Fluorescence spectroscopy, molecular docking and molecular dynamic simulation studies of HSA-Aflatoxin B1 and G1 interactions

Author’s Accepted Manuscript Fluorescence spectroscopy, molecular docking and molecular dynamic simulation studies of HSAAflatoxin B1 and G1 interactions Mohammad Bagheri, Mohammad Hossein Fatemi www.elsevier.com/locate/jlumin

PII: DOI: Reference:

S0022-2313(18)30298-9 https://doi.org/10.1016/j.jlumin.2018.05.066 LUMIN15648

To appear in: Journal of Luminescence Received date: 13 February 2018 Revised date: 23 April 2018 Accepted date: 26 May 2018 Cite this article as: Mohammad Bagheri and Mohammad Hossein Fatemi, Fluorescence spectroscopy, molecular docking and molecular dynamic simulation studies of HSA-Aflatoxin B1 and G1 interactions, Journal of Luminescence, https://doi.org/10.1016/j.jlumin.2018.05.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Fluorescence spectroscopy, molecular docking and molecular dynamic simulation studies of HSA-Aflatoxin B1 and G1 interactions Mohammad Bagheri, Mohammad Hossein Fatemi*

Address: Chemometrics Laboratory, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran

 To whom correspondence should be addressed Tel: +98-113-5302395 and +98-9111124061 Fax: +98-113-5302350

E-mail addresses: [email protected]

1

Abstract The interaction of human serum albumin (HSA) with aflatoxin B1 and G1 has been investigated by fluorescence spectroscopy, molecular docking and molecular dynamic simulation. The results of fluorescence spectroscopy demonstrated

that

the

fluorescence

emission

of HSA

was

quenched

considerably upon the addition of aflatoxin B1 and G1 through a static quenching mechanism. The thermodynamic parameters were calculated using Van’t Hoff equation which indicated that the reaction is spontaneous and hydrogen bonding and van der Waals forces played a leading role in the binding of aflatoxin B1 and G1 to HSA. The binding constants of aflatoxin B1 and G1 to HSA was determined by the modified Van’t Hoff equation. We have also performed molecular docking studies to locate the binding sites, binding mode and to determine of binding constants of aflatoxin B1 and G1 to the HSA. The results of molecular docking was shown that both aflatoxin B1 and G1 binds to subdomain IB of HSA mainly by hydrophobic interaction and hydrogen bonding which they are in good agreement with those obtained by fluorescence study. After performing the MD simulation, the interaction constant values obtained from the docking results were much closer to the experimental value of fluorescence spectroscopy. All the fluorescence spectroscopy, molecular docking and molecular dynamics results have a good correlation with each other. Keywords: fluorescence spectroscopy, molecular docking, molecular dynamic simulation, Aflatoxin, HSA 1. Introduction Binding of different ligands to plasma proteins has vital implications for ligand disposition and action. Action ligand depends on the availability of free ligand in plasma in order to bind to the receptor sites and exerting itself effect. 2

Many ligands bind reversibly to the plasma proteins. Thus, drugs are transported in the circulation, either free, dissolved in the aqueous phase of the plasma, or bounded in the form of complex with the plasma proteins [1, 2]. The protein binds a number of relatively insoluble endogenous compounds such as unesterified fatty acids, bilirubin, and bile acids; and thus facilitates their transport throughout the circulation system [3, 4]. Among the plasma proteins, ligands mostly bind with human serum albumin (HSA) [5]. Human serum albumin is the most abundant carrier protein in plasma and is able to bind a wide variety of therapeutic ligands [4]. Crystal structure analyses determine that HSA include of a single polypeptide chain of 585 amino acid residues in which the single tryptophan 214 residue measured the ligand– binding affinity. HSA molecule has three specific interaction domains, I, II and III, each of which include of two sub-domains A and B possessing common structural motifs (Fig. 1) [6]. Binding of therapeutics to one of these sites can have a significant impact on their pharmacokinetic and pharmacodynamics properties [7]. HSA is capable to binding a wide variety of exogenous systems, and much interest on this protein stems from the fact that it facilitates drug delivery [8]. Aflatoxins which are a category of mycotoxins produced by fungi species Aspergillus flavus, Aspergillus parasiticus and the rare type Aspergillus nomius [9]. These fungi produce several similar compounds of aflatoxin (around 20) but only four are naturally found in food stuffs: aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) [10] (Fig. 1). All aflatoxins absorb maximally at a wavelength centered at 360 nm [11, 12]. Under ultraviolent (UV) light illumination they fluoresce around 425 nm (blue light hence the B designation in aflatoxins B types) and around 450 nm (green-blue light for aflatoxins G types). Both types of Aspergillus flavus and Aspergillus parasiticus produce aflatoxin B (AFB) but aflatoxin G (AFG) is mainly 3

