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Exploring the binding mechanism of phosphoramidate derivative with DNA: Spectroscopy, calorimetry and modeling
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 492–496
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Exploring the binding mechanism of phosphoramidate derivative with DNA: Spectroscopy, calorimetry and modeling Gongke Wang a, Hongwei Wu b, Dongchao Wang a, Changling Yan a, Yan Lu a,⇑ a b
School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007, PR China Department of Chemistry, School of Basic Medicine, Xinxiang Medical University, Xinxiang, Henan 453003, PR 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
" Medically important
phosphoramidate derivative HMPAP is synthesized. " HMPAP is intercalative binding into ctDNA helix. " Hydrogen bonding may play an essential role in the binding of HMPAP with ctDNA. " This binding interaction is predominantly enthalpy driven.
a r t i c l e
i n f o
Article history: Received 29 June 2012 Received in revised form 11 November 2012 Accepted 16 November 2012 Available online 5 December 2012 Keywords: Amino phosphine ester derivative DNA Intercalation Spectroscopy Isothermal titration calorimetry Molecular modeling
a b s t r a c t In this study, one of the amino phosphine ester derivatives a-(3-hydroxy-4-methoxyphenyl)-N-phenyla-aminophosphonate (HMPAP) was synthesized, and the molecular interaction of HMPAP with ct-DNA has been investigated by UV–Vis absorption spectra, fluorescence spectra, isothermal titration calorimetry (ITC) and molecular modeling. The binding constant (Kb) of HMPAP to ct-DNA at different temperatures were calculated from fluorescence spectra. According to the UV–Vis absorption spectra, ethidium bromide displacement studies and ITC experimental results, we can conclude that HMPAP is an intercalator. The molecular modeling results indicated that HMPAP can slide into the G–C rich region of ct-DNA. ITC data showed that ct-DNA/HMPAP binding is enthalpy controlled. Furthermore, the results obtained from molecular modeling corroborated the experimental results obtanied from spectroscopic and ITC investigations. Ó 2012 Elsevier B.V. All rights reserved.
Introduction It is well known that deoxyribonucleic acid (DNA) is an important genetic substance in organisms. Over recent decades, many researches have been focused on interactions of small molecules with DNA [1,2]. It has been reported that DNA is generally the ⇑ Corresponding author. Address: School of Chemistry and Chemical Engineering, Henan Normal University, 46 Jian-she Road, Mu Ye District, Xinxiang 453007, PR China. Tel./fax: +86 373 3325249. E-mail address: [email protected] (Y. Lu). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.11.060
primary intracellular target of anticancer drugs which can cause DNA damage in cancer cells, blocking the division of cancer cells, and resulting in cell death [3,4]. Therefore, the binding studies of small molecules to DNA are very important in the development of DNA molecular probes and new therapeutic reagents [5,6]. Small molecules such as drugs [7], metals [8] and cationic lipids can interact with DNA through the following three non-covalent modes: groove binding, intercalation and external static electronic effects. Intercalation is one of the most important DNA-binding modes, which is related to the antitumor activity of drugs. It has been reported that the intercalating ability of the complex was
G. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 492–496
493
Fluorescence measurements
Fig. 1. Structure of HMPAP.
