Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 109–113
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Quest for the binding mode of tetrabromobisphenol A with Calf thymus DNA Yan-Qing Wang ⇑, Hong-Mei Zhang, Jian Cao Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Molecular modeling and multi-
spectroscopic methods were used. TBBPA bound to DNA by groove
mode. The hydrophobic and hydrogen
bonding forces were involved in the binding process. DNA binding changed the electron cloud of TBBPA.
a r t i c l e
i n f o
Article history: Received 24 February 2014 Received in revised form 6 April 2014 Accepted 17 April 2014 Available online 26 April 2014 Keywords: Tetrabromobisphenol A Calf thymus DNA Spectroscopy Binding mode Molecular modeling
a b s t r a c t The binding interaction of tetrabromobisphenol A with Calf thymus DNA was studied by multispectroscopic and molecular modeling methods. The UV–vis study revealed that an obvious interaction between tetrabromobisphenol A and Calf thymus DNA happened. The p–p⁄ transitions and the electron cloud of tetrabromobisphenol A might be changed by entering the groove of Calf thymus DNA. From the fluorescence spectral and thermodynamics studies, it was concluded that the hydrogen bonds and hydrophobic force played a major role in the binding of tetrabromobisphenol A to Calf thymus DNA. The molecular modeling study showed that the possible sites of tetrabromobisphenol A in the groove of DNA. Circular dichroism study also depicted that tetrabromobisphenol A bond to DNA. These above results would further advance our knowledge on the molecular mechanism of the binding interactions of brominated flame-retardants with nucleic acid. Ó 2014 Elsevier B.V. All rights reserved.
Introduction As one of the most widely used brominated flame-retardants in the world, tetrabromobisphenol A (TBBPA) is a potential environmental health problem [1,2]. It has been detected in a wide range of environmental matrices including air, dust, sewage sludge, sediment, wastewater, and aquatic biota [3,4]. Many studies have indicated that TBBPA was characterized as a suspected endocrine ⇑ Corresponding author. Tel./fax: +86 515 88233188. E-mail address: [email protected] (Y.-Q. Wang). http://dx.doi.org/10.1016/j.saa.2014.04.077 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
disruptor and had potential immune- and neurotoxin effects [5,6]. TBBPA was also shown to inhibit triiodothyronine and transthyretin binding and to modulate a number of cell signaling processes [7]. Although extensive biochemical and toxicological studies of TBBPA were reported, its interaction with DNA is not studied. Consequently, it is necessary to investigate the interaction between TBBPA and DNA to deep insight into the binding mechanism at the molecular level and to estimate the toxicology of TBBPA. As the carrier of genetic information in organisms, DNA plays essential roles in many biological processes including gene
110
Y.-Q. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 109–113
expression, transcription, mutagenesis, and carcinogenesis [8]. The diagnosis and therapy of some diseases were often associated with DNA; therefore, the binding interactions of small molecules with DNA have become the hot topics and gained major biological importance [9]. In the present work, the binding of TBBPA to Calf thymus DNA was investigated by using UV–vis absorption, fluorometric competition, circular dichroism (CD), and molecular modeling methods.
Materials and methods Materials Calf thymus DNA (ctDNA) was purchased from Sigma–Aldrich (St. Louis, UO) and used without further purification. TBBPA (98%) was obtained from Aladdin Industrial Corporation (Kaplan Ave, City of Industry). The stock solution of ctDNA was prepared by dissolving appropriate amount of ctDNA in a 0.05 mol L1 potassium phosphate buffer with pH 7.40 by gentle stirring at room temperature and stored at 4 °C. The concentration of ctDNA in stock solution was determined by UV absorption at 260 nm using e260 = 6600 L mol1 cm1. The concentration of ctDNA–ethidium bromide (EB) system was kept [DNA]/[EB] = 10 ([DNA] = 5.0 105 mol L1, [EB] = 5.0 106 mol L1), so all EB molecules were bond into DNA. The TBBPA (0.001 mol L1) solution was prepared in methanol. All other chemicals were of analytical reagent grade. During the experiment, water was purified with a Millli-Q purification system.