produced by the latter [13]. A number of adverse human health effects have been associated with dietary contamination with afltoxins, including hepatotoxicity and liver cancer. There are some reports above the relation between aflatoxin exposure and both hepatoxicity (aflatoxicosis) and liver cancer [14, 15]. Among above mentioned aflatoxin types, B1 and G1 are more human harmful [16]. Recently, some studies have been conducted on the binding of mycotoxins to HSA by spectroscopic technique and molecular modelling, e.g. Ochratoxin A (OTA) [17], Deoxynivalenol (DON) [18], Citrinin (CIT) [19] and Zearalenone (ZEN) [20]. J. Tao and coworkers study the interaction between Aflatoxin B1 and human serum albumin by fluorescence spectroscopy [21]. Based on obtained results they concluded that the main quenching mechanism between B1 and HSA was a static quenching process. In other work the interaction between the mycotoxin aflatoxin G1 (AFG1) and human serum albumin

was

investigated by molecular docking, fluorescence spectroscopy, 3D fluorescence spectrum, and circular dichroism (CD) under simulated physiological conditions (pH 7.4) by Z. Hong et al. [22]. They reports that the microenvironment of amino acid residues became more hydrophobic after binding reaction and the conformation of HSA was changed during the binding reaction as shown by an increase in α-helix based on fluorescence and circular dichroism studies. Moreover, M. Poór et al. have been investigate the non-covalent interactions of aflatoxins (B1, B2, G1, G2, and M1) with human serum albumin [23]. They suggest that Sudlow’s Site I of subdomain IIA is the high affinity binding site of aflatoxins on HAS, based on the results of modeling studies and using site markers.

4

Since the distribution of aflatoxins in human body is take place by their binding to HSA macromolecules in the blood, therefor in this study, the interaction between aflatoxin B1 and G1 with HSA was investigated by various methods such as fluorescence quenching, molecular docking and molecular dynamic simulation methods. The specific binding site of aflatoxin B1 and G1 on HSA was determined and free energy of binding was calculated by all three methods. The effect of both aflatoxins on the conformation of HSA was also investigated. The study provides an accurate and full basic data for explaining the binding mechanisms of aflatoxin B1 and G1 with HSA and is helpful for understanding its effect on protein function during the blood transportation process and its toxicity in vivo.

2. Materials and methods 2.1. Materials Human serum albumin (A1653) with 97% purity and Aflatoxin B1, G1 was purchased from Sigma Aldrich. Double distilled water was used throughout the experiments. All of the other reagents were of analytical grade and used as received without further purification. 2.2. Sample preparation HSA (30 nM) solutions were prepared in 20 µM Tris-HCL buffer solution adjusted

to

pH

7.4.

Protein

concentration

was

determined

spectrophotometrically at excitation 280 nm by using PG Instruments UV– visible spectrophotometer. Aflatoxin solution (1 ppm) was prepared in acetonitrile. All experiments were carried out in 20 µM Tris-HCL buffer (pH 7.4) at 298, 304 and 310 K. 2.3. Fluorescence quenching measurements Fluorescence measurements were performed on a JASCO FP-8300 5

fluorescence spectrophotometer equipped with a Xenon lamp source and 1 cm quartz cell. The spectral band widths of excitation and emission slits were both kept at 10 nm. Fluorescence intensity was measured by exciting HSA at 280 nm and the emission spectrums were recorded in the range of 295 to 550 nm. The scan rate was 500 nm/min. The addition of AFG1 and AFB1 (0–90 nM) to HSA (30 nM) solution was carried out at 298, 304 and 310 K. 2.4. Molecular docking Molecular docking study was carried out using AutoDock 4.2 and Lamarckian genetic algorithm in Auto Dock was applied to calculate the possible conformation of the ligand that bound to the HSA. The X-ray crystallography structure of HSA (PDB id: 1AO6) is obtained from the Protein Data Bank. All water molecules were removed with using the ArgusLab 4.0.1 software. Polar hydrogens were added to the protein structure using AutoDockTools (ADT). The saved file in the PDBQT format was used as an input in AutoDock. The 3D structure of Aflatoxin B1, G1 as ligand was generated and its geometry was optimized through HyperChem Professional program. To recognize the binding sites in BSA, docking was performed by using a box size of 50 × 50 × 50 Å with 0.375 Å grid spacing. Cluster analysis was applied on the docked results using an rmsd-tolerance of 2.0 A. The conformer with the lowest binding-free energy was used for further analysis. The LIGPLOT program was used to analyze amino acid residues involved in the binding between HSA and Aflatoxin B1, G1 in the best binding site. 2.5. Molecular dynamic simulations The molecular dynamic (MD) simulations were performed using the GROMACS 5.1.2 program, with the CHARMM27 force field. The obtained docking results of ligands complexes–HSA was used for further MD simulation. The coordinate of ligands was transferred into Gromacs topologies using the 6

