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Study of interactions of anthraquinones with DNA using ethidium bromide as a fluorescence probe
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Spectrochimica Acta Part A 70 (2008) 136–143
Study of interactions of anthraquinones with DNA using ethidium bromide as a fluorescence probe Chunyu Qiao a , Shuyun Bi b , Ying Sun a , Daqian Song a , Hanqi Zhang a , Weihong Zhou a,∗ a
College of Chemistry, Jilin University, Changchun 130012, PR China b Changchun Normal College, Changchun 130000, PR China Received 26 May 2007; accepted 12 July 2007
Abstract The interactions of fish sperm deoxyribonucleic acid (DNA) with anthraquinones, such as chrysophanol, physcion and 1,8-dihydroxy anthraquinone, were investigated by using ethidium bromide (EB) as fluorescence probe. The binding constants of anthraquinones and DNA were obtained by the fluorescence quenching technique. Further, the binding mechanisms on the reaction of the three anthraquinones with DNA and effect of ionic strength on the fluorescence property of the system have also been investigated. The results of the assay indicate that the binding modes of chrysophanol, physcion and 1,8-dihydroxy anthraquinone with DNA were evaluated to be groove binding. And the binding constants of chrysophanol, physcion and 1,8-dihydroxy anthraquinone with DNA–EB complex were 1.64 × 104 , 3.04 × 104 and 2.88 × 105 l mol−1 , respectively. © 2007 Elsevier B.V. All rights reserved. Keywords: Chrysophanol; Physcion; 1,8-Dihydroxy anthraquinone; DNA; Ethidium bromide; Fluorescence quenching
1. Introduction Deoxyribonucleic acid (DNA) is an important genetic substance in the organism. The regions of DNA involved vital processes, such as gene expression, gene transcription, mutagenesis and carcinogenesis [1]. Since DNA is an important cellular receptor, many chemicals through binding to DNA exert their antitumor effects. Therefore, the changing of DNA replication and the inhibiting growth of the tumor cells, which is the basis of designing new and more efficient antitumor drugs and their effectiveness depend on the mode and affinity of the binding [2]. Therefore, the qualitative and quantitative analysis of nucleic acids as the material base of genetic inheritance is becoming more and more important. Especially in the quantitative analysis, their natural fluorescence intensity is so weak that their fluorescence spectra have scarcely been used for studying their biological properties, and usually some fluorescent probes have been employed for the investigation [3].
∗
Corresponding author. Tel.: +86 431 85095144; fax: +86 431 85095144. E-mail address: [email protected] (W. Zhou).
1386-1425/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2007.07.038
In recent years, there have been many reports on the study of the interaction mechanism between drugs with DNA and the investigation of new effective DNA fluorescent probes [4–6]. Different probes react with DNA in different ways. Among these probes, the fluorescence intensity of some probes is enhanced by DNA, such as ethidium bromide [7–9], acridine orange (AO) [10], bisbenzimidazole (Hoechst 33258) [11], thiazole orange homodimer (TOTO) [12], and berberine [13]. While, the fluorescence of some other probes is quenched by DNA, such as 9,10-anthraquinone-2-sulfonate [14], phosphin 3R [15] and nile blue [16]. In this paper, EB (Fig. 1) was selected as a probe to investigate the interactions of chrysophanol, physcion and 1,8-dihydroxy anthraquinone with DNA by spectral methods. The structures of three analytes used in this work are presented in Fig. 2. Chrysophanol, physcion and 1,8-dihydroxy anthraquinone are the effective components in traditional herbal used in China for treating various ailments. They possess antibacterial, laxative, and antitumor functions. The accepted point is that small molecules are bound to DNA double helix by three binding modes: electrostatic binding, intercalation binding and groove binding [17]. Generally, small molecules have some extent selectivity except
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DNA by using EB as fluorescence probe were the first reported. Consequently, binding studies of anthraquinones with DNA are valuable for the understanding of the reaction mechanism. The results of various binding studies may be contributive in designing new and promising drugs for a variety of medical conditions in clinic. 2. Experimental 2.1. Reagents Fig. 1. The structure of EB.
