Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 78–83
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Spectroscopic and molecular modeling methods to investigate the interaction between 5-Hydroxymethyl-2-furfural and calf thymus DNA using ethidium bromide as a probe Jinhua Zhu a,⇑, Lanlan Chen a, Yingying Dong b, Jiazhong Li c, Xiuhua Liu a,b a b c
Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical Engineering, Henan University, Kaifeng, Henan 475004, China Key Lab of Natural Drug and Immune Engineering of Henan Province, Kaifeng, Henan 475004, China School of Pharmacy, Lanzhou University, Donggang West Road 199, 730000 Lanzhou, 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
Spectroscopic and molecular
250
Fluorescence Intensity
modeling methods were used. The quenching of DNA–EB system by 5-HMF was a static quenching. The binding of 5-HMF to DNA was a non- intercalative binding. Hydrophobic force and hydrogen bonds both played a major role.
A
200
O HO
O
150
E
100
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0
560
595
630
665
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735
Wavelength(nm)
a r t i c l e
i n f o
Article history: Received 31 October 2013 Received in revised form 20 December 2013 Accepted 23 December 2013 Available online 6 January 2014 Keywords: 5-Hydroxymethyl-2-furfural Calf thymus DNA Ethidium bromide Spectroscopy Molecular modeling
a b s t r a c t In this work, the interaction of 5-Hydroxymethyl-2-furfural (5-HMF) with calf thymus DNA (ctDNA) under simulated physiological conditions (Tris–HCl buffer of pH 7.40), was explored by UV absorption spectroscopy, fluorescence spectroscopy and molecular modeling method, using ethidium bromide (EB) as a fluorescence probe of DNA. The fluorescence quenching mechanism of EB–ctDNA by 5-HMF was confirmed to be a static quenching, which derived from the formation of a new complex. The binding constants of 5-HMF with DNA in the presence of EB were calculated to be 2.17 103, 4.24 103 and 6.95 103 L mol1 at 300, 305 and 310 K, respectively. The calculated thermodynamic parameters, enthalpy change DH and entropy change DS, suggested that both hydrophobic interactions and hydrogen bonds played a predominant role in the binding of 5-HMF to DNA. According to the UV absorption spectroscopy and melting temperature (Tm) curve results, the binding mode of 5-HMF with DNA was indicative of a non-intercalative binding, which was supposed to be a groove binding. The molecular modeling results showed that 5-HMF could bind into the hydrophobic region of ctDNA and supported the conclusions obtained from the above experiments. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Deoxyribonucleic acid (DNA) that contains the genetic instructions for the development and functioning of living organisms is an ⇑ Corresponding author. Tel./fax: +86 371 23881589. E-mail address: [email protected] (J. Zhu). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.12.091
important target of many drugs, especially anticancer and antiviral drugs [1–3]. The binding of drugs to DNA can affect its transcription, replication, the expression of genetic information in cells, and thereby influence its physiological function. Consequently, a hot subject of research surrounding drug–DNA has been carried out to obtain the effective information and explain the disease mechanism. The different binding studies have also been widely
J. Zhu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 78–83
1 O
7
6
5
OH
2 4
O
3
Fig. 1. Molecular structure of 5-HMF.
used in designing more low-toxic drugs for clinical use [1]. Because the reaction modes and binding properties during the interaction can help understand the functional mechanism of drugs, and provide theoretical basis for the development of new drugs targeted to DNA [4,5]. So studying the interaction of DNA with drugs is very important. 5-Hydroxymethyl-2-furfural (5-HMF) (structure shown in Fig. 1), an aldehyde compound produced through dehydration of monosaccharides during heat or weak acid treatment. It exists abundantly in a lot of plants and food which have carbohydrate [6,7]. It has been reported to be toxic, which can irritate respiratory tract and mucous, damage striated muscles and viscera, and so on [8,9]. So how to reduce its toxicity on human body and improve its value of application is a crucial problem. To obtain some critical information for designing newly low-toxic and efficient 5-HMF derivatives, its interactions with biomacromolecules are necessary to be investigated. By now, Zhang et al. studied the interaction of 5HMF with HSA and BSA by fluorescence spectroscopy and UV–vis absorption spectroscopy [10,11]. Guo et al. compared further the interaction mechanisms between 5-HMF and two different serum albumins by spectroscopic and molecular modeling methods [12]. However, the mechanism of the interaction between 5-HMF and DNA has not been investigated in details until now. As the intrinsic fluorescence of ctDNA itself was very weak and nearly no fluorescence was observed from 5-HMF in Tris–HCl buffer, so a fluorescence probe is necessary when investigating the interaction of 5-HMF with ctDNA. There are many fluorescence probes, of which ethidium bromide (EB) was commonly used as a fluorescent dye at present. According to the references [13,14], the fluorescence intensity of ethidium bromide (EB) can be improved up to 20–30 times after combining with DNA. Therefore, EB was selected as a fluorescence probe in this study. Obviously, the fluorescence of the DNA–EB system was much stronger and could be quenched on addition of 5-HMF, which was confirmed to be a static quenching process. UV absorption spectroscopy, fluorescence spectroscopy and melting temperature (Tm) curve results showed that the binding mode of 5-HMF–DNA might be a groove binding, mainly through hydrophobic force and hydrogen bonds. The molecular modeling results supported further the conclusions obtained from the above experiments. This study could provide a deep understanding of the side effect of 5-HMF and vital data for designing new drugs from the molecular level.