SWISSPARAM server. The simulation conditions for both protein and complexes were the same. The triclinic type of box was used for simulation. The SPC water molecules was used to complex surround and system was neutralized with Na+ and Cl‒ ions. The system was minimized by the steepest descent algorithm for 1295 steps. Canonical NVT and NPT ensemble were performed for equilibration. NVT ensemble at 298 K was used with periodic boundary conditions, and the temperature was kept constant by the Berendsen thermostat [24]. NPT ensemble at 298 K was used and pressure was kept constant by the Parrinello-Rahman. Both phase of equilibration were performed by 100,000 steps and 200 ps. Cut-off distances for the calculation of Coulomb and van der Waals interactions were 1.0 nm and time step was 2 fs for all phase. Finally 20 ns production MD was run at constant temperature and pressure. Structural comparison between structures was done using Chimera software. The results were then visualized using the VMD software.

3. Results and discussion 3.1. Fluorescence quenching of HSA The fluorescence emission spectra of HSA in the presence of different concentrations of Aflatoxin B1 and G1 are shown in Fig. 2. The emission intensity of HSA quenched with the adding of aflatoxins, which indicates the formation of complex between aflatoxins and HSA. According to Fig. 2, HSA has a fluorescence emission peak at 310 nm on excitation at 280 nm in nanomolar concentration range. After increasing the concentration of aflatoxin B1 and G1, a blue shift (5nm) in the emission spectra of HSA was observed. This shift indicated that the increasing the concentration of aflatoxin B1 and G1 induced conformational changes in protein structure [25]. Static quenching and dynamic quenching are two main mechanisms for fluorescence quenching. The dynamic quenching arising from collisional encountering and static quenching by the formation of a non-fluorescent ground state complex between the 7

fluorophore and the quencher. In dynamic quenching, the increasing of the system temperature increase the fluorescence quenching constants, the energy transfer efficiency and the effective collision times between molecules. On the other hand, by increasing the temperature, the static quenching constant decreases because the higher temperature decrease stability of complex. In order to distinction of the static and dynamic quenching mechanisms in the complex, the fluorescence quenching of HSA was performed at three temperatures (298, 304 and 310 K) and the data were analyzed by the Stern –Volmer equation (Eq. 1) [26, 27]. F0 / F = 1 + kq τ0 [Q] = 1 + Ksv [Q]

(1)

where F0 and F are the fluorescence intensities of HSA in the absence and presence of the ligands, respectively. Kq is the bimolecular quenching constant, and [Q] is the concentration of ligands. KSV is the Stern–Volmer quenching constant, τ0 is the lifetime of the fluorophore in the absence of quencher, (for HSA, approximately 1×

s) [28]. The Stern–Volmer plots at various

concentrations of ligands at 298 K and 310 K are presented in Fig. 3 and the obtained results are summarized in Table 1. For dynamic quenching, the maximum scatter collision quenching constant of various quenchers with the biopolymers is reported to 1010 M

−1

s

−1

[29].

According to Table 1, the calculated KSV and Kq values decreased by increasing the temperature, and the magnitudes of the Kq values were greater than

1010

M−1 s−1, which indicated that a static quenching mechanism occurred. In other words, quenching emission of HSA by aflatoxins B1 and G1 as a result of specific complex formation at the ground state and not through to dynamic collision effects. 3.2. Determinations of the binding constants For the determination of the binding constant and number of binding sites, 8

the modified Stern–Volmer equation was used (Eq. 2) [30]. Log (F0-F)/F = Log Kb + nLog [Q]

(2)