Spectral studies The UV–vis absorption spectra of TBBPA (1.0 104 mol L1) in presence and absence of ctDNA were measured on a SPECORD S600 (Jena, Germany) using quartz cells with a 1 cm optical path at T = 298 K. The effects of TBBPA on the fluorescence spectra of ctDNA–EB system were recorded at different temperatures with a LS-50B Spectrofluorimeter (Perkin–Elmer, USA) equipped with 1.0 cm quartz cells and a thermostat bath. For all fluorescence measurements, the excitation and emission slit widths were set at 2.5 nm with the scan speed of 500 nm min1. The fluorescence spectra were recorded in a range of 550–700 nm with the excitation wavelength 295 nm. The CD spectra of ctDNA (5.0 105 mol L1) in the absence and presence of TBBPA were acquired on a Chirascan spectrometer (Applied Photophyysics Ltd., Leatherhead, Surrey, UK) at 298 K. The instrument parameters for CD measurements were, scan speed of 20 nm min1, bandwidth of 1 nm, and spectra range from 200 nm to 300 nm.
Results and discussion UV–Visible absorption spectra of TBBPA in the presence of ctDNA UV–vis absorption spectroscopy is often used to study the binding interactions of small molecules with DNA. Fig. 1 displayed the absorption spectra of TBBPA at different concentrations of ctDNA. TBBPA showed two main absorption peaks at 220 nm and 310 nm which came from the p–p⁄ conjugation between O atom and the benzene and p–p⁄ transitions on the benzene rings, respectively. In presence of ctDNA, the red shift of 220 nm peak and the decreasing of 310 nm peak were observed. The above results indicated that a strong interaction between TBBPA and ctDNA. The obviously spectral change of 310 nm peak coming from p–p⁄ transitions of the benzene rings might be indicative of groove binding because TBBPA contains aromatic ring system. Meanwhile, the electron cloud of TBBPA might be changed by ctDNA. Molecular modeling of TBBPA–DNA interaction Molecular docking study was used to corroborate the above UV–vis spectral results and to deep insight into the interaction between TBBPA and DNA. By monitoring the docking energy of TBBPA with DNA, the possible binding sites were shown in Fig. 2. Except site 5, the other four possible sites were near the minor groove of DNA. The binding free energy values of minor groove poses of TBBPA were higher than that of the binding site 5 for the major groove binding poses (3). The result clearly indicated that TBBPA preferred to intercalate into the double helix of DNA and to sit in the minor groove of DNA [14,15]. The main reason is the minor groove of DNA has the narrower and deeper shape that offered several points of close contact with the surface of TBBPA and perfectly suited the geometry of TBBPA [16]. In addition, Fig. 3 also showed the binding energy of the docked poses and the hydrogen bonds of TBBPA with DNA. There were hydrogen bonds between TBBPA and DNA for the every possible binding site. The hydrogen bonds could help TBBPA to orient easily along the groove of DNA. Moreover, the deep penetration of TBBPA in the minor groove of DNA implied that hydrophobic force might be involved in the binding process of TBBPA with DNA [16]. Fluorescence studies (competitive binding studies) Fluorescence quenching of EB–DNA complex in the presence of TBBPA was used to analyze the binding modes of small molecules
Molecular modeling studies The crystal structure of B-DNA (PDB ID 1DCV) obtained from the Protein Data Bank was used for molecular docking [10]. The three-dimensional structure of TBBPA was optimized at DFT/ B3LYP/6-31G++(d,p) by Gaussian 09 [11]. The Autodock 4.2.3 Program was used to model the binding interaction between B-DNA and TBBPA [12]. In the autodocking, DNA was enclosed in the grid defined by Auto Grid having 0.375 Å spacing using a grid box of 70 Å 70 Å 70 Å. 20 different poses were requested. Other miscellaneous parameters were all assigned the default values given by the AutoDock program. The output from AutoDock was used for further analysis by Molegro Molecular Viewer software [13].
Fig. 1. The absorption spectra of TBBPA with the increasing concentration of ctDNA, c(TBBPA) = 1.0 104 mol L1.