79
260 nm using an extinction coefficient of 6600 mol1 cm1 [15]. The purity of DNA was verified by monitoring the ratio of absorbance at 260/280 nm (A260/A280), which was >1.80 indicating that DNA was sufficiently free from protein [16]. 5-HMF stock solution (1.0 103 mol L1) was prepared in ethanol and kept in the dark. The stock solution (1.0 103 mol L1) of EB was prepared by dissolving its crystals in buffer solution. All chemicals were of analytical reagent grade, and doubly distilled water was used throughout the experiments. Apparatus The UV–vis absorption spectra were performed on a TU-1900 spectrophotometer (Puxi Analytic Instrument Ltd., Beijing, China) equipped with 1.0 cm quartz cells. All fluorescence spectra were carried out on a Cary Eclipse fluorescence spectrophotometer (Varian, America) equipped with a 1.0 cm quartz cell. The widths of the excitation and emission slits were set at 5.0 and 10.0 nm respectively. An electronic thermostat water-bath (Yuhua Instrument Company, Henan, China) was used to control the temperature. Methods UV absorption spectra The UV absorption spectra of fixed amounts of DNA in Tris–HCl buffer solution were measured with addition of different concentrations of 5-HMF. The wavelength range of the system was from 200 to 400 nm. The blanks corresponding to the buffer were subtracted to correct the absorbance at room temperature. Fluorescence spectra In order to determine the optimal molar ratio of EB to DNA, the fluorescence spectra of a fixed concentration (9.52 lmol L1) of EB were measured with varying the concentrations of DNA from 0 to 67.80 lmol L1. Then a 3.0 mL solution, containing a certain concentration of EB–DNA ([DNA]/[EB] = 7) complex solution, was added to a 1.0 cm quartz cuvette and titrated by successive addition of 5-HMF. These solutions were allowed to stand for 5 min to equilibrate. The fluorescence spectra were measured at three different temperatures (300, 305 and 310 K) in the wavelength range of 536–800 nm, with the excitation wavelength at 526 nm. The appropriate banks corresponding to the buffer solution were subtracted to correct the background. NA melting The melting temperatures of DNA and the 5-HMF–DNA complex were determined respectively by recording the absorbance at 259 nm at different temperatures. The absorbance values were then plotted as a function of the temperature ranging from 30 C to 90 C. The values of Tm of DNA and the 5-HMF–DNA complex were obtained from the transition midpoint of the melting curves based on f ss versus temperature (T), where f ss = (A A0)/(Af A0), A0 is the initial absorbance, A is the final absorbance [17].
Experimental section Materials Calf thymus DNA was purchased from Sigma Chem. Co.; 5-Hydroxymethyl-2-furfural (5-HMF) and ethidium bromide (EB) were purchased from Aladdin Industrial Corporation (Shanghai, China). 0.05 mol L1 Tris–HCl buffer solution (containing 0.02 mol/L NaCl) was prepared and the pH was adjusted to 7.40. DNA solution was dissolved in the buffer solution and stored at 4 C for more than 24 h with occasional gentle shaking to get homogeneity. Its concentration was determined by UV absorption at
Molecular modeling In this study, the possible conformation of small molecules bound to DNA was calculated by FlexX software of the Sybyl suite. Sybyl 6.9 was applied to simulate the binding model of 5-HMF with DNA [18]. The three dimensional structure of DNA was built according to the Amber 4.0 force field with Kollman-all-atom charges. The spatial structure of 5-HMF was performed by Sybyl 6.9 and optimized by molecular mechanics in force field of CHARMm. Furthermore, hydrogen bond restraints have been added to maintain the steady duplex structure of the DNA. Thus the best binding model of the system can be found.
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Results and discussion
Binding properties of EB with DNA
UV absorption spectra
For further study of the binding mode, corresponding binding parameters and thermodynamic parameters were planned to be determined by fluorescence spectroscopy. Because DNA itself has nearly no fluorescence, EB is expected to help investigate the interaction of 5-HMF with DNA as a probe. EB is a typical mutagenic intercalating dye for DNA and it has been studied for many years [13,14]. It is found that the binding mode between them is the intercalation of the planar aromatic ring to the adjacent base pairs on the double helix. As seen in Fig. 3, there was little fluorescence for DNA, and the intrinsic fluorescence of EB in the buffer solution was quite low. But the fluorescence intensity of the complex system was greatly enhanced after adding DNA to EB. To find the best concentration ratio of DNA to EB, the fluorescence intensity of a series of assay solutions containing constant concentration of EB and various concentrations of DNA were measured. The results were shown in Fig. 4. For 9.52 lmol L1 EB solution, the maximum fluorescence intensity increased gradually with the addition of DNA, and did not increase further when the concentration of DNA was higher than 67.80 lmol L1. So based on Fig. 4, the fixed molar ratio ([DNA]/[EB]) of the system was selected to be 7 to investigate the interaction of DNA with 5-HMF.
The application of absorption spectroscopy is one of the most useful techniques in DNA-binding studies [19]. Owing to the binding of drugs to DNA, the absorbance spectrum shows hypochromism and hyperchromism, which involve a strong stacking interaction between an aromatic chromophore and the base pairs of DNA. Specifically, the hyperchromism originates from the breakage of the DNA duplex secondary structure; while the hypochromism originates from the stabilization of the DNA duplex by either the intercalation binding or the electrostatic effect [20]. Fig. 2 showed that the maximum absorption wavelength of DNA was at 258 nm. With the addition of 5-HMF, the absorption spectra increased regularly and had a red shift. The inset in Fig. 2 stated that the absorbance of the DNA-5-HMF complex was much higher than the sum value of free DNA and 5-HMF. It proved that the double helix structure of DNA changed after interacting with 5-HMF, and a hyperchromism effect happened. The results suggested that the binding mode of 5-HMF with DNA was not a typical intercalative binding, a groove binding might be more acceptable [21].
