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Binding of carbendazim to bovine serum albumin: Insights from experimental and molecular modeling studies
Journal of Molecular Structure 1139 (2017) 303e307
Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc
Binding of carbendazim to bovine serum albumin: Insights from experimental and molecular modeling studies Jinhua Li a, *, Yulei Zhang b, **, Lin Hu a, Yaling Kong a, Changqing Jin a, Zengzhe Xi a a b
School of Materials and Chemical Engineering, Xi'an Technological University, Xi'an, 710032, PR China State Key Laboratory of Solidification Processing, Northwestern Polytechnical University, Xi'an, 710072, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 October 2016 Received in revised form 10 March 2017 Accepted 11 March 2017
Carbendazim (CBZ) is a widely used benzimidazole fungicide in agriculture to control a wide range of fruit and vegetable pathogens, which may lead to potential health hazards. To evaluate the potential toxicity of CBZ, the binding mechanism of bovine serum albumin (BSA) with CBZ was investigated by the fluorescence quenching technology, UV absorbance spectra, circular dichroism (CD), and molecular modeling. The fluorescence titration and UV absorbance spectra revealed that the fluorescence quenching mechanism of BSA by CBZ was a combined quenching process. In addition, the studies of CD spectra suggested that the binding of CBZ to BSA changed the secondary structure of protein. Furthermore, the thermodynamic functions of enthalpy change (DH0) and entropy change (DS0) for the reaction were calculated to be 24.87 kJ mol1 and 162.95 J mol1 K1 according to Van't Hoff equation. These data suggested that hydrophobic interaction play a major role in the binding of CBZ to BSA, which was in good agreement with the result of molecular modeling study. © 2017 Elsevier B.V. All rights reserved.
Keywords: Carbendazim Bovine serum albumin Fluorescence quenching technology UV absorbance spectra Circular dichroism Molecular modeling
1. Introduction Carbendazim (CBZ, structure shown in Fig. 1) is a systemic fungicide of the benzimidazole (BZ) family, which has been widely used in agriculture for pre-and post-harvest treatment for the control of a wide range of fruit and vegetable pathogens in China and other countries in the world [1e4]. Generally, some benzimidazole fungicide rapidly degrade to carbendazim in water solution, such as benomyl and thiophanate [5,6]. It has been reported that carbendazim could disrupt various aspects of reproductive system, cause germ cell apoptosis, embryo toxicity, or teratogenesis in rats or human [7,8]. Serum albumins, including Bovine serum albumin (BSA) and Human serum albumin (HSA), are the main constituent of the blood plasma. It plays an important role in the binding and transport of various ligands to the target sites. The ligands are always fatty acids, dyes, drugs and pesticide residues [9e12]. It has been shown that the distribution, free concentration and the metabolism of the ligands can be influenced by their binding to protein in the bloodstream. Consequently, binding of small molecules to protein is
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (J. Li), [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.molstruc.2017.03.048 0022-2860/© 2017 Elsevier B.V. All rights reserved.
imperative importance to many biological processes. While most of these reports focused on the interactions between SA and many drugs [13e16], the binding of benzimidazole pesticides to proteins has been seldom reported previously. The binding of pesticides to serum albumin changes not only the effectiveness and action of pesticides but also the activity of serum albumins, they may produce toxic effects to the protein which will finally affect the biological function of protein. BSA and HSA display approximately 76% sequence homology which have usually been used as model proteins to investigate the interaction between bioactive component and protein. Therefore BSA was usually selected as our protein model because of its medical importance, low cost and ready availability [17,18]. BSA consists of three linearly arranged domains (IeIII): I (residues1-195), II (residues 196e383), III (residues 384e585), and each containing two subdomains (A and B). There are two tryptophans (Trp 134 and Trp 213) in BSA: Trp 134 is located on the surface of the molecule and Trp 213 resides in the hydrophobic pocket of sub-domain IIA [19,20]. The binding sites of BSA for ligands are often located in hydrophobic cavities of subdomains IIA and IIIA which are called sites I and II, respectively [21]. Since the widespread use of CBZ in agriculture may lead to potential health hazards (because of their toxicity or carcinogenicity), it is necessary to investigate the interaction of CBZ with protein. Pesticides-protein interaction experiments have a great
304
J. Li et al. / Journal of Molecular Structure 1139 (2017) 303e307
Molecular modeling was investigated through SGI FUEL WORKSTATION. The crystal structure of BSA was taken from the Brookhaven Protein Data Bank (entry code 3V03). The potential of the 3-D structure of BSA was assigned according to the Amber 4.2 force field with Kollman-all-atom charges. The initial structures of CBZ were generated by molecular modeling soft-ware SYBYL 6.9 [23]. The geometries of these compounds were subsequently optimized using the Tripos force field with Gasteiger-Marsili charges. FlexX program was applied to calculate the possible conformation of the ligands that bind to the protein. Fig. 1. The chemical structure of CBZ.