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Fig. 2. The possible binding sites of the Autodock docking runs of TBBPA in DNA.
with DNA [17,18]. If TBBPA can bind to DNA more strongly than EB and displace EB from the ctDNA–EB complex, it may induce a decrease in the fluorescence intensity of ctDNA–EB complex. The emission spectra of the EB–DNA solution in absence and presence of TBBPA were shown in Fig. 4. It can be obviously seen that the fluorescence intensity of EB–DNA complex decreased with an increase in TBBPA concentration. In addition, TBBPA caused more than 50% reduction in the fluorescence intensity of EB–ctDNA complex. This result indicated that strong interaction between TBBPA and ctDNA existed. The fluorescence quenching data was used to calculate the binding constant KA and binding sites n by equation (1) [17]
F 0 F cor 1 log ¼ nlogK A nlog ½TBBPA ðF 0 F cor Þ½EB ctDNA=F 0 F cor ð1Þ where [TBBPA] and [EB–ctDNA] are the total concentration of TBBPA and EB–ctDNA. Since n(ctDNA)/n(EB) = 10, we thought that all EB molecule bond to DNA and the fluorescence intensity of solution all came from EB–ctDNA complex. The plots of Eq. (1) were shown in Fig. 5. Seen from Fig. 5, the DNA binding constants (KA) and binding sites (n) were obtained and shown in Table 1. The results indicated that there existed binding interaction between TBBPA and ctDNA. The values of KA implied that this interaction was moderate intensity, whereas TBBPA competed efficiently with EB by the displacement of it from DNA. The values of n indicated the existence of one mainly binding site in DNA for TBBPA. In
Fig. 4. The fluorescence spectra of EB–ctDNA in the presence of different concentration of TBBPA. c(EB) = 5.0 106 mol L1, c(ctDNA) = 5.0 105 mol L1. pH = 7.40, T = 298 K, kex = 295 nm.
T = 298 K
0.0
T = 310 K
-0.2
log F0-Fcor /Fcor
Fig. 3. The cluster analysis of the Autodock docking runs of TBBPA in DNA. The red bars showed the docked poses and the green bars showed the number of H-bonds corresponding to each pose. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
-0.4 -0.6 -0.8 -1.0 -1.2 4.0
log 1/
4.2
4.4
4.6
TBBPA - F0-Fcor
4.8
5.0
EB-ctDNA /F0
Fig. 5. Fluorescence quenching plots obtained from Eq. (1), giving the binding constant (KA) and the number of binding sites (n) for TBBPA–EB–ctDNA system.
addition, the nature of binding force can be obtained from thermodynamic analysis. The hydrogen bonds, van der Waals force, hydrophobic force, and electrostatic interactions have thermodynamic properties of themselves [19]. For example, a hydrophobic interaction is often indicated by DS (entropy) > 0. The negative enthalpy (DH) values show the hydrogen binding interaction and van der
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Table 1 The binding constant (KA), the number of binding sites (n) and relative thermodynamic parameters of the TBBPA–EB–ctDNA system. T (K) 298 310 a b
KA (L mol1) 3
10.17 10 7.79 103
n
Ra
SDb
DH (kJ mol1)
DG (kJ mol1)
DS (J mol1 K1)
1.06 0.98
0.9994 0.9973
0.0137 0.0277
17.06
22.86 23.09
19.46
The correlation coefficient. The standard deviation.
Waals force. The negative values of DG reveal that the binding process is spontaneous. In order to obtain those thermodynamic parameters between TBBPA and ctDNA, the equations (Eqs. (2)– (4)) were used [20,21]. The DG, DH, and DS were shown in Table 1. From Table 1, the negative sign for DG indicated the spontaneity of the binding of TBBPA with ctDNA. The values of DH were 17.06 kJ mol1, which were the large contribution of DG value. This result showed that the binding interaction of TBBPA with ctDNA was mainly enthalpy driven by means of hydrogen binding interactions. The above conclusion was consistent with the molecular docking data. In addition, the positive values of DS also implied that hydrophobic interaction was also involved in TBBPA– ctDNA binding process. However, TBBPA has pKa values of 7.5 and 8.5 [22], the electrostatic interaction between TBBPA and ctDNA was not mainly binding force in their binding progress [23]. Thus, both hydrogen bonds and hydrophobic interaction played a major role in the binding of TBBPA to ctDNA.