In (Eq. 2), F0 and F are the steady state fluorescence intensities of HSA in the absence and presence of the ligands, respectively. Kb is the binding constant, n is the number of binding sites and Q is the concentration of ligands. The values of Kb and n was obtained from the plot of log [(F0- F)/F] versus Log [Q] (Fig. 4) and the obtained results are summarized in Table 2. This results showed that the values of n are almost equal to 1, which indicated the existence of a single binding sites for aflatoxins in HSA at both temperature. The values of binding constant (Kb) indicated a stronger binding between studied aflatoxins types and HSA, implicating that both AFB1 and AFG1 can be stored and carried by the HSA in the body. Also the values of binding constant (Kb) decreases with respect to increasing the temperature, which confirms that the quenching mechanism is static in both systems. 3.3. Thermodynamic studies Generally, four types of interactions can be occur between a small molecule and a macromolecule, which including hydrogen bonding, van der Waals forces interactions, hydrophobic and electrostatic interactions [31]. The investigation of thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS) of interaction can help us to understand the binding mode of small molecule-macromolecule interactions. This parameters can be calculated from the Van't Hoff equation: Ln Kb = ‒ ΔH / RT + ΔS / R

(3)

ΔG = ΔH ‒ TΔS

(4)

where Kb is the binding constant at the corresponding temperatures, T is temperature and R is the gas constant. The ΔH and ΔS values can be determined from the slope and intercept of the regression plot of logKb versus 1/T was 9

shown in Fig. 5 and ΔG can be calculated from Eq. 4. The obtained results are summarized in Table 3. The hydrophobic interactions occur when ΔS > 0 and ΔH > 0, also ΔS > 0 and ΔH < 0 indicated the existence of electrostatic force and ΔS < 0 and ΔH < 0 indicates the van der Waals force or hydrogen bond formation. From Table 3, the value of ΔG is negative, which means that the interaction process is spontaneous and also the value of ΔS and ΔH are negative, indicating that hydrogen bonding and van der Waals forces play the major role during the binding process. 3.4. Molecular docking study In this study, molecular docking was used to predict the binding constant and binding site on HSA-AFB1 and HSA-AFG1 complexes to confirm the results of the experimental measurements described earlier. The molecular docking was performed in two steps. The initial step was performed for detection of binding site and binding constant. In the second step, the best conformation of ligands with lowest binding-free energy was selected after performing MD, then the molecular docking was performed on the best MD selected conformer of protein and ligand. The best score ranked results of aflatoxin B1 and G1 binding with HSA before and after MD are shown in Table 4 and Table 5, respectively. The best conformation of the binding mode between aflatoxin B1 and G1 with HSA displayed in Fig. 6 and main amino acid residues involved in interaction along with amino acid responsible to formation of one hydrogen bond before and after MD are shown in Fig. 7 and Fig. 8, respectively. The obtained results indicated that both aflatoxin B1, G1 interact with the HSA at site I of sub-domain IB. also the calculated binding constant values are in good agreement with the fluorescence studies. The docking results were consistent with the findings of the thermodynamic analyses, which indicated, that the aflatoxin B1 and G1 bound with the HSA via hydrogen bonding and hydrophobic interactions. The amino acid residues in 10

HSA that indicate hydrophobic interactions with aflatoxin B1 and G1 are shown in Fig. 7 and Fig. 8, respectively. 3.5. MD simulation analysis MD simulations were performed the obtained lowest docking energy structures of HSA-AFB1 and HSA-AFG1 complex to obtain the dynamics trajectories and the best conformer structures. Root mean square deviations (RMSD) is one of the simulation results that is used to determine the system's arrival time to a stable state and to confirm the stability of the simulations [32]. In this work, the RMSD plots of the complexes and HSA protein alone were obtained from the back bone atoms of protein. The RMSD values of HSA and HSA-AFB1 and HSA-AFG1 complexes were shown in Fig. 9, which indicated that the RMSD of AFB1 is smaller than that of for AFG1. This means that AFG1 binds clearly better to HSA molecule, which confirms these results obtained by molecular docking studies. The RMSD values of back bone atoms in HSA and HSA-AFB1 and HSA-AFG1 complexes were calculated from a 5-9 ns trajectory, where the data points were fluctuated for HSA (0.230 ± 0.044 nm), HSA-AFB1 (0.288 ± 0.065 nm) and HSA-G1 (0.379 ± 0.063 nm) and the best conformer of HSA-AFB1 and HSA-AFG1 complexes were selected in this range of RMSD for molecular docking studies. These complexes were selected at 7.2 and 9.4 ns, respectively. Docking was then implemented on these structures and the results are displayed in Table 5. According to these results, the values of the binding constant of both ligands has become closer to the experimental results obtained from fluorescence experiments after the performing of MD. Which demonstrates the effect of the protein's flexibility and complex simulation in biological conditions of the body in calculating the binding energy. The match structure analysis were performed to prove that the structure of ligands obtained from the MD is the final structure and the flexibility of ligands in AutoDock did not have much effect on the calculation 11