3. Results and discussion 3.1. The mechanism of fluorescence quenching
significance in discovering the transportation and distribution of pesticides in vivo. In this paper, the interaction between CBZ and BSA has been studied using fluorescence, UVevis absorption, circular dichroism (CD) spectra and molecular modeling methods. Binding parameters, such as the binding constant, number of binding sites, and binding force were obtained from the fluorescence data. These are the first spectroscopic results on CBZ-BSA interaction. In addition, the effect of CBZ on the structure of BSA was also examined. 2. Materials and methods 2.1. Materials Bovine serum albumin (BSA) was obtained from Sigma Chemical Company. All BSA solutions were prepared in pH 7.40 buffer solution, and BSA stock solution was kept in the dark at 275 K. Carbendazim (CBZ) (analytical grade) was purchased from Aladin Ltd. (Shanghai China). NaCl (analytical grade, 1.0 mol L1) solution was used to maintain the ion strength at 0.1. The buffer (pH 7.40) consists of tris (0.2 mol L1) and HCl (0.1 mol L1). The pH was checked with a suitably standardized pH meter. Distilled water was used in the experiments.
Fluorescence quenching technique was applied to investigate whether CBZ interact with BSA. It is a powerful method to study interactions of several substances with protein which can reveal the accessibility of quenchers to albumin's fluorophore groups [24e26]. Fig. 2 shows the fluorescence emission spectra of BSA with varying concentration of CBZ. BSA has a strong fluorescence emission with a peak at 340 nm at lex 280 nm, while CBZ was almost non-fluorescent at lex 280 nm. It can be seen that the fluorescence intensity of BSA decreased regularly with increasing concentration of CBZ. In addition, the reduction of the fluorescence intensity was calculated to be 62.7% at the highest CBZ concentration. Furthermore, there was a blue shift (from 340 to 331 nm) of emission with the addition of CBZ. These results suggest that the binding of CBZ to BSA quenches the intrinsic fluorescence of BSA and affects the conformation of protein. Fluorescence quenching can be classified as static quenching and dynamic quenching which can be distinguished by their differing dependence on temperature. For dynamic quenching, the quenching constants increase with increasing temperature, while the quenching constants decrease with increasing temperature for static quenching [25]. The SternVolmer Equation (1) is often applied to recognize the quenching mechanism:
F0 =F ¼ 1 þ KSV ½Q ¼ 1 þ Kq t0 ½Q
(1)
2.2. Apparatus and methods All fluorescence spectra were recorded on a RF-5301PC Spectrofluoro-photometer (Shimadzu, Japan). The excitation and emission slit widths were both 5 nm. The excitation wavelength was 280 nm, and the emission wavelengths were red at 300e495 nm. Fluorescence titration experiments: 2.0 ml solution containing appropriate concentration of BSA was titrated manually by successive addition of a 1.0 103 mol L1 ethanol stock solution of CBZ (to give a final concentration of 7.4, 14.7, 22.0, 29.1, 36.1, 43.0, 50.0, 56.6 and 63.0 106 mol L1) with trace syringes, and the fluorescence intensity was measured (excitation at 280 nm and emission at 338 nm). All experiments were measured at different temperature (296, 303 and 310 K). The temperature of sample was kept by recycled water throughout the experiment. The UV absorbance spectra were recorded using a TU-1901 UVevis Spectrophotometer with a 1 cm quartz cell (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). Circular dichroism was made on a Jasco-20 automatic recording spectropolarimeter (Japan), using a 2 mm cell at 296 K. The spectra were recorded in the range of 200e300 nm. The results are expressed as molar ellipticity ([q]) in deg cm2 dmol1. The a-helical content of HSA was calculated from the [q] value at 208 nm using the equation: a%helix ¼ {(e[q]208e4000)/(33000e4000)} 100 as described by Lu et al. [22].
Fig. 2. The fluorescence emission spectra of CBZ-BSA system at excited 280 nm: (a) 3.0 106 mol L1 BSA; (bej) 3.0 106 mol L1 BSA in the presence of 7.4, 14.7, 22.0, 29.1, 36.1, 43.0, 50.0, 56.6 and 63.0 106 mol L1 CBZ, respectively; (k) 7.4 106 mol L1 CBZ. T ¼ 296 K, pH ¼ 7.4.