involved in the planarity of ligand, ligand donor atom type and the metal ion type. Recently, many studies involve antibiotics, transition metal complexes and heterocyclic dyes on intercalation have been performed [9]. So the development of synthetic and sequence-selective DNA binding agents for DNA and for new potential DNA-targeted antitumor drugs is essential in molecular biology and medicine fields. a-(3-Hydroxy-4-methoxyphenyl)-N-phenyl-a-aminophosphonate (HMPAP) is the analogue of a-amino acids. It is an important role in organic phosphorus chemistry in recent years [10]. The changeable structure types are the foundations of the new synthesized compound. HMPAP has the antibacterial, antioxidant, antitumor and bactericidal activity, which is similar to amino phosphate ester. The structure of HMPAP is shown in Fig. 1. In this work, HMPAP was synthesized and its interaction with calf-thymus DNA (ct-DNA) has been studied by using UV–Vis absorption spectroscopy, fluorescence spectroscopy, molecular modeling and isothermal titration calorimetry (ITC). We investigated the mode of DNA binding and demonstrate HMPAP could be bound to DNA in GC region via an intercalation mode. Materials and methods Materials HMPAP was synthesized in our laboratory. Calf thymus DNA (ct-DNA) was obtained from the Sigma Company (Sigma–Aldrich, USA) and used without further purification. The concentration of DNA was determined by absorption spectroscopy using the molar extinction coefficient of 6600 M1 cm1 at 260 nm [11]. The solutions of ct-DNA had a ratio of UV absorbance at 260 and 280 nm of more than 1.8, indicating that the DNA was sufficiently free from protein [12]. Ethidium bromide (EB) was obtained from the Sigma Company (Sigma–Aldrich, USA). The measurement of pH value of solution was performed on a pHS-2C pH-meter (Shanghai DaPu Instruments Co., Ltd., Shanghai, China). Doubly distilled water was used throughout. All the experiments were carried out in phosphate buffer (20 mM, pH 7.4) containing 0.05 M NaCl. All other chemical reagents were of analytical grade.
The fluorescence measurements were performed on Cary Eclipse fluorescence spectrophotometer (Varian, USA). The excitation wavelength was fixed and the emission range was adjusted before measurements. Fluorescence titrations were carried out by adding increasing amounts of ct-DNA directly into the solution of the HMPAP (c = 10.0 lM, containing 20 mM phosphate buffer of pH 7.4). The excitation wavelength of HMPAP was recorded by using 463 nm. Both excitation and emission bandwidths were adjusted to 5 nm. Competitive displacement experiments were conducted by adding increasing amounts of HMPAP (0–100 lM) directly into the ethidium bromide (EB)–DNA system (cEB = 5 lM, cDNA = 100 lM, 20 mM phosphate buffer of pH 7.4). Excitation wavelength was set to 500 nm. Molecular modeling studies The crystal structure of B-DNA used for docking was downloaded from the Protein Data Bank (identifier 425D) [13]. The potential of 3D structure of DNA was assigned according to the Amber 4.0 force field with Kollman-all-atom charges. The 3D structure of the molecules was generated by Sybyl 6.9. The geometries of the HMPAP were subsequently optimized to minimal energy using the Tripos force field with Gasteiger–Marsili charges. Ligand docking was carried out with the software Sybyl 6.9. PyMol was used to visualize the docked conformations and calculate the distances between possible hydrogen bonding partners. The FlexX software as a part of the Sybyl suite was used for docking of compound to DNA. An energy minimization was performed on each ligand to get the energy-minimized conformation before the docking runs. During the docking run, the ligand atoms were allowed to move within a specified region to achieve the energy conformation. Isothermal titration calorimetry ITC was performed using a Model Nano-ITC 2G biocalorimetry instrument (TA, USA) with the calorimeter cell (1.0 mL). The temperature of each cell is monitored and maintained at 25 °C through an electronic feedback loop that controls thermoelectric heaters located adjacent to each cell. HMPAP (1.0 mM) was introduced into the sample cell by means of syringes via 25 individual injections; the amount of each injection was 10 lL in 20 mM phosphate buffer. ct-DNA (0.05 mM) was maintained in the sample cell in 20 mM phosphate buffer (pH = 7.4) and the reference cell contained doubly distilled water. All the solutions used for ITC experiments were thoroughly degassed prior to use. After the peak area was integrated and the heats of dilution were subtracted, the thermogram for the binding was obtained. The titration data was fitted to an independent binding model and the intrinsic molar enthalpy change for the binding (DH0), the binding constant (Kb) and the binding stoichiometry (n) were thus obtained.
Absorbance measurements
Results and discussions
UV–Vis absorption spectra were recorded with a TU-1810 spectrophotometer (Puxi Analytic Instrument Ltd., Beijing, China) equipped with 1.0 cm quartz cells. The solutions of the blank buffer and sample were placed in the reference and sample cuvettes, respectively. The UV–Vis absorption spectra experiments were performed by keeping the concentration of the HMPAP (10.0 lM) constant while varying the concentration of ct-DNA. All measurements were performed at 25 °C.