1.0
0.35
Absorbance
Absorbance
0.8
Fluorescence quenching
1
0.30
2 0.25 0.20 0.15
0.6
0.0
0.5
1.0
1.5
2.0
2.5
c5-HMF /(10 −5 mol•L-1)
I 0.4
A
0.2
0.0 200
240
280
320
360
400
Wavelength (nm) Fig. 2. UV absorption spectra of DNA with various concentrations of 5-HMF. Inset: Comparison of absorbance at 258 nm between the DNA-5-HMF complex (1) and the sum values of separate DNA and 5-HMF (2). c(DNA) = 2.74 105 mol L1; c(5HMF)/(105 mol L1), AI: 0.00, 0.29, 0.57, 0.86, 1.14, 1.43, 2.29, 2.86, 3.43. T = 301 K.
The emission spectrum of the DNA–EB system in the absence and presence of 5-HMF was shown in Fig. 5. There was a significant maximum emission at 604 nm when the DNA– EB system was excited at 526 nm. With increasing the concentration of 5-HMF, the fluorescence intensity of the system decreased without notable changes in the wavelength of maximum emission, which might be due to three possible reasons [22,23]. First, the binding of EB with 5-HMF might appear and caused the fluorescence quenching. Second, 5-HMF competed against EB and replaced the intercalated EB from the complex, which decreased the concentration of EB binding to DNA. Third, a new complex 5-HMF–DNA–EB formed as a result of the interaction of 5-HMF with DNA–EB. In this work, 5-HMF itself could not induce obvious change on the fluorescence intensity of EB (Fig. 6), suggesting that EB could not interact with 5-HMF. In addition, it is known that the binding constant of EB with DNA is 5.16 105 L mol1 [24]. So the smaller binding constant (2.17 103 L mol1) between 5-HMF and DNA
420 250 350
c
Fluorescence Intensity
Fluorescence Intensity
200
150
100
210
140
50
b a
0 550
280
600
70 650
700
750
800
Wavelength (nm) Fig. 3. Fluorescence spectra of: (a) DNA (b) EB (c) EB–DNA in the Tris–HCl buffer solution. c(DNA) = 2.45 105 mol L1, c(EB) = 3.33 106 mol L1.
0
20
40
60
80
100
c DNA / (µmol •L-1) Fig. 4. Fluorescence intensity of EB with the addition of DNA. c(EB) = 9.52 106 mol L1.
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Fluorescence Intensity
250
that the quenching mechanism of EB–DNA by 5-HMF was most likely a static quenching process [29]. The binding constant (Kb) and the number of binding sites (n) for 5-HMF-DNA system can be determined by the following equation [30].
A
200
150
lg½ðF 0 FÞ ¼ lg K b þ n lg½Q
E
where Kb and n are the binding constant and the number of binding sites in base pairs, respectively. From the linear plot of lg[(F0 F)/F] versus lg[Q], Kb and n were calculated and listed in Table 2. The value of n was close to 1, indicating that one molecule of 5-HMF could bind to a single site of DNA. The binding constant Kb increased with the temperature increasing, indicating that rising temperature contributed to the binding of 5-HMF to DNA and increased the stability of the complex.
100
50
0
560
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ð2Þ
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Wavelength (nm)
Thermodynamic parameters and binding force
Fig. 5. Fluorescence spectra of DNA-EB with various concentrations of 5-HMF. c(DNA) = 2.74 105 mol L1, c(EB) = 3.98 106 mol L1; c(5-HMF)/(105 mol L1), AE: 0.00, 1.43, 2.38, 3.81, 5.24. T = 298 K, kex = 526 nm, kem = 604 nm.
(Data in Table 2) indicated that replacing EB from the complex was impossible. As a result, the third reason seemed more reasonable, and 5-HMF might interact with EB–DNA through groove binding or electrostatic binding [25]. The quenching constants of the system can be analyzed according to the Stern–Volmer equation [26].
F0 ¼ 1 þ K SV ½Q ¼ 1 þ K q s0 ½Q F
ð1Þ
where F0 and F are the fluorescence intensities in the absence and presence of 5-HMF, respectively, KSV is the Stern–Volmer dynamic quenching constant, Kq is the quenching rate constant, is the average lifetime of the fluorescence molecules without 5-HMF (s0 108 s) [27], and [Q] is the concentration of 5-HMF. In general, the mechanisms of fluorescence quenching are classified as either dynamic or static quenching, which can be distinguished by their different dependences on temperatures [28]. The curves of F0/F versus [Q] at three different temperatures (300, 305 and 310 K) were displayed in Fig. 7. The linear plots suggested that only one type of quenching process occurred, in either static or dynamic quenching. As shown in Table 1, the values of KSV decreased with the increasing temperatures, and the values of Kq were larger than the limiting diffusion constant of the biomacromolecules (2.0 1010 L mol1 s1), which demonstrated 80
Generally, the interaction forces between small molecules and biomacromolecules mainly include hydrogen bonds, van der Waals force, hydrophobic force, and electrostatic interactions [31]. The thermodynamic parameters in the reaction are the main evidence for confirming the binding force. If the enthalpy change (DH) does not vary significantly over the temperature range studied, then its value and that of entropy change (DS) can be determined from the van’t Hoff equation [32].
ln K ¼
DH DS þ RT R
ð3Þ
where K is the binding constant at the corresponding temperature; R is the gas constant; T is the absolute temperature; DH, DS are the enthalpy change and entropy change, respectively. The values of DH and DS were obtained from the slope and the intercept of the linear van’t Hoff plot based on lnK versus 1/T. The free energy change (DG) was then evaluated from the following equation [33].