J. Li et al. / Journal of Molecular Structure 1139 (2017) 303e307
305
3.2 1.50
f
1.38
b
2
1.32
F0 / F
2.4 2.2
1.26 1.20 1.14 8
2.0
12
-1 [Q]*10-6Mol L
16
20
1.5
b
1
1.8
2.0
f
Absorbance
2.6
Absorbance
2.8
2.5 1.44
F0 / F
3.0
1.6
210
220
230
Wavelength (nm)
a
1.4
0
1.2
200
1.0 10
20
30
40
-1 [Q]*10-6Mol L
50
60
70
Fig. 3. The SterneVolmer plots of the CBZeBSA at pH 7.40. BSA concentration: 3.0 106 mol L1; (-) 296 K; (C) 303 K; (:) 310 K; lex ¼ 280 nm, lem ¼ 338 nm.
where F and F0 are the relative fluorescence intensities in the presence and absence of quencher, respectively. Ksv is the SterneVolmer quenching constant, [Q] is the concentration of quencher. Kq is the quenching constant of bimolecular fluorescence, t0 is the lifetime of the fluorophore in the absence of quencher (BSA: 108 s), Kq ¼ KSV/t0. The SterneVolmer quenching plots of CBZ with BSA at different temperatures (296, 303, and 310 K) are displayed in Fig. 3. The plots show a good linear relationship, and the slopes increase with increasing temperature (shown in Table 1) revealing that dynamic quenching interaction exists between CBZ and BSA [27,28]. Moreover, the values Kq were all greater than 2.0 1010 L mol1 S1 (the maximum quenching constant for dynamic quenching) which indicated that the static quenching may also occur between them [29]. For static quenching, the absorption spectra of fluorescence substance would be changed because of the formation of ground-state complex [30]. To confirm the quenching mechanism, we measured the UV absorbance spectra of BSA with various amounts of CBZ. Fig. 4 shows that the absorption spectra of CBZ, BSA and CBZ-BSA were different. As can be seen in Fig. 4, BSA has a strong absorbance with peaks at 214 nm and 277 nm. With the addition of CBZ, the intensity of the peak was changed. In addition, the position peak at 214 nm of BSA has a red shift (1.5 nm). The results indicated that the static quenching exists in the binding process. Collectively, the fluorescence quenching mechanism of BSA by CBZ should be a combined quenching process (including dynamic and static quenching) [31]. 3.2. Binding parameters and number of binding sites The binding constants and number of binding sites of small molecules interaction with protein can be obtained by the Scatchard equation [32]:
250
Wavelength (nm)
300
Fig. 4. UV absorption spectra obtained in Tris buffer solution (pH 7.4): (a) CBZ 6.0 106 mol L1; (b) BSA, 3.0 106 mol L1; (cef) CBZ-BSA, 3.0 106 mol L1 BSA in the presence of 6.0, 12.0, 18.0, 24.0 106 mol L1 CBZ.
Fig. 5. The Scatchard plots of CBZ-BSA at pH 7.40, BSA concentration: 3.0 106 mol L1; (-) 296 K; (C) 303 K; (:) 310 K. lex ¼ 280 nm, lem ¼ 338 nm.
r=Df ¼ nKrK;
(2)
where r is the number of moles of bound drug per mole of protein, Df is the concentration of free drug, K and n are the binding constant and number of binding sites, respectively. The results are presented in Fig. 5 and Table 1. Fig. 5 displays the Scatchard plots for the BSACBZ system at three different temperatures (296 K, 303 K and 310 K). The Scatchard plots obtained from this procedure show a straight line for different temperatures. The linearity indicates that CBZ binds to one class of site on BSA, and the binding constants
Table 1 Binding parameters and thermodynamic parameters of BSA-CBZ. T(K)
KSV (L mol1)
K (L mol1)
n
DG0 (kJ mol1)
DS0 (J mol1 K1)
DH0 (kJ mol1)
296 303 310
2.77 104 3.06 104 3.15 104
1.34 104 1.65 104 2.09 104
1.37 1.28 1.15
23.38 24.52 25.66
162.95 162.95 162.95
24.87 24.87 24.87
T is the absolute temperature, Ksv is the SterneVolmer quenching constant, K is the binding constant; n is number of binding site; DG0 is the Gibbs' free-energy change; DS0 is the entropy change; DH0 is the enthalpy change.