of binding constant. The obtained results were shown in Fig. 10, Fig. 11 and Table 6. According to Table 6, in the final stage, the changes in the structure of ligands are very small, which indicates that the structure of ligands obtained from the MD is the final structure. The Root mean square fluctuation (RMSF) values is other criteria obtained from MD simulation that use for investigation of the protein mobility [33]. The RMSF of all residue over all time scale was calculated for HSA and HSAaflatoxin B1, G1 complexes and results was shown in Fig. 12. As can be seen in the Fig. 12, After the complex formation, the ligand’s binding site in sub domain IB have a smaller fluctuation compared to other sites. This results indicates that there is a good interaction between the ligands and the binding site sub domain IB and confirms the results obtained from docking. The Radius of gyration (Rg) was used to investigation of stability of the back bone atoms of HSA during the MD simulations [34]. The Rg values of HSA and HSA-aflatoxin B1, G1 complexes are shown in Fig. 13. In all systems, the Rg values were stabilized at about 11 ns, indicating that the MD simulation achieved to the equilibrium condition after this time. The Initial values of Rg for all HSA, HSA-AFB1 and HSA-AFG1 complexes was 2.80 nm. The unliganded HSA, HSA-AFB1 and HSA-AFG1 complexes were stabilized at 2.745 ± 0.015, 2.741 ± 0.019 and 2.781 ± 0.014 nm, respectively after 11 ns of trajectories. The obtained results showed that the Rg value of HSA-AFB1 complex decrease after complex formation, while in the case of HSA-AFG1, the Rg value was increase, which indicates that the HSA structure has become more compact after interaction with aflatoxin B1. In the case of HSA-AFG1, the higher mean value of Rg represents the reducing of protein compactness due to interaction with HSA. The reported Rg value of HSA determined experimentally from neutron scattering in aqueous solution was 2.74 ± 0.035 nm [35], it showed that the value was obtained by our work is identical to the experimental values. 12

4. Conclusions This paper presents fluorescence spectroscopy, molecular docking and molecular dynamics simulation investigation on the interaction of aflatoxin B1 and G1 complexes with HSA protein. The fluorescence spectroscopy results indicate that the HSA emission was quenched by AFB1 and AFG1 through static quenching. The negative values of ΔH and ΔS indicate that hydrogen bond and van der Waals force play major roles in the binding of the AFB1 and AFG1 to and HSA macromolecule [36]. The binding constants and number of binding sites of HSA were calculated using the modified Stern–Volmer equation. According to the molecular docking results, AFB1 and AFG1 can bind to HSA at site I in subdomain IB. The binding process is dominated by hydrogen bonds and hydrophobic interactions, which is consistent with the fluorescence studies. The binding constant of aflatoxin B1 and G1 to HSA was determined by molecular docking, and the obtained results were very close to those obtained by fluorescence spectroscopic studies. MD simulation results suggested that AFB1, G1 can interact with HSA, without affecting the secondary structure of HSA but probably with a little change in compaction of its tertiary structure. All approaches used in this study indicated that aflatoxin G1 has a higher binding affinity to HSA than the aflatoxin B1. This type of study can be helpful for understanding ligands effect on protein function during its transportation and distribution in blood.

Acknowledgment The financial support of this project by Mazandaran University is gratefully acknowledged.

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Table 1 Stern–Volmer quenching constants (KSV) and bimolecular quenching constant (Kq) for the HSA–Aflatoxin B1 and G1 complexes at two different temperatures.

Complex

T (K)

Ksv (× 106 M–1)

Kq (× 1014 M–1 S–1)

Ra

HSA–Aflatoxin B1

298 310

7.9 3.3

7.9 3.3

0.9801 0.9893

HSA–Aflatoxin G1

298 310

7.2 3.9

7.2 3.9

0.9994 0.9945

a

R is the linear correlated coefficient

18

Table 2 Binding constants and the number of binding sites for the interaction of HSA–Aflatoxin B1 and G1 at two different temperatures.