for its binding with DNA by the mode of electrostatic binding. Electrostatic interaction is along the external DNA double helix. In this work, the binding mechanism on the reaction of the anthraquinones with DNA was investigated by UV–vis spectrophotometry and fluorescence quenching technique. And the effects of the melting temperatures and ionic strength were also obtained. These studies suggest that the groove-binding mode appears to be more acceptable. Here, the interactions of chrysophanol, physcion and 1,8-dihydroxy anthraquinone with
Deoxyribonucleic acid (DNA) was purchased from Sigma Chem. Co. Ethidium bromide (EB, 5 ml mg−1 ) was purchased from Changchun Baotaike Company. Chrysophanol, physcion and 1,8-dihydroxy anthraquinone were obtained from China Drug Biological Products Qualifying Institute. DNA used in this work was double stranded DNA (dsDNA) unless specified. All the chemicals used were of analytical reagent grade and doubly distilled water was used throughout the experiment. A stock solution of DNA was prepared by dissolving an appropriate amount of DNA in doubly distilled water overnight and hoard at 4 ◦ C in the dark for about a week. The concentration of DNA solution was determined from UV absorption at 260 nm using a molar absorption coefficient ε260 = 6600 mol−1 cm−1 . Concentration of DNA in the stock solution was 4.22 × 10−4 mol l−1 in the experiment. Purity of the DNA was checked by monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of A260/A280 > 1.8, indicating that DNA was sufficiently free from protein [18,19]. A stock solution of EB was prepared by adding 0.4 ml liquid EB into flasks of 50 ml and diluted to mark with doubly distilled water and mixed thoroughly. Then the concentration of EB was determined by UV–vis spectrophotometer based on ε480 = 5450 mol−1 cm−1 . The concentration of EB stock solution was 7.94 × 10−5 mol l−1 and the solution was stored in cool and dark place. Chrysophanol, physcion and 1,8-dihydroxy anthraquinone were prepared into 1.00 × 10−3 mol−1 l−1 stock solutions by dissolving an appropriate amount of the drugs in boil ethanol. Britton–Robinson (B–R) buffer (pH 7.4, 2.66 ml 85% H3 PO4 , 2.36 ml acetic acid, 2.4700 g boric acid were dissolved in 1000 ml doubly distilled water) was used to control the pH of the reaction system. 2.2. Apparatus
Fig. 2. Molecular structure of chrysophanol (a), physcion (b) and 1,8-dihydroxy anthraquinone (c).
All fluorescence measurements were conducted by using a Shimadzu RF-5301PC fluorophotometer (Kyoto, Japan) equipped with a xenon lamp source and quartz cells of 1 cm path length. All absorption spectra were measured on a GBC Cintra 10e UV–vis spectrophotometer (Australia) equipped with quartz cells. pH measurement was carried out with a pHS3C digital pH-meter (Hangzhou Dongxing Instrument Works, Hangzhou, China) with a combined glass–calomel electrode. An electronic thermostat water-bath (Tianjin Taisite Instrument Company, Tianjin, China) was used for controlling the temperature.
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2.3. Experimental procedures 2.3.1. The binding of EB with DNA The EB fluorescence spectra in the absence and presence DNA was measured by fluorophotometer. All fluorescence measurements were under a pH 7.4 Britton–Robinson buffer. Under the conditions, the excitation and emission wavelength were at 525 nm at 595 nm, respectively, and the slits width of the excitation and emission were all 5 nm. Then the change of absorption spectra was measured when DNA with increasing concentration were added. The slit width was 1.5 nm, the scan rate was 442 nm min−1 and limits of scan wavelength were from 220 nm to 600 nm. At room temperature, UV–vis absorption spectras were measured against the blank solution, which consists of a pH 7.4 Britton–Robinson buffer. 2.3.2. Binding of anthraquinones with DNA in the presence of EB When DNA with increasing concentration was added into the solution containing three anthraquinones, the UV–vis spectra were determined. All fluorescence measurements were carried out in a buffer by adding a various concentrations of chrysophanol, physcion and 1,8-dihydroxy anthraquinone into the 5 ml test tubes, which containing fixed concentration of DNA–EB. The mixture was diluted to mark with buffer and mixed thoroughly. The test tubes were placed in the thermostat water-bath for 5 min for equilibrium. Then the above solution was transferred into the quartz cell and the fluorescence measurement was performed. 2.3.3. Effect of ionic strength on the fluorescence properties The fluorescence measurements were performed with a series of admixture solutions containing constant concentration of DNA–EB and NaCl, and various concentrations of anthraquinones. Then when the concentration of NaCl in the solutions was changed and the concentration of other reagents was fixed, the fluorescence intensity was determined and was recorded at selected experimental conditions.