Fluorescence spectroscopic studies The binding of HMPAP to DNA has been characterized classically through fluorescence spectroscopy. Fig. 2 shows the fluorescence emission spectra of HMPAP in the absence and presence of ct-DNA. HMPAP has an emission maximum at 521 nm when excited at 463 nm. The fluorescence intensity increased with the addition of ct-DNA without wavelength shift was observed. The
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Fig. 2. The change of fluorescence emission spectra of HMPAP (1 105 M) in 20 mM phosphate buffer of pH 7.4, in the absence and presence of increasing concentration ct-DNA (from bottom to top, 0–20 lM), kex ¼ 463 nm. Arrow shows the intensity increased with increasing DNA concentration.
stronger enhancement of fluorescence emission spectra may be due to the planar aromatic group of HMPAP stacking between adjacent base pairs of ct-DNA. As a result, the compound is protected from solvent water molecules by the hydrophobic environment inside the DNA helix; the accessibility of solvent water molecules to these compounds is reduced [14]. The spectral characteristics also agree with those observed for other intercalators [15,16]. In order to further investigate the binding mode of HMPAP to DNA, the competitive binding experiment was carried out using EB as a probe. EB is one of the most sensitive fluorescence probes having a planar structure that binds to DNA by an intercalative mode [17,18]. EB emits weak fluorescence in aqueous solution, however the fluorescence intensity of EB remarkably increases while in the presence of DNA due to its strong intercalation between the adjacent DNA base pairs [19]. This enhanced fluorescence of the DNA–EB complex can be quenched by the addition of a second ligand molecule. If the ligand intercalates into DNA, it leads to a decrease in the binding sites of DNA available for EB, which in turn decreases the fluorescence intensity of the EB–DNA system [20]. The fluorescent emission of EB bound to DNA in the absence and presence of complex is shown in Fig. 3. It is shown that a regular decrease in the fluorescence intensity of DNA–EB complex but no shift of fluorescence emission took place upon increasing the concentration of HMPAP. It has been reported that small organic molecules interact with DNA by intercalation when the concentration ratio of them to DNA is less than 100 and the fluorescence intensity of EB–DNA system decreases by 50% [21]. Herein, the quenching extent has reached up to 43.2%. This phenomenon indicates that HMPAP can compete with EB in binding to DNA. According to the classical Stern–Volmer equation [22]:
I0 ¼ 1 þ Kqr I
Fig. 3. Emission spectra of DNA–EB system (100 lM DNA and 10 lM EB), in the presence of HMPAP (from top to bottom 0–100 lM), kex ¼ 500 nm. Arrow shows the emission intensity changes upon increasing HMPAP concentration. The inset is Stern–Volmer quenching plot of DNA–EB system by HMPAP.
the value of Kq of HMPAP binding to DNA is 0.9, which suggests that the interaction of HMPAP with DNA is moderate [23].
UV–Vis absorption spectroscopy To understand the interaction pattern of HMPAP with DNA more clearly, the absorption experiment was carried out. The absorption spectra of HMPAP in the absence and presence of different concentrations of DNA is given in Fig. 4. The electronic absorption spectra of HMPAP exhibited broad absorption bands in the region 200–300 nm. The addition of ct-DNA to the solution of HMPAP resulted in a decrease in the absorption at 235 nm. The hypochromicity suggests HMPAP may bind to DNA by intercalation mode, due to a strong interaction between the electronic states of the intercalation chromophore and those of DNA bases [24].
ð1Þ
where I0 and I represent the fluorescence intensities in the absence and presence of HMPAP, respectively. Kq is a linear Stern–Volmer quenching constant dependent on the concentration ratio of the bound of EB to DNA, and r is the concentration ratio of HMPAP to DNA. The Kq value is obtained as the slope of I0/I versus [Q] linear plot. The fluorescence quenching curve of DNA–bound EB by HMPAP is given in the inset of Fig. 3. From Fig. 3, we know that
Fig. 4. UV/Vis absorption spectra of HMPAP (1 105 M) in the absence (top) and presence of ct-DNA (0–100 lM) in 20 mM phosphate buffer (pH = 7.4). The arrow shows the intensity decreased with increasing DNA concentration.