ln
ðK A Þ2 DH 1 1 ¼ ðK A Þ1 R T1 T2
ð2Þ
DG ¼ RT ln K A
ð3Þ
DH DG T
ð4Þ
DS ¼
Circular dichroism studies The circular dichroism (CD) spectral method is often used to investigate the binding interactions of small molecules with DNA [24,25]. The changes in CD signals of DNA in the absence and presence of TBBPA were shown in Fig. 6. ctDNA shows two main bands at 247 nm and 275 nm which come from base stacking and righthanded helicity of DNA, respectively [26]. It can be seen from 6 that the negative band had a slight blue shift and the positive band
decreased in presence of TBBPA. This result depicted that TBBPA bond to DNA, which was also proved by molecular docking study and other spectroscopic experiments. Conclusion In conclusion, we have reported a comprehensive study on the binding interaction of TBBPA with DNA by the spectral and theoretical methods in this paper. The UV–vis and molecular docking data indicated that TBBPA bond to DNA by groove mode. TBBPA entered into the double helix of the DNA and sit in the minor groove of DNA. This binding interaction changed the p–p⁄ transitions and the electron cloud of TBBPA. The fluorescence spectral data showed that TBBPA could displace EB from the ctDNA–EB complex and induce a decrease in the fluorescence intensity of ctDNA–EB complex. The result from fluorescence quenching analysis showed that the binding interaction of TBBPA with ctDNA was mainly enthalpy driven by means of hydrogen binding interactions. In addition, the hydrophobic interaction was also involved in TBBPA–ctDNA binding process. Multi-binding forces pushed the interaction of TBBPA with DNA. According to these methods, we successfully analyze the binding mode of TBBPA with DNA from the experimental and theoretical viewpoint. Although in vitro results could not completely represent the fully biochemical properties of TBBPA, these results not only provided the binding mechanism of TBBPA with DNA but also possessed potential applications in analyzing the binding mechanism of other brominated flame retardants with nucleic acid. Acknowledgements We gratefully acknowledge financial support of the Fund for the National Natural Science Foundation of China (Project No. 21201147), the Natural Science Foundation of Jiangsu Province (Grant Nos. BK2011422 and BK2012671), the Natural Science Foundation of Education Department of Jiangsu Province (Grant No. 11KJB150019), the Jiangsu Fundament of ‘‘Qilan Project’’ and ‘‘333 Project’’, and the sponsorship of Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents. References
Fig. 6. Circular dichroism spectra of DNA (50.0 105 mol L1) in the absence and presence of TBBPA.
[1] S.K. Han, P. Bilski, B. Karriker, R.H. Sik, C.F. Chignell, Environ. Sci. Technol. 42 (2008) 166–172. [2] K.D. Lin, W.P. Liu, J. Gan, Environ. Sci. Technol. 43 (2009) 4480–4486. [3] J. Xu, W. Meng, Y. Zhang, L. Li, C.S. Guo, Appl. Catal. B: Environ. 107 (2011) 355– 362. [4] Y.H. Zhang, Y.L. Tang, S.Y. Li, S.L. Yu, Chem. Eng. J. 222 (2013) 94–100. [5] C.C. Carignan, M.A. Abdallah, N. Wu, W. Heiger-Bernays, M.D. McClean, S. Harrad, T.F. Webster, Environ. Sci. Technol. 46 (2012) 12146–12153. [6] T. Xu, J. Wang, S.Z. Liu, C. Lü, W.L. Shelver, Q.X. Li, J. Li, Anal. Chim. Acta 751 (2012) 119–127. [7] O.A. Ogunbayo, K.T. Jensen, F. Michelangeli, Biochim. Biophys. Acta 1768 (2007) 1559–1566. [8] G.W. Zhang, Y. Ma, Food Chem. 141 (2013) 41–47. [9] W. Hu, S. Deng, J. Huang, Y. Lu, X. Le, W. Zheng, J. Inorg. Biochem. 127 (2013) 90–98. [10] http://www.rcsb.org/pdb/explore.do?structureId=1DCV.