Fig. 3. The fluorescence spectrum of EB (a) and DNA–EB (b). The concentration of EB and DNA is 3.18 and 25.30 mol l−1 , respectively.
intensity of the system markedly increased when EB is added into the DNA solution (Fig. 3). Thus it is a sensitive reagent to determine diversified character of DNA. The absorption spectrum of EB and DNA are shown in Fig. 4. It can be seen from Fig. 4 that the absorption peaks of EB are at 284.48 and 479.36 nm. After DNA was added into the solution containing the EB, the absorption peaks of EB at 284.48 and 479.36 nm gradually decrease along with the increasing concentration of DNA, and the absorption peak at 479.36 nm of EB has a red-shifts. Generally, bathochromic shift and hypochromic effects are the spectral effect when the small molecules intercalate with DNA [20]. The experimental outcomes validate again that EB intercalates into the base pairs in a DNA double helix and the complex formed is stabilized by the stacking interaction between EB and the DNA bases. The optimal ratio of DNA to EB in the system was determined by a fluorescence method. Result is shown in Fig. 5. It is seen from Fig. 5 that the fluorescence intensity of EB was increased along with the increasing concentration of DNA.
2.3.4. DNA melting studies When the solution was in the absence and presence of anthraquinones, the fluorescence intensity was separately measured in the temperature range from 20 to 100 ◦ C. The data was recorded every other 2–5 ◦ C. 3. Results and discussion 3.1. Binding properties of EB with DNA In recent years, many literatures reported that EB was one of the most frequently used probe molecule in the binding studies of small molecules to DNA. It is well known that the binding of EB to DNA is representative intercalation binding, and it can insert between two adjacent base pairs in a DNA double helix. EB has the characteristics of high sensitivity and selectivity. Fluorescence intensity of EB is so weak, while the fluorescence
Fig. 4. The effect of DNA on the absorption spectrum of EB. DNA concentrations are 0, 16.87, 33.74, 50.61, 67.48, 84.35, 101.22 and 118.09 mol l−1 for absorption spectra (a)–(h), respectively. EB concentration is 15.87 mol l−1 .
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Fig. 5. Fluorescence intensity of EB with increasing concentration of DNA. EB concentration is 3.18 mol l−1 .
And for 3.18 × 10−6 mol l−1 EB, when the DNA concentration was higher than 33.76 × 10−6 mol l−1 , the fluorescence intensity of the system was not enhanced any more. This indicates that binding of EB to DNA near upon saturation. Based on this curve, for 3.18 × 10−6 mol l−1 EB, the DNA concentration in the experiment was selected as 25.30 × 10−6 mol l−1 . It is said that for 5.00 ml solution, 200 l of EB and 300 l of DNA were added. 3.2. Binding characteristics of anthraquinones with DNA in the presence of EB 3.2.1. UV–vis spectra UV–vis spectra are shown in Fig. 6, which is the results of chrysophanol, physcion and 1,8-dihydroxy anthraquinone binding with DNA. Compared with spectra of DNA–EB binding, bathochromic shift and hypochromic effect are not observed for the bining of DNA and anthraquinones. It is say that anthraquinones not intercalate with DNA. When increasing concentration of DNA was continuously added to the solution, maximum absorption wavelength of chrysophanol at 410 nm, physcion at 410 nm and 1,8-dihydroxy anthraquinone at 431 nm almost did not change. Therefore, this analysis of UV–vis spectra suggests that the binding modes of chrysophanol, physcion and 1,8-dihydroxy anthraquinone with DNA are not the classical intercalation binding. 3.2.2. Fluorescence studies When chrysophanol, physcion and 1,8-dihydroxy anthraquinone were added into the DNA–EB solution, the shape and position of the fluorescence peak did not change. While their fluorescence intensity decreased regularly with the increasing concentration of the three anthraquinones. The results are shown in Fig. 7. This indicated that the interaction occurred between the drugs and system of DNA–EB. There are three imaginable reasons for such phenomenon. First, the petition of anthraquinones compound compete binding to DNA on binding sites with EB. And along with concen-
Fig. 6. The absorption spectra of chrysophanol (1), physcion (2) and 1,8dihydroxy anthraquinone (3) in the absence of DNA (a) and in the presence of DNA (b)–(f). DNA concentrations are 16.87, 33.74, 50.61, 67.48 and 84.35 mol l−1 for spectrum (b)–(f), respectively. Concentrations of chrysophanol and physcion are 20.00 mol l−1 and 1,8-dihydroxy anthraquinone is 2.00 mol l−1 .