DG ¼ DH T DS
ð4Þ
The thermodynamic parameters for the interaction of DNA with 5-HMF were listed in Table 2. The negative values of DG revealed that the binding process was spontaneous. The positive value of DS was frequently regarded as evidence for the hydrophobic interaction [34,35]. The positive value of DH showed that the binding process was mainly enthalpy driven and by means of hydrogen bonds [36]. So both hydrophobic interaction and hydrogen bonds might play a major role in the binding of 5-HMF to DNA and improve the stability of the complex. This result was in good accordance with the information coming from the molecular modeling. DNA melting analysis
Fluorescence Intensity
70
Another evidence for the interaction of 5-HMF with DNA was the effect of 5-HMF on the melting temperature of DNA (Tm), at which half of dsDNA was dissociated into single strands. Because the interaction of DNA with small molecules could influence the corresponding melting temperature. Therefore, it was studied to estimate the binding mode of 5-HMF to DNA. In general, the inter-
60
50
Table 1 Stern–Volmer temperatures.
40
30 0.0
0.6
1.2
1.8
2.4
3.0
c 5-HMF / (10 -5mol •L-1) Fig. 6. Fluorescence intensity c(EB) = 1.0 105 mol L1.
of
EB
with
the
addition
of
5-HMF.
quenching
constants
of
5-HMFDNA–EB
system
at
various
T (K)
KSV (103 L mol1)
Kq (1012 L mol1 s1)
R
300 305 310
3.52 3.19 2.78
3.52 3.19 2.78
0.9920 0.9973 0.9929
R is the correlation coefficient.
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Table 2 Binding constants and thermodynamic parameters of 5-HMFDNA at various temperatures. T (K)
Kb (103 L mol1)
R
n
DH (kJ mol1)
DG (kJ mol1)
DS (J mol1 K1)
300 305 310
2.17 4.24 6.95
0.9990 0.9957 0.9918
0.94 1.03 1.09
3.77
19.17 21.18 22.80
76.37
R is the correlation coefficient.
1.25
1.20
F0/F
1.15
1.10
1.05
1.00 0.0
1.3
2.6
3.9 -5
5.2
6.5
-1
c5-HMF / (10 mol •L ) Fig. 7. Stern–Volmer plots for the fluorescence quenching of DNA-EB by 5-HMF at different temperatures. (j) 300 K; (d) 305 K; (N) 310 K. kex = 526 nm, kem = 604 nm.
1.0
a
0.8
b
Fig. 9. Molecular modeling of the interaction between 5-HMF and DNA. The double helix structure of DNA is represented using the line model, while the 5-HMF structure is represented using a ball and stick model. The hydrogen bonds between 5-HMF and DNA are represented by dashed line.
ƒ ss
0.6
0.4
0.2
0.0 33
44
55
66
77
88
Temperature / ( 0C) Fig. 8. Melting curves of DNA in the absence (b) and presence (a) of 5-HMF. c(DNA) = 7.11 105 mol L1, c(5-HMF) = 2.86 105 mol L1.
calation binding could stabilize the double helix structure of DNA and increase the Tm about 5—8 C, while the non-intercalation binding caused no obvious increase in Tm [37]. As seen from Fig. 8, the Tm of DNA was 82 1 C, upon the addition of 5-HMF, its Tm was found to be 80 1 C. This result suggested further that the binding mode between 5-HMF and DNA was non-intercalation, and the stabilization of DNA decreased. It was speculated that the groove binding of 5-HMF with DNA might change the conformation of DNA and decreased the Tm [38]. Molecular modeling Molecular modeling has played an important role in studying the interaction of small molecules with biomacromolecules and designing new drugs in recent years [39,40]. The application of
molecular modeling by computer methods could be employed to confirm the binding mode of 5-HMF to DNA directly. The best energy docking result was shown in Fig. 9. It could be seen that 5-HMF bound to the hydrophobic environment inside the double strands of DNA, suggesting the existence of hydrophobic interaction between 5-HMF and DNA. On the other hand, there were some hydrogen bonds between them (DG-10 was suitable to form intermolecular H-bonds with 6-C@O and 7-OH, DC-9 with 7-OH). The length of hydrogen bonds was 2.64, 3.42 and 1.99 Å, respectively. This indicated again that hydrogen bonds played an important role in the binding of 5-HMF to DNA. Therefore, the results obtained from the above molecular modeling verified the experimental results from the spectroscopic studies.