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Table 1 Binding constants Kb and binding sites n of the system of HMPAP–DNA at different temperatures.
a
pH
T (K)
Kb (104 M1)
n
Ra
7.4
288 298 310
3.04 1.91 1.01
0.88 0.86 0.82
0.9987 0.9988 0.9974
R is the correlation coefficient.
Binding equilibrium analysis When small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant Kb can be determined by the following equation [25]:
lg
F0 F ¼ lgK b þ nlg½Q F
ð2Þ
where F0 and F represent the steady-state fluorescence intensities in the absence and presence of enhancer. Kb is the binding constant and n is the number of binding sites per DNA, which can be determined, respectively, by the intercept and slope of double logarithm regression curve of log (F F0)/F versus log [Q] based on the equation. The Kb values for HMPAP to DNA at different temperatures are presented in Table 1. From Table 1, we can see that the binding constants of HMPAP to DNA are similar with other intercalators. Therefore, the binding constants determined here also confirm that HMPAP may intercalate into ct-DNA helix. Molecular modeling studies Docking studies provide further insight into the interactions between the ligand and macromolecule [26,27]. The docked structure as presented in Fig. S1 (shown in supplementary materials) exhibits the optimal energy ranked results of HMPAP interaction with ct-DNA. It suggests that the chromophore of HMPAP can slide into the G–C rich region of ct-DNA. In addition, there are hydrogen interactions of the atom O of hydroxyl group for HMPAP with the O atom of C21, and the length of the hydrogen bond is 2.05 Å. The results of molecular modeling indicate that the interaction between HMPAP and DNA is intercalation dominated and hydrogen bonding may play an essential role in the binding. Isothermal titration calorimetry To obtain further insight into the thermodynamics of the DNA binding process, the interaction of HMPAP with ct-DNA was investigated using ITC. A typical titration is shown in Fig. 5. Each addition of the HMPAP solution (1.0 mM) to the calorimeter cell, which contained a solution of ct-DNA (0.05 mM), resulted in the release of heat until the binding was saturated. The data must be corrected for the dilution heats associated with the addition of buffer into ct-DNA and drug into buffer solution. The area under each peak was integrated, heats of dilution are subtracted, and the thermogram for the binding of HMPAP to ct-DNA has been obtained. The heat released during the binding is related to the number of binding sites available, binding constant and thermodynamic parameters of the binding equilibrium [28]. These data were best fitted to independent binding model and yielding the binding parameters DH0 = 86.21 kJ mol1, n = 0.80 and Kb = 2.92 104 M1. Additionally, the entropy and free energy of binding can be calculated from the following equations:
DG0 ¼ RT ln K b
ð3Þ
DG0 ¼ DH0 T DS0
ð4Þ
Fig. 5. ITC curve of the binding of HMPAP to ct-DNA at 25 °C in 20 mM phosphate buffer (pH = 7.4).
According to Eqs. (3) and (4), we calculated DS0 = 222.96 J mol1 K1 and DG0 = 19.77 kJ mol1. In general, the binding of an intercalator to DNA is driven entirely by a large favorable enthalpy reduction but with an unfavorable entropy decrease [29]. The results indicated that the binding of HMPAP to ct-DNA is driven by a large favorable enthalpy along with unfavorable entropy contributions. So we can conclude that HMPAP binds to ct-DNA through intercalation binding. In addition, the binding enthalpy and binding entropy are both negative, thus we can conjecture that hydrogen bonds and van der Waals force may play an important role in the binding.
Conclusions In this paper, the interaction of HMPAP with ct-DNA was studied by spectroscopic, ITC and molecular modeling techniques. The results obtained from the fluorescence spectroscopy and UV–Vis absorption spectroscopy, we concluded that HMPAP can bind to ct-DNA with high affinity through intercalation binding. Moreover, the molecular modeling and ITC studies further confirm their intercalative binding mode. The binding study of drugs to DNA is greatly important in understanding chemico-biological interactions for drug design, pharmacology and biochemistry. Furthermore, these studies are expected to provide important insight into the design of new HMPAP drugs.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant Nos. 21173071, 21273061) and the Research Fund for the Doctoral Program of Higher Education of China (No.20114104110002) for their financial supports. In addition, we thank Lanzhou University for supporting the molecular modeling software (Sybyl 6.9) and SGI FUEL workstations.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2012.11.060.