tration of anthraquinones ceaseless increases, EB was crushed continuously from the DNA double helix, following with the decreases of the system fluorescence intensity. Secondly, probe molecule and anthraquinones compound bound, which made
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concentration of EB binding with DNA reduces, and fluorescence intensity of system decreases, consequently. Thirdly, after anthraquinones compound binding to DNA–EB to form a new shape of non-fluorescence complex DNA–EB–anthraquinones, and following with the decrease of the fluorescence intensity of DNA–EB. In the UV–vis experiment, it has been already indicated that the binding mode of these anthraquinones compound to DNA was not classical intercalation binding. So, there was no competition between anthraquinones compound and EB. Also the first reason is not true of interpreting the fluorescence intensity decrease of DNA–EB. When anthraquinones compound were added into the EB solution, the change of EB fluorescence intensity did not observed in the investigation. This indicates that there were no reaction between anthraquinones compound and EB. Therefore, the second reason also seems not the real reason. Summarize, the third reason seems to be more reasonable, that the binding of anthraquinones compound to DNA–EB conduces fluorescence quenching phenomenon. And the binding modes of DNA and anthraquinones compound have already proven not the intercalation binding by UV–vis experimentation. The experiment result concludes that groove binding is more primarily binding mode rather than intercalation binding. The addition of small molecule to biomacromolecule solution can cause quenching which was divided into static quenching and dynamic quenching. It was assumed that the quenching of anthraquinones compound to DNA–EB were dynamic quenching, which was caused by molecule collision. According to the equation of Stern–Volmer [21,22]: F0 = 1 + Kq τ0 [Q] = 1 + Ksv [Q] (1) F where F0 is the fluorescence intensity in the absence of the quenching reagent, F is the one after adding the quenching reagent, Kq the quenching rate constant of biomacromolecule, τ 0 the average lifetime of the fluorescence molecule in the absence of quenching reagent and its value is about 10−8 s [23]. [Q] is the concentration of quenching reagent, Ksv is the Stern–Volmer dynamic quenching constant, and Ksv = Kq τ 0 . Primary data were transferred to the ORIGIN graphic program for plotting and analysis. For correcting anthraquinones compound absorption influence at excitation wavelength (525 nm) and emission wavelength (595 nm), all fluorescence data was corrected by following formula [24]: Fc = F0 e(A1 +A2 )/2 Fig. 7. Fluorescence spectra of DNA–EB in the presence of chrysophanol (1), physcion (2) and 1,8-dihydroxy anthraquinone (3). The total concentrations of chrysophanol are 0.00 (a), 2.00 (b), 4.00 (c), 6.00 (d), 8.00 (e), 10.00 (f), 12.00 (g) and 14.00 mol l−1 (h); the total concentrations of physcion are 0.00 (a), 2.00 (b), 4.00 (c), 6.00 (d), 8.00 (e), 10.00 (f), 12.00 (g) and 14.00 mol l−1 (h); the total concentrations of 1,8-dihydroxy anthraquinone are 0.00 (a), 0.20 (b), 0.40 (c), 0.60 (d), 0.80 (e), 1.00 (f), 1.20 (g) and 1.40 mol l−1 (h). EB and DNA concentrations are 3.18 and 25.30 mol l−1 , respectively.
(2)
where Fc and F0 are the corrected and measured fluorescence intensity, respectively. A1 and A2 are absorbance of anthraquinones compound at excitation wavelength and emission wavelength, respectively. Fig. 8 was obtained based on Stern–Volmer equation. The Stern–Volmer plots of three anthraquinones binding to DNA–EB were obtained at 15, 25, 35 and 45 ◦ C, respectively. The value of Ksv decreases along with the temperature increases. This indicated that the quenching of anthraquinones to DNA–EB is not controlled by diffusion. Also, the quenching rate constant Kq for chrysophanol, physcion and 1,8-dihydroxy anthraquinone were
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Fig. 9. The plots of 1 /(F0 − F) vs. [Q]−1 for chrysophanol (a), physcion (b) and 1,8-dihydroxy anthraquinone (c).