Conclusions In this work, the interaction of 5-HMF with DNA was studied by UV absorption, fluorescence spectroscopy and molecular modeling methods using EB as a probe. The absorption and melting results indicated that the interaction between 5-HMF and DNA was nonintercalation and probably a groove binding. The binding constants and the number of binding sites were calculated from the fluorescence quenching data at different temperatures. The obtained thermodynamic parameters DH and DS suggested that hydrophobic interaction and hydrogen bonds played an important role in the binding of 5-HMF to DNA. Moreover, molecular modeling studies provided direct evidence for the interaction and supported the
J. Zhu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 78–83
experimental results further. This research can help clarify the molecular mechanism in vivo and design new drugs in the future. Acknowledgments We are grateful for financial support from the Joint Fund of National Natural Science Foundation of China and Henan province (No. U1204304), China Postdoctoral Science Foundation (Nos. 20110490992 and 2013T60698), and the National Twelfth FiveYear Science and Technology Support Program (No. 2011B AI06B04). References [1] M.P. Singh, T. Joseph, S. Kumar, Y. Bathini, J.W. Lown, Chem. Res. Toxicol. 5 (1992) 597–607. [2] J. Sparks, C. Scholz, Biomacromolecules 10 (2009) 1715–1719. [3] J.W. Lown, Anti-Cancer Drug Des. 3 (1988) 25–40. [4] E. Froehlich, J.S. Mandeville, C.M. Weinert, L. Kreplak, H.A. Tajmir-Riahi, Biomacromolecules 12 (2011) 511–517. [5] M. Liang, X. Liu, G. Liu, S. Dou, D. Cheng, Y. Liu, M. Rusckowski, D.J. Hnatowich, Mol. Pharm. 8 (2011) 126–132. [6] A.T. Nguyen, J. Fontaine, H. Malonne, M. Claeys, M. Luhmer, P. Duez, Phytochemistry 66 (2005) 1186–1191. [7] C.D. Wu, J. Nutr. 139 (2009) 1818S–1823S. [8] Z.Q. Fu, M.Y. Wang, B.Q. Cai, Chin. Arch. Trad. Chin. Med. 3 (2008) 508–510. [9] R. Pamplona, M.J. Bellmunt, M. Portero, D. Riba, J. Prat, Life Sci. 9 (1995) 873– 879. [10] Y. Zhang, Q.Q. Wang, L.J. Yang, J.L. Zhang, D.X.J. Sun, Anal. Sci. 27 (2011) 639– 642. [11] Y. Zhang, Q.Q. Wang, L.J. Yang, J.L. Zhang, D.X. Sun, China Pharmacy. 22 (2011) 3095–3098. [12] M. Guo, L. He, X.W. Lu, Acta Pharm. Sinica 47 (2012) 385–392. [13] W. Fuller, M.J. Warning, Ber. Bunsenges. Phys. Chem. 68 (1964) 805.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Spectroscopic and molecular modeling methods to investigate the interaction between 5-Hydroxymethyl-2-furfural and calf thymus DNA using ethidium bromide as a probe Jinhua Zhu a,⇑, Lanlan Chen a, Yingying Dong b, Jiazhong Li c, Xiuhua Liu a,b a b c
Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical Engineering, Henan University, Kaifeng, Henan 475004, China Key Lab of Natural Drug and Immune Engineering of Henan Province, Kaifeng, Henan 475004, China School of Pharmacy, Lanzhou University, Donggang West Road 199, 730000 Lanzhou, 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
Spectroscopic and molecular
250
Fluorescence Intensity
modeling methods were used. The quenching of DNA–EB system by 5-HMF was a static quenching. The binding of 5-HMF to DNA was a non- intercalative binding. Hydrophobic force and hydrogen bonds both played a major role.
A
200
O HO
O
150
E
100
50
0
560
595
630
665
700
735
Wavelength(nm)
a r t i c l e
i n f o
Article history: Received 31 October 2013 Received in revised form 20 December 2013 Accepted 23 December 2013 Available online 6 January 2014 Keywords: 5-Hydroxymethyl-2-furfural Calf thymus DNA Ethidium bromide Spectroscopy Molecular modeling
a b s t r a c t In this work, the interaction of 5-Hydroxymethyl-2-furfural (5-HMF) with calf thymus DNA (ctDNA) under simulated physiological conditions (Tris–HCl buffer of pH 7.40), was explored by UV absorption spectroscopy, fluorescence spectroscopy and molecular modeling method, using ethidium bromide (EB) as a fluorescence probe of DNA. The fluorescence quenching mechanism of EB–ctDNA by 5-HMF was confirmed to be a static quenching, which derived from the formation of a new complex. The binding constants of 5-HMF with DNA in the presence of EB were calculated to be 2.17 103, 4.24 103 and 6.95 103 L mol1 at 300, 305 and 310 K, respectively. The calculated thermodynamic parameters, enthalpy change DH and entropy change DS, suggested that both hydrophobic interactions and hydrogen bonds played a predominant role in the binding of 5-HMF to DNA. According to the UV absorption spectroscopy and melting temperature (Tm) curve results, the binding mode of 5-HMF with DNA was indicative of a non-intercalative binding, which was supposed to be a groove binding. The molecular modeling results showed that 5-HMF could bind into the hydrophobic region of ctDNA and supported the conclusions obtained from the above experiments. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Deoxyribonucleic acid (DNA) that contains the genetic instructions for the development and functioning of living organisms is an ⇑ Corresponding author. Tel./fax: +86 371 23881589. E-mail address: [email protected] (J. Zhu). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.12.091
important target of many drugs, especially anticancer and antiviral drugs [1–3]. The binding of drugs to DNA can affect its transcription, replication, the expression of genetic information in cells, and thereby influence its physiological function. Consequently, a hot subject of research surrounding drug–DNA has been carried out to obtain the effective information and explain the disease mechanism. The different binding studies have also been widely
J. Zhu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 78–83
1 O
7
6
5
OH
2 4
O
3
Fig. 1. Molecular structure of 5-HMF.
used in designing more low-toxic drugs for clinical use [1]. Because the reaction modes and binding properties during the interaction can help understand the functional mechanism of drugs, and provide theoretical basis for the development of new drugs targeted to DNA [4,5]. So studying the interaction of DNA with drugs is very important. 5-Hydroxymethyl-2-furfural (5-HMF) (structure shown in Fig. 1), an aldehyde compound produced through dehydration of monosaccharides during heat or weak acid treatment. It exists abundantly in a lot of plants and food which have carbohydrate [6,7]. It has been reported to be toxic, which can irritate respiratory tract and mucous, damage striated muscles and viscera, and so on [8,9]. So how to reduce its toxicity on human body and improve its value of application is a crucial problem. To obtain some critical information for designing newly low-toxic and efficient 5-HMF derivatives, its interactions with biomacromolecules are necessary to be investigated. By now, Zhang et al. studied the interaction of 5HMF with HSA and BSA by fluorescence spectroscopy and UV–vis absorption spectroscopy [10,11]. Guo et al. compared further the interaction mechanisms between 5-HMF and two different serum albumins by spectroscopic and molecular modeling methods [12]. However, the mechanism of the interaction between 5-HMF and DNA has not been investigated in details until now. As the intrinsic fluorescence of ctDNA itself was very weak and nearly no fluorescence was observed from 5-HMF in Tris–HCl buffer, so a fluorescence probe is necessary when investigating the interaction of 5-HMF with ctDNA. There are many fluorescence probes, of which ethidium bromide (EB) was commonly used as a fluorescent dye at present. According to the references [13,14], the fluorescence intensity of ethidium bromide (EB) can be improved up to 20–30 times after combining with DNA. Therefore, EB was selected as a fluorescence probe in this study. Obviously, the fluorescence of the DNA–EB system was much stronger and could be quenched on addition of 5-HMF, which was confirmed to be a static quenching process. UV absorption spectroscopy, fluorescence spectroscopy and melting temperature (Tm) curve results showed that the binding mode of 5-HMF–DNA might be a groove binding, mainly through hydrophobic force and hydrogen bonds. The molecular modeling results supported further the conclusions obtained from the above experiments. This study could provide a deep understanding of the side effect of 5-HMF and vital data for designing new drugs from the molecular level.