Complex

T (K)

Kb (× 106 M–1)

ΔGb (kcal mol‒ 1)

n

R

HSA–Aflatoxin B1

298 310

6.733 1.03

‒ 9.24 ‒ 8.49

0.98 0.92

0.9910 0.9959

HSA–Aflatoxin G1

298 310

10.23 1.435

‒ 9.52 ‒ 8.69

1.02 0.93

0.9994 0.9959

19

Table 3 Thermodynamic parameters obtained from the interaction of Aflatoxin B1, G1 and HSA.

Complex 1 )

T (K)

ΔS (J mol ‒ 1 K‒ 1)

HSA–Aflatoxin B1

298 304 310

‒ 271.85

‒ 120.08

‒ 39.07 ‒ 37.44 ‒ 35.8

HSA–Aflatoxin G1

298 304 310

‒ 286.4

‒ 125.52

‒ 40.17 ‒ 38.45 ‒ 36.74

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ΔH (kJ mol‒ 1)

ΔG (kJ mol‒

Table 4 Docking results obtained from the interaction of Aflatoxin B1, G1 and HSA.

docking results Complex interactions HSA–AFB1 Leu135,

ΔGb (kcal mol‒ 1) ‒ 8.58

Kb (×106 M‒ 1)

2.07

Hydrogen bonds

Arg117

Hydrophobic

Ala158, Leu139, Ile142, Tyr138, Phe134, Phe165, Leu182,

Met123, HSA–AFG1 ‒ 8.88 Phe165,

Tyr161. 3.45

Arg117

Ile142, Ala158, Phe134, Leu135, Tyr138, Met123, Leu182,

Tyr161.

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Table 5 Post-docking results after performing MD between of Aflatoxin B1, G1 and HSA.

post-docking results Complex interactions

ΔGb (kcal mol‒ 1)

HSA–AFB1 ‒ 9.03 HSA–AFG1 ‒ 9.69 Leu135,

Kb (×106 M‒ 1)

4.45

Hydrogen bonds

Leu154

13.56

Leu154

Hydrophobic

Tyr161, Leu139, Ile142, Phe134, Phe165, Leu135, Tyr138, Ala158. Leu139, Ala158, Phe134, Tyr161, Phe165, Tyr138, Leu182,

Met123.

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Table 6 RMSD values of aflatoxin B1, G1 structures obtained from docking, MD simulation and docking after MD

RMSD (A0) Ligand

HyperChem‒ Docking

Docking‒ MD

MD‒ post docking

AFB1

1.854

3.593

0.443

AFG1

2.525

2.696

0.0665

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Figures Captions Fig. 1. Left: Stracture of human serum albumin (HSA) and its different binding and its main sites. Right: The stractures of Aflatoxin B1 and G1 Fig. 2. Fluorescence emission spectra of HSA in the presence of various concentrations of aflatoxin B1 and G1 in two temperature. [HSA]=30 nM, [Aflatoxin B1, G1]=0, 15, 30, 45, 60, 75 and 90 nM. T=298 and 310 K, PH=7/4 Fig. 3. The Stern–Volmer plots for the fluorescence quenching of HSA by aflatoxin B1 and G1 at two temperatures (298 and 310 K) Fig. 4. Modified Stern–Volmer plot for the HSA–Aflatoxin B1, G1 interactions at 298 and 310 K Fig. 5. Van’t Hoff plot for the interaction of HSA and Aflatoxin B1, G1 in Tris buffer, pH=7.4, T= 298, 304 and 310 K Fig. 6. The best conformation of the binding mode between aflatoxin B1 and G1 with HSA Fig. 7. The main amino acid residues involved in hydrophobic interactions along with amino acid responsible to formation of one hydrogen bond Fig. 8. The main amino acid residues involved in hydrophobic interactions along with amino acid responsible to formation of one hydrogen bond after performing MD Fig. 9. Root mean square deviation (nm) of backbone atoms of HSA in the absence and presence of Aflatoxin B1 and G1 Fig. 10. Match structure analysis on A: HyperChem‒ Docking, B: Docking‒ MD and C: MD‒ post docking structures of aflatoxin B1 Fig. 11. Match structure analysis on A: HyperChem‒ Docking, B: Docking‒ MD and C: MD‒ post docking structures of aflatoxin G1 Fig. 12. The RMSF values of HSA and HSA-AFB1, G1 complexs were plotted against residue numbers. Fig. 13. Time dependence of the radius of gyration (Rg) for the backbone atoms of HSA during the simulation in the absence and presence of Aflatoxin B1 and G1.

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Fig. 7

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Fig. 8

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Fig. 9

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Fig. 10

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Fig. 11

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Fig. 12

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Fig. 13

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