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[19] P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096–3102. [20] N. Shahabadi, S.M. Fili, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 118 (2014) 422–429. [21] S.Y. Bi, B. Pang, T.J. Wang, T.T. Zhao, W. Yu, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 120 (2014) 456–461. [22] EFSA Panel on Contaminants in the Food Chain (CONTAM), Scientific opinion on tetrabromobisphenol A (TBBPA) and its derivatives in food, EFSA J. 9 (2011) 2477–2544. [23] H.M. Zhang, Q.H. Zhou, J. Cao, Y.Q. Wang, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 116 (2013) 251–257. [24] N. Shahabadi, Z.M. Kalar, N.H. Moghadam, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 96 (2012) 723–728. [25] G.W. Zhang, X. Hu, L. Wang, M.M. Hu, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 78 (2011) 687–694. [26] G.W. Zhang, P. Fu, L. Wang, M.M. Hu, J. Agric. Food Chem. 59 (2011) 8944– 8952.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Quest for the binding mode of tetrabromobisphenol A with Calf thymus DNA Yan-Qing Wang ⇑, Hong-Mei Zhang, Jian Cao Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Molecular modeling and multi-
spectroscopic methods were used. TBBPA bound to DNA by groove
mode. The hydrophobic and hydrogen
bonding forces were involved in the binding process. DNA binding changed the electron cloud of TBBPA.
a r t i c l e
i n f o
Article history: Received 24 February 2014 Received in revised form 6 April 2014 Accepted 17 April 2014 Available online 26 April 2014 Keywords: Tetrabromobisphenol A Calf thymus DNA Spectroscopy Binding mode Molecular modeling
a b s t r a c t The binding interaction of tetrabromobisphenol A with Calf thymus DNA was studied by multispectroscopic and molecular modeling methods. The UV–vis study revealed that an obvious interaction between tetrabromobisphenol A and Calf thymus DNA happened. The p–p⁄ transitions and the electron cloud of tetrabromobisphenol A might be changed by entering the groove of Calf thymus DNA. From the fluorescence spectral and thermodynamics studies, it was concluded that the hydrogen bonds and hydrophobic force played a major role in the binding of tetrabromobisphenol A to Calf thymus DNA. The molecular modeling study showed that the possible sites of tetrabromobisphenol A in the groove of DNA. Circular dichroism study also depicted that tetrabromobisphenol A bond to DNA. These above results would further advance our knowledge on the molecular mechanism of the binding interactions of brominated flame-retardants with nucleic acid. Ó 2014 Elsevier B.V. All rights reserved.
Introduction As one of the most widely used brominated flame-retardants in the world, tetrabromobisphenol A (TBBPA) is a potential environmental health problem [1,2]. It has been detected in a wide range of environmental matrices including air, dust, sewage sludge, sediment, wastewater, and aquatic biota [3,4]. Many studies have indicated that TBBPA was characterized as a suspected endocrine ⇑ Corresponding author. Tel./fax: +86 515 88233188. E-mail address: [email protected] (Y.-Q. Wang). http://dx.doi.org/10.1016/j.saa.2014.04.077 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
disruptor and had potential immune- and neurotoxin effects [5,6]. TBBPA was also shown to inhibit triiodothyronine and transthyretin binding and to modulate a number of cell signaling processes [7]. Although extensive biochemical and toxicological studies of TBBPA were reported, its interaction with DNA is not studied. Consequently, it is necessary to investigate the interaction between TBBPA and DNA to deep insight into the binding mechanism at the molecular level and to estimate the toxicology of TBBPA. As the carrier of genetic information in organisms, DNA plays essential roles in many biological processes including gene
110
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expression, transcription, mutagenesis, and carcinogenesis [8]. The diagnosis and therapy of some diseases were often associated with DNA; therefore, the binding interactions of small molecules with DNA have become the hot topics and gained major biological importance [9]. In the present work, the binding of TBBPA to Calf thymus DNA was investigated by using UV–vis absorption, fluorometric competition, circular dichroism (CD), and molecular modeling methods.