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260 nm using an extinction coefficient of 6600 mol1 cm1 [15]. The purity of DNA was verified by monitoring the ratio of absorbance at 260/280 nm (A260/A280), which was >1.80 indicating that DNA was sufficiently free from protein [16]. 5-HMF stock solution (1.0 103 mol L1) was prepared in ethanol and kept in the dark. The stock solution (1.0 103 mol L1) of EB was prepared by dissolving its crystals in buffer solution. All chemicals were of analytical reagent grade, and doubly distilled water was used throughout the experiments. Apparatus The UV–vis absorption spectra were performed on a TU-1900 spectrophotometer (Puxi Analytic Instrument Ltd., Beijing, China) equipped with 1.0 cm quartz cells. All fluorescence spectra were carried out on a Cary Eclipse fluorescence spectrophotometer (Varian, America) equipped with a 1.0 cm quartz cell. The widths of the excitation and emission slits were set at 5.0 and 10.0 nm respectively. An electronic thermostat water-bath (Yuhua Instrument Company, Henan, China) was used to control the temperature. Methods UV absorption spectra The UV absorption spectra of fixed amounts of DNA in Tris–HCl buffer solution were measured with addition of different concentrations of 5-HMF. The wavelength range of the system was from 200 to 400 nm. The blanks corresponding to the buffer were subtracted to correct the absorbance at room temperature. Fluorescence spectra In order to determine the optimal molar ratio of EB to DNA, the fluorescence spectra of a fixed concentration (9.52 lmol L1) of EB were measured with varying the concentrations of DNA from 0 to 67.80 lmol L1. Then a 3.0 mL solution, containing a certain concentration of EB–DNA ([DNA]/[EB] = 7) complex solution, was added to a 1.0 cm quartz cuvette and titrated by successive addition of 5-HMF. These solutions were allowed to stand for 5 min to equilibrate. The fluorescence spectra were measured at three different temperatures (300, 305 and 310 K) in the wavelength range of 536–800 nm, with the excitation wavelength at 526 nm. The appropriate banks corresponding to the buffer solution were subtracted to correct the background. NA melting The melting temperatures of DNA and the 5-HMF–DNA complex were determined respectively by recording the absorbance at 259 nm at different temperatures. The absorbance values were then plotted as a function of the temperature ranging from 30 C to 90 C. The values of Tm of DNA and the 5-HMF–DNA complex were obtained from the transition midpoint of the melting curves based on f ss versus temperature (T), where f ss = (A A0)/(Af A0), A0 is the initial absorbance, A is the final absorbance [17].
Experimental section Materials Calf thymus DNA was purchased from Sigma Chem. Co.; 5-Hydroxymethyl-2-furfural (5-HMF) and ethidium bromide (EB) were purchased from Aladdin Industrial Corporation (Shanghai, China). 0.05 mol L1 Tris–HCl buffer solution (containing 0.02 mol/L NaCl) was prepared and the pH was adjusted to 7.40. DNA solution was dissolved in the buffer solution and stored at 4 C for more than 24 h with occasional gentle shaking to get homogeneity. Its concentration was determined by UV absorption at
Molecular modeling In this study, the possible conformation of small molecules bound to DNA was calculated by FlexX software of the Sybyl suite. Sybyl 6.9 was applied to simulate the binding model of 5-HMF with DNA [18]. The three dimensional structure of DNA was built according to the Amber 4.0 force field with Kollman-all-atom charges. The spatial structure of 5-HMF was performed by Sybyl 6.9 and optimized by molecular mechanics in force field of CHARMm. Furthermore, hydrogen bond restraints have been added to maintain the steady duplex structure of the DNA. Thus the best binding model of the system can be found.
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Results and discussion
Binding properties of EB with DNA
UV absorption spectra
For further study of the binding mode, corresponding binding parameters and thermodynamic parameters were planned to be determined by fluorescence spectroscopy. Because DNA itself has nearly no fluorescence, EB is expected to help investigate the interaction of 5-HMF with DNA as a probe. EB is a typical mutagenic intercalating dye for DNA and it has been studied for many years [13,14]. It is found that the binding mode between them is the intercalation of the planar aromatic ring to the adjacent base pairs on the double helix. As seen in Fig. 3, there was little fluorescence for DNA, and the intrinsic fluorescence of EB in the buffer solution was quite low. But the fluorescence intensity of the complex system was greatly enhanced after adding DNA to EB. To find the best concentration ratio of DNA to EB, the fluorescence intensity of a series of assay solutions containing constant concentration of EB and various concentrations of DNA were measured. The results were shown in Fig. 4. For 9.52 lmol L1 EB solution, the maximum fluorescence intensity increased gradually with the addition of DNA, and did not increase further when the concentration of DNA was higher than 67.80 lmol L1. So based on Fig. 4, the fixed molar ratio ([DNA]/[EB]) of the system was selected to be 7 to investigate the interaction of DNA with 5-HMF.
