panel represents heat changes in each titration. The bottom panel shows an integrated plot of heat liberated per injection. The data points ( ) represent the heats of injection whereas the solid line as the calculated fit of data.

▪

Table 2 Binding energy and thermodynamic parameters of PND-HSA complex obtained by ITC. Binding site (n)

Kb  104 (M1)

DH (KJ mol1)

TDS (KJ mol1)

DG (KJ mol1)

1.1

12.34 ± 0.13

25.68 ± 1.4

6.32

32.00

Synchronous fluorescence is a technique to investigate the alteration of microenvironment around Trp and Tyr residues. The characteristic information of Trp and Tyr residues can be obtained by examining the excitation and emission simultaneously while keeping a constant wavelength interval at Dl ¼ 60 nm for tryptophan and Dl ¼ 15 nm for tyrosine residue. Shifting in location of the emission maxima corresponds to the alteration of the polarity throughout the chromophore molecules (Rehman et al., 2014). Fig. 4, shows the synchronous fluorescence spectra of HSA obtained with different amounts of PND. It was clear from the Fig. 4 (a) that the fluorescence emission peak of HSA showed insignificant alterations with PND, suggesting that the microenvironment of Tyr residues stand unchanged. Whereas, the emission peak of Trp-214 of HSA decreased significantly with 1 nm red-shift by addition of PND, implying altered microenvironment throughout Trp residue [Fig. 4 (b)]. It is deduced that, after linking with PND the polarity throughout Trp-214 of HSA was elevated markedly which could lead to the change in conformation of HSA.

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Fig. 3. Binding site identification of PND on HSA by site-specific markers. PND persuaded fluorescence quenching of HSA:warfarin (a), HSA:ibuprofen (b), and HSA:diazepam (c) preincubated at 1:1 M ratios.

Table 3 The quenching constants for the site marker displacement study of the PNDeHSA system. Site marker of HSA

Ksv (  104)

R2

PND alone IBU þ PND DIA þ PND WAR þ PND

13.64 14.33 11.96 07.55

0.997 0.993 0.997 0.995

3.8. Effect of PND on esterase-like activity of HSA The esterase-like activity of human serum album reveals for the hydrolysis of various compounds, such as amides, esters, p-NPA and phosphates (Kragh-Hansen, 2013). In the detoxication pathway, the esterase enzymes play an important role. The balance between toxic activation and detoxification is a key factor in their toxicity and species sensitivity (Sogorb and Vilanova, 2002). The catalytic esterase-like activity of HSA was examined by comparing the closely related binding sites of drugs/xenobiotics with p-nitrophenyl acetate as substrate. For hydrolysis of p-NPA, the kinetic parameters, such as Kcat and Km, were calculated by fixing initial velocity versus substrate (p-NPA) concentration as

shown in Fig. 5 (a) and the reciprocated values of the initial velocity and substrate (p-NPA) concentration was graphed in Fig. 5 (b). The computed values of all kinetic variables are recorded in Table 4. The crucial amino acid residues of HSA which are included in its esterase activity are Arg 410 and Tyr411 whereas Lys199, Lys 402, Lys 519 and Lys 545 form the pseudo-esterase activity pocket (Watanabe et al., 2000; Liyasova et al., 2010). The kinetic parameters, Km, Vmax and Kcat values for p-NPA hydrolysis at different HSA:PND concentrations (1:0, 1:5, 1:10 and 1:15 M ratios) were calculated with the help of Michaelis-Menten equation given in Fig. 4 and Table 4. The Km values for the hydrolysis of p-NPA of HSA were increased ranging from 227.6 to 428.0 mM at HSA:PND molar ratio of 1:0 to 1:15, respectively. The significant elevation in Km and insignificant alteration in Vmax shows that competitive inhibitor (PND) competes with the active site of HSA substrate. At all molar ratios the Vmax remained the same further suggested that PND interaction takes place in the same catalytic part of HSA. After binding of PND to HSA causes a significant increase in Km which further illustrates the conformational alterations occurs in the secondary and tertiary structure, as is obvious by the far UV-, fluorescence and CD-spectral measurements. The kinetic data were also plotted with the help of Lineweaver-Burk plots to know the mechanism of esterase activity of HSA (Fig. 5). The increase slope in