Materials and methods Materials Calf thymus DNA (ctDNA) was purchased from Sigma–Aldrich (St. Louis, UO) and used without further purification. TBBPA (98%) was obtained from Aladdin Industrial Corporation (Kaplan Ave, City of Industry). The stock solution of ctDNA was prepared by dissolving appropriate amount of ctDNA in a 0.05 mol L1 potassium phosphate buffer with pH 7.40 by gentle stirring at room temperature and stored at 4 °C. The concentration of ctDNA in stock solution was determined by UV absorption at 260 nm using e260 = 6600 L mol1 cm1. The concentration of ctDNA–ethidium bromide (EB) system was kept [DNA]/[EB] = 10 ([DNA] = 5.0 105 mol L1, [EB] = 5.0 106 mol L1), so all EB molecules were bond into DNA. The TBBPA (0.001 mol L1) solution was prepared in methanol. All other chemicals were of analytical reagent grade. During the experiment, water was purified with a Millli-Q purification system.
Spectral studies The UV–vis absorption spectra of TBBPA (1.0 104 mol L1) in presence and absence of ctDNA were measured on a SPECORD S600 (Jena, Germany) using quartz cells with a 1 cm optical path at T = 298 K. The effects of TBBPA on the fluorescence spectra of ctDNA–EB system were recorded at different temperatures with a LS-50B Spectrofluorimeter (Perkin–Elmer, USA) equipped with 1.0 cm quartz cells and a thermostat bath. For all fluorescence measurements, the excitation and emission slit widths were set at 2.5 nm with the scan speed of 500 nm min1. The fluorescence spectra were recorded in a range of 550–700 nm with the excitation wavelength 295 nm. The CD spectra of ctDNA (5.0 105 mol L1) in the absence and presence of TBBPA were acquired on a Chirascan spectrometer (Applied Photophyysics Ltd., Leatherhead, Surrey, UK) at 298 K. The instrument parameters for CD measurements were, scan speed of 20 nm min1, bandwidth of 1 nm, and spectra range from 200 nm to 300 nm.
Results and discussion UV–Visible absorption spectra of TBBPA in the presence of ctDNA UV–vis absorption spectroscopy is often used to study the binding interactions of small molecules with DNA. Fig. 1 displayed the absorption spectra of TBBPA at different concentrations of ctDNA. TBBPA showed two main absorption peaks at 220 nm and 310 nm which came from the p–p⁄ conjugation between O atom and the benzene and p–p⁄ transitions on the benzene rings, respectively. In presence of ctDNA, the red shift of 220 nm peak and the decreasing of 310 nm peak were observed. The above results indicated that a strong interaction between TBBPA and ctDNA. The obviously spectral change of 310 nm peak coming from p–p⁄ transitions of the benzene rings might be indicative of groove binding because TBBPA contains aromatic ring system. Meanwhile, the electron cloud of TBBPA might be changed by ctDNA. Molecular modeling of TBBPA–DNA interaction Molecular docking study was used to corroborate the above UV–vis spectral results and to deep insight into the interaction between TBBPA and DNA. By monitoring the docking energy of TBBPA with DNA, the possible binding sites were shown in Fig. 2. Except site 5, the other four possible sites were near the minor groove of DNA. The binding free energy values of minor groove poses of TBBPA were higher than that of the binding site 5 for the major groove binding poses (3). The result clearly indicated that TBBPA preferred to intercalate into the double helix of DNA and to sit in the minor groove of DNA [14,15]. The main reason is the minor groove of DNA has the narrower and deeper shape that offered several points of close contact with the surface of TBBPA and perfectly suited the geometry of TBBPA [16]. In addition, Fig. 3 also showed the binding energy of the docked poses and the hydrogen bonds of TBBPA with DNA. There were hydrogen bonds between TBBPA and DNA for the every possible binding site. The hydrogen bonds could help TBBPA to orient easily along the groove of DNA. Moreover, the deep penetration of TBBPA in the minor groove of DNA implied that hydrophobic force might be involved in the binding process of TBBPA with DNA [16]. Fluorescence studies (competitive binding studies) Fluorescence quenching of EB–DNA complex in the presence of TBBPA was used to analyze the binding modes of small molecules
Molecular modeling studies The crystal structure of B-DNA (PDB ID 1DCV) obtained from the Protein Data Bank was used for molecular docking [10]. The three-dimensional structure of TBBPA was optimized at DFT/ B3LYP/6-31G++(d,p) by Gaussian 09 [11]. The Autodock 4.2.3 Program was used to model the binding interaction between B-DNA and TBBPA [12]. In the autodocking, DNA was enclosed in the grid defined by Auto Grid having 0.375 Å spacing using a grid box of 70 Å 70 Å 70 Å. 20 different poses were requested. Other miscellaneous parameters were all assigned the default values given by the AutoDock program. The output from AutoDock was used for further analysis by Molegro Molecular Viewer software [13].