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were close to 104 L mol1. The results indicated that the binding between CBZ and BSA was strong and CBZ could be transported to some organs by protein in body. 3.3. Thermodynamic analyses (binding mode) Generally, there are mainly four types binding forces in the binding process of small molecular substrates with proteins, which are Van' der Waals interaction, hydrophobic force, electrostatic interaction, hydrogen bond [33]. The thermodynamic parameters, enthalpy change (DH0), entropy change (DS0) and free energy change (DG0) of reaction can be calculated using the following equations:
Fig. 6. Van't Hoff plots of CBZ-BSA (pH 7.40).
lnKT ¼ DH0 =RT þ DS0 =R
(3)
DG0 ¼ DH 0 T DS0
(4)
where K is the binding constant, T is the experimental temperature and R is the gas constant. By plotting the binding constants according to Van't Hoff equation, the thermodynamic parameters were determined from linear Van't Hoff plot (spectrum shown in Fig. 6) and are presented in Table 1. As shown in Table 1, the negative value of DG0 meant that the binding process between CBZ and BSA is spontaneous. The positive values of DS0 and DH0 revealed that hydrophobic interaction plays a major role in the binding of CBZ to BSA [33]. From the structure of CBZ and BSA, Hbond was easily formed in the atoms oxygen and nitrogen. Therefore, H-bond also existed between them. 3.4. Changes of the BSA secondary structure induced by CBZ binding
Fig. 7. CD spectra of the BSA-CBZ system: (a) 3.0 106 mol L1 BSA; (b) 3.0 106 mol L1 BSA in the presence of 1.2 105 mol L1 CBZ; pH ¼ 7.4.
To investigate the possible influence of CBZ on the secondary structure of BSA, CD studies of BSA were performed in absence and presence of CBZ. The CD spectra of BSA and CBZ-BSA complex were shown in Fig. 7. It can be found that CD spectrum of BSA exhibits two negative bands in the ultraviolet region at 207 and 218 nm characteristic of the a-helical structure of protein. The binding of CBZ to BSA causes an increase in band intensity, indicating the
(b)
(a)
CBZ
Fig. 8. The best energy ranked results of docking. (a) Binding site of BSA. CBZ is shown in green sticks; BSA is represented using cartoon. (b) Docking details showing the interaction between CBZ and amino acid residues of BSA; CBZ is shown in green sticks, main frame of residues are in gray. The hydrogen bond between CBZ and BSA is represented using dot line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
J. Li et al. / Journal of Molecular Structure 1139 (2017) 303e307
considerable changes in the protein secondary structure with the decrease of a-helical structure, and it may be the result of the formation of CBZ-BSA complex. The calculated result exhibited that the content of the a-helical structure in BSA decreased from 59.5% to 57.5% at molar ratio of CBZ/BSA of 4:1. The results reveal that the binding of CBZ to BSA has changed the secondary structure of BSA. 3.5. Study of molecular modeling Molecular modeling method has been applied to improve the understanding about the interaction of CBZ and BSA. The best energy ranked result is shown in Fig. 8. From Fig. 8a and b, it can be seen that CBZ binds into the pocket of sub-domain IIA (sites I) where Trp 213 locates, and has close contact with the amino acid residues Trp 213, Val 342, Ala 341, Arg 194, Pro 446, Asp 450 etc. In addition, there are also two hydrogen bonding interactions of CBZ with Asp 450 and Trp 213, and the distance between two atoms forming hydrogen bond is about 2.1 Å, which were shown in Fig. 8b. The formation of hydrogen bond decreased the hydrophilicity and increased the hydrophobicity to stability the CBZ-BSA system [34]. The results obtained from modeling indicate that the interaction between CBZ and BSA is dominated by hydrophobic force, and there are also hydrogen bond interactions between the pesticide and the residues of BSA. Meanwhile, the calculated binding free energy was 23.7 kJ mol1 for CBZeBSA system which agreed with the result of the binding mode (23.38 kJ mol1). In a word, the results of molecular modeling are in accordance with the results of spectroscopic and thermodynamic analysis. 4. Conclusions In this study, the interaction between CBZ and BSA has been studied thoroughly by spectroscopic and molecular docking methods. The results have clearly indicated that CBZ is a strong quencher and can bind to the protein with changing its molecular conformation. Meanwhile, the predominance of hydrophobic interaction has been concluded from both experimental results and molecular docking model. The study is relevant to offer a better understanding of CBZ's toxicity, pesticides reasonable use, and design a new type of low toxicity pesticides.
of the State Key Laboratory of Solidification Processing in NWPU (SKLSP201506), Chinese Postdoctoral Science Foundation (2016M602938XB) and Shaanxi Provincial Education Department Programme (2013JK0678).