Md.I. Ahmad et al. / Chemosphere 243 (2020) 125270

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Fig. 4. Synchronous fluorescence spectra of HSA selecting: (a) Dl ¼ 15 nm and (b) Dl ¼ 60 nm. The reaction was run taking HSA (2.4 mM) and PND ranging from (0.33e3.0 mM) in sodium phosphate buffer, pH 7.4 at 298 K.

Fig. 5. (a) Relationship between initial velocity (v0) and substrate molar concentration [p-NPA]. (b) Line-weaverBurk plots of HSA (alone) or different molar ratio of HSA-PND concentartions.

Table 4 Kinetic parameters of HSA and different molecular ratio of HSA:PND. HSA/PND

Vmax (mM

1:0 1:5 1:10 1:15

0.306 0.294 0.301 0.308

s1

)

Km (mM)

Kcat (s1)

Kcat/Km (mM1 s1)

227.6 273.1 357.3 428.0

2.55  102 2.45  102 2.50  102 2.57  102

112.04 89.71 69.96 60.04

all the studied molar ratios with constant intercept shows that the competitive inhibition takes place in esterase-like activity of HSA by PND. Subsequent addition of PND to HSA, a decline in catalytic efficiency (Kcat/Km) suggests that PND binds within the active site of HSA and also exerted conformational change in protein molecule. Hence, possibly affect the target binding site of p-NPA which may lead to expression in esterase activity of HSA. In the light of the above results of drug displacement assay we suggest that inherent esterase activity of HSA essentially requires Lys199 which is positioned within the active site of its subdomain IIA. This could be further confirmed by molecular docking studies.

3.9. Molecular docking of PND and HSA Molecular docking investigations were made to obtain the detailed information about the interactions between PND and HSA. The docking calculation generated 4 conformers of PND having rmsd of <0.5 between them, meaning that they have almost the same conformations and binds to similar residues. Therefore, the best scoring pose from docking was used for further studies. These findings noticeably suggested that PND binds to HSA at subdomain IIA (sudlow’s site I) [Fig. 6 (a)]. PND was predicted to bind specifically within the hydrophobic cavity of subdomain IIA, and is surrounded by the following amino acid residues: Lys199, Arg222, Tyr150, His242, Arg257, Leu238, Ala291 and Lue 219 (Fig. 6). Docking results showed that hydrogen bonding and hydrophobic interactions played a significant role in PND-HSA interactions as shown in Table 1S (Supplementary material). One nitro group of PND form two hydrogen bonds with the Lys199 and Arg222 residues at a distance of 3.01 and 2.99 Å, respectively. Whereas, another nitro group forms two hydrogen bonds with the same Arg257 of 2.86 and 3.30 Å. Further Ala291, Lys 219, Leu238 and