Fig. 1. The absorption spectra of TBBPA with the increasing concentration of ctDNA, c(TBBPA) = 1.0 104 mol L1.
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Fig. 2. The possible binding sites of the Autodock docking runs of TBBPA in DNA.
with DNA [17,18]. If TBBPA can bind to DNA more strongly than EB and displace EB from the ctDNA–EB complex, it may induce a decrease in the fluorescence intensity of ctDNA–EB complex. The emission spectra of the EB–DNA solution in absence and presence of TBBPA were shown in Fig. 4. It can be obviously seen that the fluorescence intensity of EB–DNA complex decreased with an increase in TBBPA concentration. In addition, TBBPA caused more than 50% reduction in the fluorescence intensity of EB–ctDNA complex. This result indicated that strong interaction between TBBPA and ctDNA existed. The fluorescence quenching data was used to calculate the binding constant KA and binding sites n by equation (1) [17]
F 0 F cor 1 log ¼ nlogK A nlog ½TBBPA ðF 0 F cor Þ½EB ctDNA=F 0 F cor ð1Þ where [TBBPA] and [EB–ctDNA] are the total concentration of TBBPA and EB–ctDNA. Since n(ctDNA)/n(EB) = 10, we thought that all EB molecule bond to DNA and the fluorescence intensity of solution all came from EB–ctDNA complex. The plots of Eq. (1) were shown in Fig. 5. Seen from Fig. 5, the DNA binding constants (KA) and binding sites (n) were obtained and shown in Table 1. The results indicated that there existed binding interaction between TBBPA and ctDNA. The values of KA implied that this interaction was moderate intensity, whereas TBBPA competed efficiently with EB by the displacement of it from DNA. The values of n indicated the existence of one mainly binding site in DNA for TBBPA. In
Fig. 4. The fluorescence spectra of EB–ctDNA in the presence of different concentration of TBBPA. c(EB) = 5.0 106 mol L1, c(ctDNA) = 5.0 105 mol L1. pH = 7.40, T = 298 K, kex = 295 nm.
T = 298 K
0.0
T = 310 K
-0.2
log F0-Fcor /Fcor
Fig. 3. The cluster analysis of the Autodock docking runs of TBBPA in DNA. The red bars showed the docked poses and the green bars showed the number of H-bonds corresponding to each pose. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
-0.4 -0.6 -0.8 -1.0 -1.2 4.0
log 1/
4.2
4.4
4.6
TBBPA - F0-Fcor
4.8
5.0
EB-ctDNA /F0
Fig. 5. Fluorescence quenching plots obtained from Eq. (1), giving the binding constant (KA) and the number of binding sites (n) for TBBPA–EB–ctDNA system.
addition, the nature of binding force can be obtained from thermodynamic analysis. The hydrogen bonds, van der Waals force, hydrophobic force, and electrostatic interactions have thermodynamic properties of themselves [19]. For example, a hydrophobic interaction is often indicated by DS (entropy) > 0. The negative enthalpy (DH) values show the hydrogen binding interaction and van der
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Table 1 The binding constant (KA), the number of binding sites (n) and relative thermodynamic parameters of the TBBPA–EB–ctDNA system. T (K) 298 310 a b
KA (L mol1) 3
10.17 10 7.79 103
n
Ra
SDb
DH (kJ mol1)
DG (kJ mol1)
DS (J mol1 K1)
1.06 0.98
0.9994 0.9973
0.0137 0.0277
17.06
22.86 23.09
19.46
The correlation coefficient. The standard deviation.