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Acknowledgements This work was financially supported by the National Science Foundation of China (Grant No. 51502233 and 11404251), the fund
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Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc
Binding of carbendazim to bovine serum albumin: Insights from experimental and molecular modeling studies Jinhua Li a, *, Yulei Zhang b, **, Lin Hu a, Yaling Kong a, Changqing Jin a, Zengzhe Xi a a b
School of Materials and Chemical Engineering, Xi'an Technological University, Xi'an, 710032, PR China State Key Laboratory of Solidification Processing, Northwestern Polytechnical University, Xi'an, 710072, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 October 2016 Received in revised form 10 March 2017 Accepted 11 March 2017
Carbendazim (CBZ) is a widely used benzimidazole fungicide in agriculture to control a wide range of fruit and vegetable pathogens, which may lead to potential health hazards. To evaluate the potential toxicity of CBZ, the binding mechanism of bovine serum albumin (BSA) with CBZ was investigated by the fluorescence quenching technology, UV absorbance spectra, circular dichroism (CD), and molecular modeling. The fluorescence titration and UV absorbance spectra revealed that the fluorescence quenching mechanism of BSA by CBZ was a combined quenching process. In addition, the studies of CD spectra suggested that the binding of CBZ to BSA changed the secondary structure of protein. Furthermore, the thermodynamic functions of enthalpy change (DH0) and entropy change (DS0) for the reaction were calculated to be 24.87 kJ mol1 and 162.95 J mol1 K1 according to Van't Hoff equation. These data suggested that hydrophobic interaction play a major role in the binding of CBZ to BSA, which was in good agreement with the result of molecular modeling study. © 2017 Elsevier B.V. All rights reserved.
Keywords: Carbendazim Bovine serum albumin Fluorescence quenching technology UV absorbance spectra Circular dichroism Molecular modeling
1. Introduction Carbendazim (CBZ, structure shown in Fig. 1) is a systemic fungicide of the benzimidazole (BZ) family, which has been widely used in agriculture for pre-and post-harvest treatment for the control of a wide range of fruit and vegetable pathogens in China and other countries in the world [1e4]. Generally, some benzimidazole fungicide rapidly degrade to carbendazim in water solution, such as benomyl and thiophanate [5,6]. It has been reported that carbendazim could disrupt various aspects of reproductive system, cause germ cell apoptosis, embryo toxicity, or teratogenesis in rats or human [7,8]. Serum albumins, including Bovine serum albumin (BSA) and Human serum albumin (HSA), are the main constituent of the blood plasma. It plays an important role in the binding and transport of various ligands to the target sites. The ligands are always fatty acids, dyes, drugs and pesticide residues [9e12]. It has been shown that the distribution, free concentration and the metabolism of the ligands can be influenced by their binding to protein in the bloodstream. Consequently, binding of small molecules to protein is
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (J. Li), [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.molstruc.2017.03.048 0022-2860/© 2017 Elsevier B.V. All rights reserved.
imperative importance to many biological processes. While most of these reports focused on the interactions between SA and many drugs [13e16], the binding of benzimidazole pesticides to proteins has been seldom reported previously. The binding of pesticides to serum albumin changes not only the effectiveness and action of pesticides but also the activity of serum albumins, they may produce toxic effects to the protein which will finally affect the biological function of protein. BSA and HSA display approximately 76% sequence homology which have usually been used as model proteins to investigate the interaction between bioactive component and protein. Therefore BSA was usually selected as our protein model because of its medical importance, low cost and ready availability [17,18]. BSA consists of three linearly arranged domains (IeIII): I (residues1-195), II (residues 196e383), III (residues 384e585), and each containing two subdomains (A and B). There are two tryptophans (Trp 134 and Trp 213) in BSA: Trp 134 is located on the surface of the molecule and Trp 213 resides in the hydrophobic pocket of sub-domain IIA [19,20]. The binding sites of BSA for ligands are often located in hydrophobic cavities of subdomains IIA and IIIA which are called sites I and II, respectively [21]. Since the widespread use of CBZ in agriculture may lead to potential health hazards (because of their toxicity or carcinogenicity), it is necessary to investigate the interaction of CBZ with protein. Pesticides-protein interaction experiments have a great
304
J. Li et al. / Journal of Molecular Structure 1139 (2017) 303e307
Molecular modeling was investigated through SGI FUEL WORKSTATION. The crystal structure of BSA was taken from the Brookhaven Protein Data Bank (entry code 3V03). The potential of the 3-D structure of BSA was assigned according to the Amber 4.2 force field with Kollman-all-atom charges. The initial structures of CBZ were generated by molecular modeling soft-ware SYBYL 6.9 [23]. The geometries of these compounds were subsequently optimized using the Tripos force field with Gasteiger-Marsili charges. FlexX program was applied to calculate the possible conformation of the ligands that bind to the protein. Fig. 1. The chemical structure of CBZ.