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Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Exploring the binding mechanism of phosphoramidate derivative with DNA: Spectroscopy, calorimetry and modeling Gongke Wang a, Hongwei Wu b, Dongchao Wang a, Changling Yan a, Yan Lu a,⇑ a b
School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007, PR China Department of Chemistry, School of Basic Medicine, Xinxiang Medical University, Xinxiang, Henan 453003, PR 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
" Medically important
phosphoramidate derivative HMPAP is synthesized. " HMPAP is intercalative binding into ctDNA helix. " Hydrogen bonding may play an essential role in the binding of HMPAP with ctDNA. " This binding interaction is predominantly enthalpy driven.
a r t i c l e
i n f o
Article history: Received 29 June 2012 Received in revised form 11 November 2012 Accepted 16 November 2012 Available online 5 December 2012 Keywords: Amino phosphine ester derivative DNA Intercalation Spectroscopy Isothermal titration calorimetry Molecular modeling
a b s t r a c t In this study, one of the amino phosphine ester derivatives a-(3-hydroxy-4-methoxyphenyl)-N-phenyla-aminophosphonate (HMPAP) was synthesized, and the molecular interaction of HMPAP with ct-DNA has been investigated by UV–Vis absorption spectra, fluorescence spectra, isothermal titration calorimetry (ITC) and molecular modeling. The binding constant (Kb) of HMPAP to ct-DNA at different temperatures were calculated from fluorescence spectra. According to the UV–Vis absorption spectra, ethidium bromide displacement studies and ITC experimental results, we can conclude that HMPAP is an intercalator. The molecular modeling results indicated that HMPAP can slide into the G–C rich region of ct-DNA. ITC data showed that ct-DNA/HMPAP binding is enthalpy controlled. Furthermore, the results obtained from molecular modeling corroborated the experimental results obtanied from spectroscopic and ITC investigations. Ó 2012 Elsevier B.V. All rights reserved.
Introduction It is well known that deoxyribonucleic acid (DNA) is an important genetic substance in organisms. Over recent decades, many researches have been focused on interactions of small molecules with DNA [1,2]. It has been reported that DNA is generally the ⇑ Corresponding author. Address: School of Chemistry and Chemical Engineering, Henan Normal University, 46 Jian-she Road, Mu Ye District, Xinxiang 453007, PR China. Tel./fax: +86 373 3325249. E-mail address: [email protected] (Y. Lu). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.11.060
primary intracellular target of anticancer drugs which can cause DNA damage in cancer cells, blocking the division of cancer cells, and resulting in cell death [3,4]. Therefore, the binding studies of small molecules to DNA are very important in the development of DNA molecular probes and new therapeutic reagents [5,6]. Small molecules such as drugs [7], metals [8] and cationic lipids can interact with DNA through the following three non-covalent modes: groove binding, intercalation and external static electronic effects. Intercalation is one of the most important DNA-binding modes, which is related to the antitumor activity of drugs. It has been reported that the intercalating ability of the complex was
G. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 492–496
493
Fluorescence measurements
Fig. 1. Structure of HMPAP.
involved in the planarity of ligand, ligand donor atom type and the metal ion type. Recently, many studies involve antibiotics, transition metal complexes and heterocyclic dyes on intercalation have been performed [9]. So the development of synthetic and sequence-selective DNA binding agents for DNA and for new potential DNA-targeted antitumor drugs is essential in molecular biology and medicine fields. a-(3-Hydroxy-4-methoxyphenyl)-N-phenyl-a-aminophosphonate (HMPAP) is the analogue of a-amino acids. It is an important role in organic phosphorus chemistry in recent years [10]. The changeable structure types are the foundations of the new synthesized compound. HMPAP has the antibacterial, antioxidant, antitumor and bactericidal activity, which is similar to amino phosphate ester. The structure of HMPAP is shown in Fig. 1. In this work, HMPAP was synthesized and its interaction with calf-thymus DNA (ct-DNA) has been studied by using UV–Vis absorption spectroscopy, fluorescence spectroscopy, molecular modeling and isothermal titration calorimetry (ITC). We investigated the mode of DNA binding and demonstrate HMPAP could be bound to DNA in GC region via an intercalation mode. Materials and methods Materials HMPAP was synthesized in our laboratory. Calf thymus DNA (ct-DNA) was obtained from the Sigma Company (Sigma–Aldrich, USA) and used without further purification. The concentration of DNA was determined by absorption spectroscopy using the molar extinction coefficient of 6600 M1 cm1 at 260 nm [11]. The solutions of ct-DNA had a ratio of UV absorbance at 260 and 280 nm of more than 1.8, indicating that the DNA was sufficiently free from protein [12]. Ethidium bromide (EB) was obtained from the Sigma Company (Sigma–Aldrich, USA). The measurement of pH value of solution was performed on a pHS-2C pH-meter (Shanghai DaPu Instruments Co., Ltd., Shanghai, China). Doubly distilled water was used throughout. All the experiments were carried out in phosphate buffer (20 mM, pH 7.4) containing 0.05 M NaCl. All other chemical reagents were of analytical grade.