The application of absorption spectroscopy is one of the most useful techniques in DNA-binding studies [19]. Owing to the binding of drugs to DNA, the absorbance spectrum shows hypochromism and hyperchromism, which involve a strong stacking interaction between an aromatic chromophore and the base pairs of DNA. Specifically, the hyperchromism originates from the breakage of the DNA duplex secondary structure; while the hypochromism originates from the stabilization of the DNA duplex by either the intercalation binding or the electrostatic effect [20]. Fig. 2 showed that the maximum absorption wavelength of DNA was at 258 nm. With the addition of 5-HMF, the absorption spectra increased regularly and had a red shift. The inset in Fig. 2 stated that the absorbance of the DNA-5-HMF complex was much higher than the sum value of free DNA and 5-HMF. It proved that the double helix structure of DNA changed after interacting with 5-HMF, and a hyperchromism effect happened. The results suggested that the binding mode of 5-HMF with DNA was not a typical intercalative binding, a groove binding might be more acceptable [21].
1.0
0.35
Absorbance
Absorbance
0.8
Fluorescence quenching
1
0.30
2 0.25 0.20 0.15
0.6
0.0
0.5
1.0
1.5
2.0
2.5
c5-HMF /(10 −5 mol•L-1)
I 0.4
A
0.2
0.0 200
240
280
320
360
400
Wavelength (nm) Fig. 2. UV absorption spectra of DNA with various concentrations of 5-HMF. Inset: Comparison of absorbance at 258 nm between the DNA-5-HMF complex (1) and the sum values of separate DNA and 5-HMF (2). c(DNA) = 2.74 105 mol L1; c(5HMF)/(105 mol L1), AI: 0.00, 0.29, 0.57, 0.86, 1.14, 1.43, 2.29, 2.86, 3.43. T = 301 K.
The emission spectrum of the DNA–EB system in the absence and presence of 5-HMF was shown in Fig. 5. There was a significant maximum emission at 604 nm when the DNA– EB system was excited at 526 nm. With increasing the concentration of 5-HMF, the fluorescence intensity of the system decreased without notable changes in the wavelength of maximum emission, which might be due to three possible reasons [22,23]. First, the binding of EB with 5-HMF might appear and caused the fluorescence quenching. Second, 5-HMF competed against EB and replaced the intercalated EB from the complex, which decreased the concentration of EB binding to DNA. Third, a new complex 5-HMF–DNA–EB formed as a result of the interaction of 5-HMF with DNA–EB. In this work, 5-HMF itself could not induce obvious change on the fluorescence intensity of EB (Fig. 6), suggesting that EB could not interact with 5-HMF. In addition, it is known that the binding constant of EB with DNA is 5.16 105 L mol1 [24]. So the smaller binding constant (2.17 103 L mol1) between 5-HMF and DNA
420 250 350
c
Fluorescence Intensity
Fluorescence Intensity
200
150
100
210
140
50
b a
0 550
280
600
70 650
700
750
800
Wavelength (nm) Fig. 3. Fluorescence spectra of: (a) DNA (b) EB (c) EB–DNA in the Tris–HCl buffer solution. c(DNA) = 2.45 105 mol L1, c(EB) = 3.33 106 mol L1.
0
20
40
60
80
100
c DNA / (µmol •L-1) Fig. 4. Fluorescence intensity of EB with the addition of DNA. c(EB) = 9.52 106 mol L1.
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Fluorescence Intensity
250
that the quenching mechanism of EB–DNA by 5-HMF was most likely a static quenching process [29]. The binding constant (Kb) and the number of binding sites (n) for 5-HMF-DNA system can be determined by the following equation [30].
A
200
150
lg½ðF 0 FÞ ¼ lg K b þ n lg½Q
E
where Kb and n are the binding constant and the number of binding sites in base pairs, respectively. From the linear plot of lg[(F0 F)/F] versus lg[Q], Kb and n were calculated and listed in Table 2. The value of n was close to 1, indicating that one molecule of 5-HMF could bind to a single site of DNA. The binding constant Kb increased with the temperature increasing, indicating that rising temperature contributed to the binding of 5-HMF to DNA and increased the stability of the complex.
100
50
0
560
595
630
665
700
ð2Þ
735
Wavelength (nm)
Thermodynamic parameters and binding force
Fig. 5. Fluorescence spectra of DNA-EB with various concentrations of 5-HMF. c(DNA) = 2.74 105 mol L1, c(EB) = 3.98 106 mol L1; c(5-HMF)/(105 mol L1), AE: 0.00, 1.43, 2.38, 3.81, 5.24. T = 298 K, kex = 526 nm, kem = 604 nm.
(Data in Table 2) indicated that replacing EB from the complex was impossible. As a result, the third reason seemed more reasonable, and 5-HMF might interact with EB–DNA through groove binding or electrostatic binding [25]. The quenching constants of the system can be analyzed according to the Stern–Volmer equation [26].
F0 ¼ 1 þ K SV ½Q ¼ 1 þ K q s0 ½Q F
ð1Þ
where F0 and F are the fluorescence intensities in the absence and presence of 5-HMF, respectively, KSV is the Stern–Volmer dynamic quenching constant, Kq is the quenching rate constant, is the average lifetime of the fluorescence molecules without 5-HMF (s0 108 s) [27], and [Q] is the concentration of 5-HMF. In general, the mechanisms of fluorescence quenching are classified as either dynamic or static quenching, which can be distinguished by their different dependences on temperatures [28]. The curves of F0/F versus [Q] at three different temperatures (300, 305 and 310 K) were displayed in Fig. 7. The linear plots suggested that only one type of quenching process occurred, in either static or dynamic quenching. As shown in Table 1, the values of KSV decreased with the increasing temperatures, and the values of Kq were larger than the limiting diffusion constant of the biomacromolecules (2.0 1010 L mol1 s1), which demonstrated 80
Generally, the interaction forces between small molecules and biomacromolecules mainly include hydrogen bonds, van der Waals force, hydrophobic force, and electrostatic interactions [31]. The thermodynamic parameters in the reaction are the main evidence for confirming the binding force. If the enthalpy change (DH) does not vary significantly over the temperature range studied, then its value and that of entropy change (DS) can be determined from the van’t Hoff equation [32].
ln K ¼
DH DS þ RT R
ð3Þ
where K is the binding constant at the corresponding temperature; R is the gas constant; T is the absolute temperature; DH, DS are the enthalpy change and entropy change, respectively. The values of DH and DS were obtained from the slope and the intercept of the linear van’t Hoff plot based on lnK versus 1/T. The free energy change (DG) was then evaluated from the following equation [33].