3. Results and discussion 3.1. The mechanism of fluorescence quenching
significance in discovering the transportation and distribution of pesticides in vivo. In this paper, the interaction between CBZ and BSA has been studied using fluorescence, UVevis absorption, circular dichroism (CD) spectra and molecular modeling methods. Binding parameters, such as the binding constant, number of binding sites, and binding force were obtained from the fluorescence data. These are the first spectroscopic results on CBZ-BSA interaction. In addition, the effect of CBZ on the structure of BSA was also examined. 2. Materials and methods 2.1. Materials Bovine serum albumin (BSA) was obtained from Sigma Chemical Company. All BSA solutions were prepared in pH 7.40 buffer solution, and BSA stock solution was kept in the dark at 275 K. Carbendazim (CBZ) (analytical grade) was purchased from Aladin Ltd. (Shanghai China). NaCl (analytical grade, 1.0 mol L1) solution was used to maintain the ion strength at 0.1. The buffer (pH 7.40) consists of tris (0.2 mol L1) and HCl (0.1 mol L1). The pH was checked with a suitably standardized pH meter. Distilled water was used in the experiments.
Fluorescence quenching technique was applied to investigate whether CBZ interact with BSA. It is a powerful method to study interactions of several substances with protein which can reveal the accessibility of quenchers to albumin's fluorophore groups [24e26]. Fig. 2 shows the fluorescence emission spectra of BSA with varying concentration of CBZ. BSA has a strong fluorescence emission with a peak at 340 nm at lex 280 nm, while CBZ was almost non-fluorescent at lex 280 nm. It can be seen that the fluorescence intensity of BSA decreased regularly with increasing concentration of CBZ. In addition, the reduction of the fluorescence intensity was calculated to be 62.7% at the highest CBZ concentration. Furthermore, there was a blue shift (from 340 to 331 nm) of emission with the addition of CBZ. These results suggest that the binding of CBZ to BSA quenches the intrinsic fluorescence of BSA and affects the conformation of protein. Fluorescence quenching can be classified as static quenching and dynamic quenching which can be distinguished by their differing dependence on temperature. For dynamic quenching, the quenching constants increase with increasing temperature, while the quenching constants decrease with increasing temperature for static quenching [25]. The SternVolmer Equation (1) is often applied to recognize the quenching mechanism:
F0 =F ¼ 1 þ KSV ½Q ¼ 1 þ Kq t0 ½Q
(1)
2.2. Apparatus and methods All fluorescence spectra were recorded on a RF-5301PC Spectrofluoro-photometer (Shimadzu, Japan). The excitation and emission slit widths were both 5 nm. The excitation wavelength was 280 nm, and the emission wavelengths were red at 300e495 nm. Fluorescence titration experiments: 2.0 ml solution containing appropriate concentration of BSA was titrated manually by successive addition of a 1.0 103 mol L1 ethanol stock solution of CBZ (to give a final concentration of 7.4, 14.7, 22.0, 29.1, 36.1, 43.0, 50.0, 56.6 and 63.0 106 mol L1) with trace syringes, and the fluorescence intensity was measured (excitation at 280 nm and emission at 338 nm). All experiments were measured at different temperature (296, 303 and 310 K). The temperature of sample was kept by recycled water throughout the experiment. The UV absorbance spectra were recorded using a TU-1901 UVevis Spectrophotometer with a 1 cm quartz cell (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). Circular dichroism was made on a Jasco-20 automatic recording spectropolarimeter (Japan), using a 2 mm cell at 296 K. The spectra were recorded in the range of 200e300 nm. The results are expressed as molar ellipticity ([q]) in deg cm2 dmol1. The a-helical content of HSA was calculated from the [q] value at 208 nm using the equation: a%helix ¼ {(e[q]208e4000)/(33000e4000)} 100 as described by Lu et al. [22].
Fig. 2. The fluorescence emission spectra of CBZ-BSA system at excited 280 nm: (a) 3.0 106 mol L1 BSA; (bej) 3.0 106 mol L1 BSA in the presence of 7.4, 14.7, 22.0, 29.1, 36.1, 43.0, 50.0, 56.6 and 63.0 106 mol L1 CBZ, respectively; (k) 7.4 106 mol L1 CBZ. T ¼ 296 K, pH ¼ 7.4.