The fluorescence measurements were performed on Cary Eclipse fluorescence spectrophotometer (Varian, USA). The excitation wavelength was fixed and the emission range was adjusted before measurements. Fluorescence titrations were carried out by adding increasing amounts of ct-DNA directly into the solution of the HMPAP (c = 10.0 lM, containing 20 mM phosphate buffer of pH 7.4). The excitation wavelength of HMPAP was recorded by using 463 nm. Both excitation and emission bandwidths were adjusted to 5 nm. Competitive displacement experiments were conducted by adding increasing amounts of HMPAP (0–100 lM) directly into the ethidium bromide (EB)–DNA system (cEB = 5 lM, cDNA = 100 lM, 20 mM phosphate buffer of pH 7.4). Excitation wavelength was set to 500 nm. Molecular modeling studies The crystal structure of B-DNA used for docking was downloaded from the Protein Data Bank (identifier 425D) [13]. The potential of 3D structure of DNA was assigned according to the Amber 4.0 force field with Kollman-all-atom charges. The 3D structure of the molecules was generated by Sybyl 6.9. The geometries of the HMPAP were subsequently optimized to minimal energy using the Tripos force field with Gasteiger–Marsili charges. Ligand docking was carried out with the software Sybyl 6.9. PyMol was used to visualize the docked conformations and calculate the distances between possible hydrogen bonding partners. The FlexX software as a part of the Sybyl suite was used for docking of compound to DNA. An energy minimization was performed on each ligand to get the energy-minimized conformation before the docking runs. During the docking run, the ligand atoms were allowed to move within a specified region to achieve the energy conformation. Isothermal titration calorimetry ITC was performed using a Model Nano-ITC 2G biocalorimetry instrument (TA, USA) with the calorimeter cell (1.0 mL). The temperature of each cell is monitored and maintained at 25 °C through an electronic feedback loop that controls thermoelectric heaters located adjacent to each cell. HMPAP (1.0 mM) was introduced into the sample cell by means of syringes via 25 individual injections; the amount of each injection was 10 lL in 20 mM phosphate buffer. ct-DNA (0.05 mM) was maintained in the sample cell in 20 mM phosphate buffer (pH = 7.4) and the reference cell contained doubly distilled water. All the solutions used for ITC experiments were thoroughly degassed prior to use. After the peak area was integrated and the heats of dilution were subtracted, the thermogram for the binding was obtained. The titration data was fitted to an independent binding model and the intrinsic molar enthalpy change for the binding (DH0), the binding constant (Kb) and the binding stoichiometry (n) were thus obtained.
Absorbance measurements
Results and discussions
UV–Vis absorption spectra were recorded with a TU-1810 spectrophotometer (Puxi Analytic Instrument Ltd., Beijing, China) equipped with 1.0 cm quartz cells. The solutions of the blank buffer and sample were placed in the reference and sample cuvettes, respectively. The UV–Vis absorption spectra experiments were performed by keeping the concentration of the HMPAP (10.0 lM) constant while varying the concentration of ct-DNA. All measurements were performed at 25 °C.