Double reciprocal chart were obtained using the equation of the static quenching [26]: (F0 − F )−1 = F0−1 + KD F0−1 [Q]−1
(3)
where KD is the dissociation constants for the reaction of quenching reagent and fluorophore. It is defined as the ratio of the slope KD F0 −1 and the intercept F0 −1 . Then the binding constants KA (KA = 1/KD ) can be obtained. Fig. 9 is the curve of 1/(F0 − F) versus [Q]−1 at 25 ◦ C. The binding constants KA of chrysophanol, physcion and 1,8-dihydroxy anthraquinone to DNA–EB at various temperatures are listed in Table 1. In the assay, the binding of three anthraquinones and single stranded DNA (ssDNA) was investigated. The ssDNA was obtained by heating the dsDNA in a boiling water bath for 30 min and quickly cooling in an ice-water bath for 10 min. The binding constants of anthraquinone compounds with dsDNA and ssDNA under the same conditions are also listed in Table 2. The results indicate that binding constants of anthraquinones with dsDNA are much higher than those of anthraquinones with ssDNA. Because the probability of ssDNA binding to anthraquinones is much lower than that of dsDNA, it is reasonable that the anthraquinones binding to DNA is groove binding. 3.3. Effect of ionic strength on the fluorescence properties
Fig. 8. Stern–Volmer curves at 15 (a), 25 (b), 35 (c) and 45 ◦ C (d) of chrysophanol–DNA–EB (A), physcion–DNA–EB (B) and 1,8-dihydroxy anthraquinone–DNA–EB (C), respectively.
1.69 × 1012 , 1.36 × 1012 and 2.01 × 1013 l (mol s)−1 at 25 ◦ C, which was much more than the maximum collision quenching rate constants 2.0 × 1010 l (mol s)−1 [25]. These indicated the process was static quenching rather than the dynamic collision quenching.
Under different concentrations of NaCl, the effect of ionic environment on system of anthraquinones compound and DNA–EB was studied. When the concentration of NaCl ranged from 0 to 0.3 mol l−1 , the fluorescence intensity of system continuously decreased and binding constants of DNA–EB–anthraquinones increased with the concentration of NaCl (Table 3). Because NaCl is not an anionic quencher of DNA [27], the influence comes mostly from ionic strength. When three anthraquinone compounds is absent from the solution of DNA–EB, it was discovered that concentration of NaCl had no effect on the system. Due to the protection of base pairs above and below, EB is not sensitive to surrounding change, when EB insert between two adjacent base pairs in a DNA double helix
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Table 1 The binding constants (KA ) of the anthraquinones with DNA–EB at various temperatures Drug
15 ◦ C
25 ◦ C
KA (l mol−1 ) Chrysophanol Physcion 1,8-Dihydroxy anthraquinone
6.11 × 103 1.96 × 104 1.61 × 105
r
KA (l mol−1 )
0.9987 0.9987 0.9974
1.64 × 104 3.04 × 104 2.88 × 105
Table 2 Binding constants of anthraquinones and DNA–EB at 25 ◦ C Drug
dsDNA KA (l mol−1 )
ssDNA KA (l mol−1 )
Chrysophanol Physcion 1,8-Dihydroxy anthraquinone
1.64 × 104 3.04 × 104 2.88 × 105
4.26 × 103 1.96 × 104 1.42 × 105
[28]. Nevertheless, small molecule binds DNA in the mode of groove binding, and it was exposed in the solution much more than it does in the mode of the intercalation [28,29]. So, the groove binder is much more sensitive to the surrounding change. DNA is an anionic polyelectrolyte, so Na+ ions can exhibit a tendency to coordinate with the phosphate groups of DNA by electrostatic interaction. It was much more in favor of compacting with DNA–EB configuration, narrowing the minor groove and forming the van der Waals, hydrogen bond and hydrophobic force. Accordingly, binding constants were increased. 