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Fig. 6. Molecular docking study of PND with HSA. (a) Cartoon representation for the structure of HSA along with the location of bound PND. (b) The ligand (PND) structure is represented by purple color. The important active site residues of HSA and PND are shown by ligplot. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Tyr150 bring about clear hydrophobic interactions with PND. These findings were also support by the ITC results, which showing involvement of hydrogen bonds and hydrophobic interactions between PND and HSA molecules. As shown in Fig. 6, the aromatic part of the PND seems to get accommodated in the vicinity of Trp214 residue which further leads us to infer that PND has higher binding affinity to HSA probably near or at site I. The free binding energy to be 7.5 kcal mol1 evaluated by Autodock was comparable to observational values i.e. 7.64 kcal mol1 as obtained by ITC and 7.12 kcal mol1 by fluorescence quenching respectively. Molecular docking results showed that PND occupies the best place in the hydrophobic cavity of Sudlow’s site I, subdomain II A of HSA. Interestingly, these results are in good agreement with the in vitro results (shown in Fig. 3) which revealed that PND binds to sudlows’s site I of HSA. These molecular docking studies lead us to elucidate the exact phenomenon or process of binding of PND and -vis the binding energy and preferable binding site for HSA vis-a PND. 3.10. Molecular dynamics simulations of PND and HSA Molecular dynamic study was carried out to study the behavior of the predicted PND-HSA complex, considering their protein flexibility. The stability, rigidity and micro-environment of the protein complex was also evaluated by computing the Ca root mean-squared deviation (RMSD). The solvent accessible surface (SASA) area and radius of gyration (Rg) were watched over the course of the simulation time. Further, the stability of the molecular interactions predicted by docking method was examined by monitoring the percentage occurrence of the interactions during the simulation period. The reliability of the simulation system was examined by analyzing the RMSDs of the proteins from the starting structure. As shown in Fig. 7a, initially, the C alpha RMSD of apo enzymes and PND-HSA complex showed slight variation, however,

the RMSD value has arrived a relatively stable confirmation after 10 ns, at around 2.5e3 Å and 3e3.5 Å respectively. The radius of gyration (Rg) quantifies the compactness of a protein structure. The Rg of native HSA fluctuate at around 26.5e26.8 Å and maintains throughout the simulation. However, the PND-HSA complexes fluctuate at lower Rg value 26.2 Å, indicating a slightly more compact structure in presence of PND (Fig. 7b). Corroboration were found for other ligands bound to HSA (Kallubai et al., 2015; Malleda et al., 2012; Yaseen et al., 2018). The calculated solvent-accessible surface area (SASA) for the complex showed comparison to that of the native HSA pointing towards the fact that binding of PND ligands has minimal impact on the exposed areas (Fig. 7c). To explore the detail atomic fluctuations of PND-HSA binding sites, the RMSF value of each amino acid residue at the particular site was calculated. A total of ten surrounding residues of the binding pocket of the PND in site I, were monitored which were actively involved in different types of interaction fractions during the 20 ns simulations runs, namely Tyr150, Lys199, Trp214, Arg222, His242, Arg257, Leu238, Ala291, Leu 219 and Arg 218 (Fig. 7d and e). The overlap RMSF values of PND-HSA complex and HSA (Apo) are plotted against residue numbers (Fig. 7e). Our results indicated that very few atomic fluctuations were observed at ligand binding sites I with respect to other domain (Fig. 7d and e). This study also confirmed that the PND, when bound to in site I (subdomain IIA) exhibited pronounced conformational adjustments in the HSA structure, in concurrence with PND conformational adaptation to these binding sites. Further, to assess the effect of non-covalent interaction on the flexibility of HSA protein, hydrogen bond formation during the simulation period was monitored for each trajectory. Initially, PNDHSA complex showed decreased in total hydrogen bonds as compared to the apo form as shown in Fig. S3 (Supplementary material) suggests the changes in conformation of the protein due to binding. However, it increases after 6ns perhaps due to conformational adaptation of PND at the HSA binding site. The average

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Fig. 7. (a) Root mean-squared deviation (RMSD (Å)), (b) Radius of gyration (RG (Å)), (c) Solvent accessible surface area (SASA (Å2)) and (d) root mean square fluctuation (RMSF (Å))(e) The RMSF values and (f) types of interaction fraction values of HSA (Apo) and PND-HSA complex calculated from the simulations (Green: Hydrogen bonds; Light violet: Hydrophobic and Blue: Water bridges). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

hydrogen bonds throughout the simulation were calculated to be 138.62 for the apo protein and 135.23 for PND-HSA complex. The above results suggest that PND changes the microenvironment of HSA upon binding and further leads to the conformational changes. The above results also support the changes observed in CD data (Fig. S1). The insight into the molecular interactions was studied with simulation interactions diagram. The interactions with the ligand are monitored along the simulation period and are categorized accordingly as shown in Fig. 7f. There were about 15 contacts found in between PND-HSA complex with 7 hydrogen bonds in between them, with residues TYR150; LYS199; ARG218; ARG222; HIS242; ARG257 and GLU292. Among them TYR150,