Fluorescence spectroscopic studies The binding of HMPAP to DNA has been characterized classically through fluorescence spectroscopy. Fig. 2 shows the fluorescence emission spectra of HMPAP in the absence and presence of ct-DNA. HMPAP has an emission maximum at 521 nm when excited at 463 nm. The fluorescence intensity increased with the addition of ct-DNA without wavelength shift was observed. The
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G. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 492–496
Fig. 2. The change of fluorescence emission spectra of HMPAP (1 105 M) in 20 mM phosphate buffer of pH 7.4, in the absence and presence of increasing concentration ct-DNA (from bottom to top, 0–20 lM), kex ¼ 463 nm. Arrow shows the intensity increased with increasing DNA concentration.
stronger enhancement of fluorescence emission spectra may be due to the planar aromatic group of HMPAP stacking between adjacent base pairs of ct-DNA. As a result, the compound is protected from solvent water molecules by the hydrophobic environment inside the DNA helix; the accessibility of solvent water molecules to these compounds is reduced [14]. The spectral characteristics also agree with those observed for other intercalators [15,16]. In order to further investigate the binding mode of HMPAP to DNA, the competitive binding experiment was carried out using EB as a probe. EB is one of the most sensitive fluorescence probes having a planar structure that binds to DNA by an intercalative mode [17,18]. EB emits weak fluorescence in aqueous solution, however the fluorescence intensity of EB remarkably increases while in the presence of DNA due to its strong intercalation between the adjacent DNA base pairs [19]. This enhanced fluorescence of the DNA–EB complex can be quenched by the addition of a second ligand molecule. If the ligand intercalates into DNA, it leads to a decrease in the binding sites of DNA available for EB, which in turn decreases the fluorescence intensity of the EB–DNA system [20]. The fluorescent emission of EB bound to DNA in the absence and presence of complex is shown in Fig. 3. It is shown that a regular decrease in the fluorescence intensity of DNA–EB complex but no shift of fluorescence emission took place upon increasing the concentration of HMPAP. It has been reported that small organic molecules interact with DNA by intercalation when the concentration ratio of them to DNA is less than 100 and the fluorescence intensity of EB–DNA system decreases by 50% [21]. Herein, the quenching extent has reached up to 43.2%. This phenomenon indicates that HMPAP can compete with EB in binding to DNA. According to the classical Stern–Volmer equation [22]:
I0 ¼ 1 þ Kqr I
Fig. 3. Emission spectra of DNA–EB system (100 lM DNA and 10 lM EB), in the presence of HMPAP (from top to bottom 0–100 lM), kex ¼ 500 nm. Arrow shows the emission intensity changes upon increasing HMPAP concentration. The inset is Stern–Volmer quenching plot of DNA–EB system by HMPAP.
the value of Kq of HMPAP binding to DNA is 0.9, which suggests that the interaction of HMPAP with DNA is moderate [23].
UV–Vis absorption spectroscopy To understand the interaction pattern of HMPAP with DNA more clearly, the absorption experiment was carried out. The absorption spectra of HMPAP in the absence and presence of different concentrations of DNA is given in Fig. 4. The electronic absorption spectra of HMPAP exhibited broad absorption bands in the region 200–300 nm. The addition of ct-DNA to the solution of HMPAP resulted in a decrease in the absorption at 235 nm. The hypochromicity suggests HMPAP may bind to DNA by intercalation mode, due to a strong interaction between the electronic states of the intercalation chromophore and those of DNA bases [24].
ð1Þ
where I0 and I represent the fluorescence intensities in the absence and presence of HMPAP, respectively. Kq is a linear Stern–Volmer quenching constant dependent on the concentration ratio of the bound of EB to DNA, and r is the concentration ratio of HMPAP to DNA. The Kq value is obtained as the slope of I0/I versus [Q] linear plot. The fluorescence quenching curve of DNA–bound EB by HMPAP is given in the inset of Fig. 3. From Fig. 3, we know that
Fig. 4. UV/Vis absorption spectra of HMPAP (1 105 M) in the absence (top) and presence of ct-DNA (0–100 lM) in 20 mM phosphate buffer (pH = 7.4). The arrow shows the intensity decreased with increasing DNA concentration.