Waals force. The negative values of DG reveal that the binding process is spontaneous. In order to obtain those thermodynamic parameters between TBBPA and ctDNA, the equations (Eqs. (2)– (4)) were used [20,21]. The DG, DH, and DS were shown in Table 1. From Table 1, the negative sign for DG indicated the spontaneity of the binding of TBBPA with ctDNA. The values of DH were 17.06 kJ mol1, which were the large contribution of DG value. This result showed that the binding interaction of TBBPA with ctDNA was mainly enthalpy driven by means of hydrogen binding interactions. The above conclusion was consistent with the molecular docking data. In addition, the positive values of DS also implied that hydrophobic interaction was also involved in TBBPA– ctDNA binding process. However, TBBPA has pKa values of 7.5 and 8.5 [22], the electrostatic interaction between TBBPA and ctDNA was not mainly binding force in their binding progress [23]. Thus, both hydrogen bonds and hydrophobic interaction played a major role in the binding of TBBPA to ctDNA.
ln
ðK A Þ2 DH 1 1 ¼ ðK A Þ1 R T1 T2
ð2Þ
DG ¼ RT ln K A
ð3Þ
DH DG T
ð4Þ
DS ¼
Circular dichroism studies The circular dichroism (CD) spectral method is often used to investigate the binding interactions of small molecules with DNA [24,25]. The changes in CD signals of DNA in the absence and presence of TBBPA were shown in Fig. 6. ctDNA shows two main bands at 247 nm and 275 nm which come from base stacking and righthanded helicity of DNA, respectively [26]. It can be seen from 6 that the negative band had a slight blue shift and the positive band
decreased in presence of TBBPA. This result depicted that TBBPA bond to DNA, which was also proved by molecular docking study and other spectroscopic experiments. Conclusion In conclusion, we have reported a comprehensive study on the binding interaction of TBBPA with DNA by the spectral and theoretical methods in this paper. The UV–vis and molecular docking data indicated that TBBPA bond to DNA by groove mode. TBBPA entered into the double helix of the DNA and sit in the minor groove of DNA. This binding interaction changed the p–p⁄ transitions and the electron cloud of TBBPA. The fluorescence spectral data showed that TBBPA could displace EB from the ctDNA–EB complex and induce a decrease in the fluorescence intensity of ctDNA–EB complex. The result from fluorescence quenching analysis showed that the binding interaction of TBBPA with ctDNA was mainly enthalpy driven by means of hydrogen binding interactions. In addition, the hydrophobic interaction was also involved in TBBPA–ctDNA binding process. Multi-binding forces pushed the interaction of TBBPA with DNA. According to these methods, we successfully analyze the binding mode of TBBPA with DNA from the experimental and theoretical viewpoint. Although in vitro results could not completely represent the fully biochemical properties of TBBPA, these results not only provided the binding mechanism of TBBPA with DNA but also possessed potential applications in analyzing the binding mechanism of other brominated flame retardants with nucleic acid. Acknowledgements We gratefully acknowledge financial support of the Fund for the National Natural Science Foundation of China (Project No. 21201147), the Natural Science Foundation of Jiangsu Province (Grant Nos. BK2011422 and BK2012671), the Natural Science Foundation of Education Department of Jiangsu Province (Grant No. 11KJB150019), the Jiangsu Fundament of ‘‘Qilan Project’’ and ‘‘333 Project’’, and the sponsorship of Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents. References
Fig. 6. Circular dichroism spectra of DNA (50.0 105 mol L1) in the absence and presence of TBBPA.
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