J. Li et al. / Journal of Molecular Structure 1139 (2017) 303e307
305
3.2 1.50
f
1.38
b
2
1.32
F0 / F
2.4 2.2
1.26 1.20 1.14 8
2.0
12
-1 [Q]*10-6Mol L
16
20
1.5
b
1
1.8
2.0
f
Absorbance
2.6
Absorbance
2.8
2.5 1.44
F0 / F
3.0
1.6
210
220
230
Wavelength (nm)
a
1.4
0
1.2
200
1.0 10
20
30
40
-1 [Q]*10-6Mol L
50
60
70
Fig. 3. The SterneVolmer plots of the CBZeBSA at pH 7.40. BSA concentration: 3.0 106 mol L1; (-) 296 K; (C) 303 K; (:) 310 K; lex ¼ 280 nm, lem ¼ 338 nm.
where F and F0 are the relative fluorescence intensities in the presence and absence of quencher, respectively. Ksv is the SterneVolmer quenching constant, [Q] is the concentration of quencher. Kq is the quenching constant of bimolecular fluorescence, t0 is the lifetime of the fluorophore in the absence of quencher (BSA: 108 s), Kq ¼ KSV/t0. The SterneVolmer quenching plots of CBZ with BSA at different temperatures (296, 303, and 310 K) are displayed in Fig. 3. The plots show a good linear relationship, and the slopes increase with increasing temperature (shown in Table 1) revealing that dynamic quenching interaction exists between CBZ and BSA [27,28]. Moreover, the values Kq were all greater than 2.0 1010 L mol1 S1 (the maximum quenching constant for dynamic quenching) which indicated that the static quenching may also occur between them [29]. For static quenching, the absorption spectra of fluorescence substance would be changed because of the formation of ground-state complex [30]. To confirm the quenching mechanism, we measured the UV absorbance spectra of BSA with various amounts of CBZ. Fig. 4 shows that the absorption spectra of CBZ, BSA and CBZ-BSA were different. As can be seen in Fig. 4, BSA has a strong absorbance with peaks at 214 nm and 277 nm. With the addition of CBZ, the intensity of the peak was changed. In addition, the position peak at 214 nm of BSA has a red shift (1.5 nm). The results indicated that the static quenching exists in the binding process. Collectively, the fluorescence quenching mechanism of BSA by CBZ should be a combined quenching process (including dynamic and static quenching) [31]. 3.2. Binding parameters and number of binding sites The binding constants and number of binding sites of small molecules interaction with protein can be obtained by the Scatchard equation [32]:
250
Wavelength (nm)
300
Fig. 4. UV absorption spectra obtained in Tris buffer solution (pH 7.4): (a) CBZ 6.0 106 mol L1; (b) BSA, 3.0 106 mol L1; (cef) CBZ-BSA, 3.0 106 mol L1 BSA in the presence of 6.0, 12.0, 18.0, 24.0 106 mol L1 CBZ.
Fig. 5. The Scatchard plots of CBZ-BSA at pH 7.40, BSA concentration: 3.0 106 mol L1; (-) 296 K; (C) 303 K; (:) 310 K. lex ¼ 280 nm, lem ¼ 338 nm.
r=Df ¼ nKrK;
(2)
where r is the number of moles of bound drug per mole of protein, Df is the concentration of free drug, K and n are the binding constant and number of binding sites, respectively. The results are presented in Fig. 5 and Table 1. Fig. 5 displays the Scatchard plots for the BSACBZ system at three different temperatures (296 K, 303 K and 310 K). The Scatchard plots obtained from this procedure show a straight line for different temperatures. The linearity indicates that CBZ binds to one class of site on BSA, and the binding constants
Table 1 Binding parameters and thermodynamic parameters of BSA-CBZ. T(K)
KSV (L mol1)
K (L mol1)
n
DG0 (kJ mol1)
DS0 (J mol1 K1)
DH0 (kJ mol1)
296 303 310
2.77 104 3.06 104 3.15 104
1.34 104 1.65 104 2.09 104
1.37 1.28 1.15
23.38 24.52 25.66
162.95 162.95 162.95
24.87 24.87 24.87
T is the absolute temperature, Ksv is the SterneVolmer quenching constant, K is the binding constant; n is number of binding site; DG0 is the Gibbs' free-energy change; DS0 is the entropy change; DH0 is the enthalpy change.