ARG218 and GLU292 have higher interaction fraction. PND also formed hydrophobic interactions with residues TYR150, TRP214, LEU219, LEU238 and ALA291. The rest of the interactions are found to be participated through water-bridging bonds. 4. Conclusion Present work was carried out to examine the process of PNDHSA complex formation. Human serum albumin was an ideal macro-molecule for studying the binding associations among biomolecules, chemicals, drug and fatty acids. The Uv- and CDspectroscopy showed that the binding of PND leads to alteration

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in the secondary structures of HSA. The results from molecular docking and dynamics simulation investigations reveal that the PND molecule sets foot in the hydrophobic cleft of Sudlow’s site I, subdomain IIA near Trp214. Which, form distinct hydrogen bonds with Arg222, Arg257 and Lys199, thus lead to static fluorescence quenching of Trp214. The molecular docking and thermodynamic studies both suggest that linking of HSA and PND molecule is managed by the hydrophobic and hydrogen bond interactions. Radius of gyration (Rg) and root mean square fluctuations (RMSF) indicates that the PND, when bound to site I exhibits conformational changes in microenvironment of HSA by conformational arrangements of the protein, mutually with PND conformational modification to these active sites. This study would help in making the future prospects for better application of herbicides in general and PND analogues in particular, and also provides a deeper insight into the possible mechanism of HSA linked esterase activity alteration with particular reference to the toxicological effects of this herbicide. The inclusive results are in agreement with the experimental inspection that binding of PND accomplish a conformational change in HSA molecule, which can help the better understanding of risks in human caused by PND exposure. Author contribution statements M.I.A. carried out the main research work and wrote the main manuscript text. A.M.P performed simulation studies. M.J. helps during the experimental work and textual work. M.A. provided guidance. All authors reviewed the final draft of manuscript. Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article. Acknowledgements MIA is thankful to University Grants Commission, New Delhi, for the award of Maulana Azad National Fellowship. Financial support from the DBT-RA Program in Biotechnology and Life Sciences is gratefully acknowledged by AMP. The authors acknowledge the Bioinformatics Infrastructure Facility, Manipur University for providing computational facilities. The authors also thank A. M. U., Aligarh, for providing the necessary facilities. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125270. References U.S. EPA, 1999. Persistent bioaccumulative toxic (PBT) chemicals, final rule. Fed. Regist. 64, 58666e58753. Ahmad, M.I., et al., 2018. Pendimethalin-induced oxidative stress, DNA damage and activation of anti-inflammatory and apoptotic markers in male rats. Sci. reports. 8, 17139. https://doi.org/10.1038/s41598-018-35484-3. Ahmad, I., Ahmad, M., 2015. Fresh water fish, Channa punctatus, as a model for pendimethalin genotoxicity testing: a new approach toward aquatic environmental contaminants. Environ. Toxicol. 31, 1520e1529. Ahmad, I., Ahmad, A., Ahmad, M., 2016. Binding properties of pendimethalin herbicide to DNA: multispectroscopic and molecular docking approaches. Phys. Chem. Chem. Phys. 18, 6476e6485. Ahmad, M.I., Usman, A., Ahmad, M., 2017. Computational study involving identification of endocrine disrupting potential of herbicides: its implication in TDS and cancer progression in CRPC patients. Chemos 173, 395e403. Alavanja, M.C.R., et al., 2004. Pesticides and lung cancer risk in the agricultural health study cohort. Am. J. Epidemiol. 160, 876e885. Asman, W.A., et al., 2005. Wet deposition of pesticides and nitrophenols at two sites in Denmark: measurements and contributions from regional sources. Chemos

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