DG ¼ DH T DS
ð4Þ
The thermodynamic parameters for the interaction of DNA with 5-HMF were listed in Table 2. The negative values of DG revealed that the binding process was spontaneous. The positive value of DS was frequently regarded as evidence for the hydrophobic interaction [34,35]. The positive value of DH showed that the binding process was mainly enthalpy driven and by means of hydrogen bonds [36]. So both hydrophobic interaction and hydrogen bonds might play a major role in the binding of 5-HMF to DNA and improve the stability of the complex. This result was in good accordance with the information coming from the molecular modeling. DNA melting analysis
Fluorescence Intensity
70
Another evidence for the interaction of 5-HMF with DNA was the effect of 5-HMF on the melting temperature of DNA (Tm), at which half of dsDNA was dissociated into single strands. Because the interaction of DNA with small molecules could influence the corresponding melting temperature. Therefore, it was studied to estimate the binding mode of 5-HMF to DNA. In general, the inter-
60
50
Table 1 Stern–Volmer temperatures.
40
30 0.0
0.6
1.2
1.8
2.4
3.0
c 5-HMF / (10 -5mol •L-1) Fig. 6. Fluorescence intensity c(EB) = 1.0 105 mol L1.
of
EB
with
the
addition
of
5-HMF.
quenching
constants
of
5-HMFDNA–EB
system
at
various
T (K)
KSV (103 L mol1)
Kq (1012 L mol1 s1)
R
300 305 310
3.52 3.19 2.78
3.52 3.19 2.78
0.9920 0.9973 0.9929
R is the correlation coefficient.
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Table 2 Binding constants and thermodynamic parameters of 5-HMFDNA at various temperatures. T (K)
Kb (103 L mol1)
R
n
DH (kJ mol1)
DG (kJ mol1)
DS (J mol1 K1)
300 305 310
2.17 4.24 6.95
0.9990 0.9957 0.9918
0.94 1.03 1.09
3.77
19.17 21.18 22.80
76.37
R is the correlation coefficient.
1.25
1.20
F0/F
1.15
1.10
1.05
1.00 0.0
1.3
2.6
3.9 -5
5.2
6.5
-1
c5-HMF / (10 mol •L ) Fig. 7. Stern–Volmer plots for the fluorescence quenching of DNA-EB by 5-HMF at different temperatures. (j) 300 K; (d) 305 K; (N) 310 K. kex = 526 nm, kem = 604 nm.
1.0
a
0.8
b
Fig. 9. Molecular modeling of the interaction between 5-HMF and DNA. The double helix structure of DNA is represented using the line model, while the 5-HMF structure is represented using a ball and stick model. The hydrogen bonds between 5-HMF and DNA are represented by dashed line.
ƒ ss
0.6
0.4
0.2
0.0 33
44
55
66
77
88
Temperature / ( 0C) Fig. 8. Melting curves of DNA in the absence (b) and presence (a) of 5-HMF. c(DNA) = 7.11 105 mol L1, c(5-HMF) = 2.86 105 mol L1.
calation binding could stabilize the double helix structure of DNA and increase the Tm about 5—8 C, while the non-intercalation binding caused no obvious increase in Tm [37]. As seen from Fig. 8, the Tm of DNA was 82 1 C, upon the addition of 5-HMF, its Tm was found to be 80 1 C. This result suggested further that the binding mode between 5-HMF and DNA was non-intercalation, and the stabilization of DNA decreased. It was speculated that the groove binding of 5-HMF with DNA might change the conformation of DNA and decreased the Tm [38]. Molecular modeling Molecular modeling has played an important role in studying the interaction of small molecules with biomacromolecules and designing new drugs in recent years [39,40]. The application of
molecular modeling by computer methods could be employed to confirm the binding mode of 5-HMF to DNA directly. The best energy docking result was shown in Fig. 9. It could be seen that 5-HMF bound to the hydrophobic environment inside the double strands of DNA, suggesting the existence of hydrophobic interaction between 5-HMF and DNA. On the other hand, there were some hydrogen bonds between them (DG-10 was suitable to form intermolecular H-bonds with 6-C@O and 7-OH, DC-9 with 7-OH). The length of hydrogen bonds was 2.64, 3.42 and 1.99 Å, respectively. This indicated again that hydrogen bonds played an important role in the binding of 5-HMF to DNA. Therefore, the results obtained from the above molecular modeling verified the experimental results from the spectroscopic studies.
Conclusions In this work, the interaction of 5-HMF with DNA was studied by UV absorption, fluorescence spectroscopy and molecular modeling methods using EB as a probe. The absorption and melting results indicated that the interaction between 5-HMF and DNA was nonintercalation and probably a groove binding. The binding constants and the number of binding sites were calculated from the fluorescence quenching data at different temperatures. The obtained thermodynamic parameters DH and DS suggested that hydrophobic interaction and hydrogen bonds played an important role in the binding of 5-HMF to DNA. Moreover, molecular modeling studies provided direct evidence for the interaction and supported the
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