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were close to 104 L mol1. The results indicated that the binding between CBZ and BSA was strong and CBZ could be transported to some organs by protein in body. 3.3. Thermodynamic analyses (binding mode) Generally, there are mainly four types binding forces in the binding process of small molecular substrates with proteins, which are Van' der Waals interaction, hydrophobic force, electrostatic interaction, hydrogen bond [33]. The thermodynamic parameters, enthalpy change (DH0), entropy change (DS0) and free energy change (DG0) of reaction can be calculated using the following equations:
Fig. 6. Van't Hoff plots of CBZ-BSA (pH 7.40).
lnKT ¼ DH0 =RT þ DS0 =R
(3)
DG0 ¼ DH 0 T DS0
(4)
where K is the binding constant, T is the experimental temperature and R is the gas constant. By plotting the binding constants according to Van't Hoff equation, the thermodynamic parameters were determined from linear Van't Hoff plot (spectrum shown in Fig. 6) and are presented in Table 1. As shown in Table 1, the negative value of DG0 meant that the binding process between CBZ and BSA is spontaneous. The positive values of DS0 and DH0 revealed that hydrophobic interaction plays a major role in the binding of CBZ to BSA [33]. From the structure of CBZ and BSA, Hbond was easily formed in the atoms oxygen and nitrogen. Therefore, H-bond also existed between them. 3.4. Changes of the BSA secondary structure induced by CBZ binding
Fig. 7. CD spectra of the BSA-CBZ system: (a) 3.0 106 mol L1 BSA; (b) 3.0 106 mol L1 BSA in the presence of 1.2 105 mol L1 CBZ; pH ¼ 7.4.
To investigate the possible influence of CBZ on the secondary structure of BSA, CD studies of BSA were performed in absence and presence of CBZ. The CD spectra of BSA and CBZ-BSA complex were shown in Fig. 7. It can be found that CD spectrum of BSA exhibits two negative bands in the ultraviolet region at 207 and 218 nm characteristic of the a-helical structure of protein. The binding of CBZ to BSA causes an increase in band intensity, indicating the
(b)
(a)
CBZ
Fig. 8. The best energy ranked results of docking. (a) Binding site of BSA. CBZ is shown in green sticks; BSA is represented using cartoon. (b) Docking details showing the interaction between CBZ and amino acid residues of BSA; CBZ is shown in green sticks, main frame of residues are in gray. The hydrogen bond between CBZ and BSA is represented using dot line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
J. Li et al. / Journal of Molecular Structure 1139 (2017) 303e307
considerable changes in the protein secondary structure with the decrease of a-helical structure, and it may be the result of the formation of CBZ-BSA complex. The calculated result exhibited that the content of the a-helical structure in BSA decreased from 59.5% to 57.5% at molar ratio of CBZ/BSA of 4:1. The results reveal that the binding of CBZ to BSA has changed the secondary structure of BSA. 3.5. Study of molecular modeling Molecular modeling method has been applied to improve the understanding about the interaction of CBZ and BSA. The best energy ranked result is shown in Fig. 8. From Fig. 8a and b, it can be seen that CBZ binds into the pocket of sub-domain IIA (sites I) where Trp 213 locates, and has close contact with the amino acid residues Trp 213, Val 342, Ala 341, Arg 194, Pro 446, Asp 450 etc. In addition, there are also two hydrogen bonding interactions of CBZ with Asp 450 and Trp 213, and the distance between two atoms forming hydrogen bond is about 2.1 Å, which were shown in Fig. 8b. The formation of hydrogen bond decreased the hydrophilicity and increased the hydrophobicity to stability the CBZ-BSA system [34]. The results obtained from modeling indicate that the interaction between CBZ and BSA is dominated by hydrophobic force, and there are also hydrogen bond interactions between the pesticide and the residues of BSA. Meanwhile, the calculated binding free energy was 23.7 kJ mol1 for CBZeBSA system which agreed with the result of the binding mode (23.38 kJ mol1). In a word, the results of molecular modeling are in accordance with the results of spectroscopic and thermodynamic analysis. 4. Conclusions In this study, the interaction between CBZ and BSA has been studied thoroughly by spectroscopic and molecular docking methods. The results have clearly indicated that CBZ is a strong quencher and can bind to the protein with changing its molecular conformation. Meanwhile, the predominance of hydrophobic interaction has been concluded from both experimental results and molecular docking model. The study is relevant to offer a better understanding of CBZ's toxicity, pesticides reasonable use, and design a new type of low toxicity pesticides.
of the State Key Laboratory of Solidification Processing in NWPU (SKLSP201506), Chinese Postdoctoral Science Foundation (2016M602938XB) and Shaanxi Provincial Education Department Programme (2013JK0678).
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Acknowledgements This work was financially supported by the National Science Foundation of China (Grant No. 51502233 and 11404251), the fund
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