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Table 1 Binding constants Kb and binding sites n of the system of HMPAP–DNA at different temperatures.
a
pH
T (K)
Kb (104 M1)
n
Ra
7.4
288 298 310
3.04 1.91 1.01
0.88 0.86 0.82
0.9987 0.9988 0.9974
R is the correlation coefficient.
Binding equilibrium analysis When small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant Kb can be determined by the following equation [25]:
lg
F0 F ¼ lgK b þ nlg½Q F
ð2Þ
where F0 and F represent the steady-state fluorescence intensities in the absence and presence of enhancer. Kb is the binding constant and n is the number of binding sites per DNA, which can be determined, respectively, by the intercept and slope of double logarithm regression curve of log (F F0)/F versus log [Q] based on the equation. The Kb values for HMPAP to DNA at different temperatures are presented in Table 1. From Table 1, we can see that the binding constants of HMPAP to DNA are similar with other intercalators. Therefore, the binding constants determined here also confirm that HMPAP may intercalate into ct-DNA helix. Molecular modeling studies Docking studies provide further insight into the interactions between the ligand and macromolecule [26,27]. The docked structure as presented in Fig. S1 (shown in supplementary materials) exhibits the optimal energy ranked results of HMPAP interaction with ct-DNA. It suggests that the chromophore of HMPAP can slide into the G–C rich region of ct-DNA. In addition, there are hydrogen interactions of the atom O of hydroxyl group for HMPAP with the O atom of C21, and the length of the hydrogen bond is 2.05 Å. The results of molecular modeling indicate that the interaction between HMPAP and DNA is intercalation dominated and hydrogen bonding may play an essential role in the binding. Isothermal titration calorimetry To obtain further insight into the thermodynamics of the DNA binding process, the interaction of HMPAP with ct-DNA was investigated using ITC. A typical titration is shown in Fig. 5. Each addition of the HMPAP solution (1.0 mM) to the calorimeter cell, which contained a solution of ct-DNA (0.05 mM), resulted in the release of heat until the binding was saturated. The data must be corrected for the dilution heats associated with the addition of buffer into ct-DNA and drug into buffer solution. The area under each peak was integrated, heats of dilution are subtracted, and the thermogram for the binding of HMPAP to ct-DNA has been obtained. The heat released during the binding is related to the number of binding sites available, binding constant and thermodynamic parameters of the binding equilibrium [28]. These data were best fitted to independent binding model and yielding the binding parameters DH0 = 86.21 kJ mol1, n = 0.80 and Kb = 2.92 104 M1. Additionally, the entropy and free energy of binding can be calculated from the following equations:
DG0 ¼ RT ln K b
ð3Þ
DG0 ¼ DH0 T DS0
ð4Þ
Fig. 5. ITC curve of the binding of HMPAP to ct-DNA at 25 °C in 20 mM phosphate buffer (pH = 7.4).
According to Eqs. (3) and (4), we calculated DS0 = 222.96 J mol1 K1 and DG0 = 19.77 kJ mol1. In general, the binding of an intercalator to DNA is driven entirely by a large favorable enthalpy reduction but with an unfavorable entropy decrease [29]. The results indicated that the binding of HMPAP to ct-DNA is driven by a large favorable enthalpy along with unfavorable entropy contributions. So we can conclude that HMPAP binds to ct-DNA through intercalation binding. In addition, the binding enthalpy and binding entropy are both negative, thus we can conjecture that hydrogen bonds and van der Waals force may play an important role in the binding.
Conclusions In this paper, the interaction of HMPAP with ct-DNA was studied by spectroscopic, ITC and molecular modeling techniques. The results obtained from the fluorescence spectroscopy and UV–Vis absorption spectroscopy, we concluded that HMPAP can bind to ct-DNA with high affinity through intercalation binding. Moreover, the molecular modeling and ITC studies further confirm their intercalative binding mode. The binding study of drugs to DNA is greatly important in understanding chemico-biological interactions for drug design, pharmacology and biochemistry. Furthermore, these studies are expected to provide important insight into the design of new HMPAP drugs.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant Nos. 21173071, 21273061) and the Research Fund for the Doctoral Program of Higher Education of China (No.20114104110002) for their financial supports. In addition, we thank Lanzhou University for supporting the molecular modeling software (Sybyl 6.9) and SGI FUEL workstations.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2012.11.060.
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