3.4. Studies of DNA melting When solutions of DNA are exposed to extremes of pH or heat, the double helical structure of DNA undergoes a transition into a randomly single-stranded form at the melting temperature (Tm ). The intercalation of small molecules into the double helix is known to increase the DNA melting temperature, at which the double helix denatures into single stranded DNA [30]. Intercalation binding can stabilize the double helix structure and Tm increases by about 5–8 ◦ C, but the non-intercalation binding causes no obvious increase in Tm [28]. Tm of DNA–EB, chrysophanol–DNA–EB, physcion– DNA–EB and 1,8-dihydroxy anthraquinone–DNA–EB were determined by fluorescence method. When the temperature ranged from 25 to 100 ◦ C, the maximum fluorescence intensity of the systems was noted, and by the curve on the dependence of F25◦ C /F versus T was obtained. The results were shown in Fig. 10. Transition midpoint of the melting curve was Table 3 The effect of NaCl concentrations on the binding constants (KA ) of the anthraquinones with DNA (25 ◦ C) Drug
Chrysophanol Physcion 1,8-Dihydroxy anthraquinone
35 ◦ C
CNaCl (mol l−1 ) 0.1
0.2
0.3
2.35 × 104 3.93 × 104 3.99 × 105
3.05 × 104 4.14 × 104 4.11 × 105
4.05 × 104 5.41 × 104 4.99 × 105
r
KA (l mol−1 )
0.9976 0.9993 0.9995
5.93 × 104 4.19 × 104 3.51 × 105
45 ◦ C r
KA (l mol−1 )
r
0.9960 0.9995 0.9972
1.29 × 105
0.9990 0.9979 0.9997
8.09 × 104 3.62 × 105
the Tm of the mixed solution. After three anthraquinones was added into the solution containing the DNA–EB, Tm of the system was no increases. The results once more proved that the binding modes of these anthraquinones with DNA were nonintercalated. It can be seen from the decrease of Tm , that due to the binding of anthraquinones with DNA, the conformation of DNA changed in some degree and the stabilization of systems decreased. 3.5. Determination of the thermodynamics parameter The binding constants of three anthraquinones with DNA were determined at 25, 35, and 45 ◦ C. It is seen from Table 1 that the binding constants of three anthraquinones increase along with temperature. This result indicates that the binding action of anthraquinones and DNA is endothermal reaction. There are several acting force between small molecular and biomacromolecule, such as hydrogen bond, van der Waals, electrostatic force, hydrophobic force, etc. When there is little change of temperature, the enthalpy change can be seen as a constant. The formulas which reflect the relationship to the change of enthalpy, free energy and entropy are as follows: ln
K2 (1/T1 − 1/T2 ) = H K1 R
(4)
G = H − TS = −RT ln K
(5)
with KA value at different temperatures, H, G and S between three anthraquinones and DNA–EB were obtained at 25 ◦ C, respectively (Table 4). It can be seen from the results obtained, that for the binding systems of chrysophanol, physcion and 1,8-dihydroxy anthraquinone to DNA–EB, H > 0, binding action is endothermal. When H < 0 or H ≈ 0, S > 0, the mainly acting force is electrostatic force; when H < 0, S < 0, the mainly acting force is van der Waals or hydrogen bond and when H > 0, S > 0, the mainly force is hydrophobic [31]. For all the binding system of chrysophanol, physcion and 1,8-dihydroxy with DNA–EB, H > 0, S > 0. Therefore, the hydrophobic forces Table 4 Thermodynamic parameters of the binding reactions of the anthraquinones with DNA at 25 ◦ C Drug
H (kJ mol−1 )
G (kJ mol−1 )
S (J mol−1 k−1 )
Chrysophanol Physcion 1,8-Dihydroxy anthraquinone
98.08 31.64 19.51
−24.04 −25.57 −31.14
409.82 191.99 169.97
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are the main acting force in the binding of three anthraquinones and DNA–EB. 4. Conclusion In this work, the binding of chrysophanol, physcion and 1,8dihydroxy anthraquinone with DNA was in detail investigated by fluorescence quenching and UV–vis spectrophotometry techniques using ethidium bromide as a DNA probe. The binding constants of the three anthraquinones with DNA in the presence of EB were obtained. The effects of the melting temperatures and ionic strength on the binding systems were also studied. All the results revealed that the binding of anthraquinones and DNA–EB are mainly groove binding mode. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] Fig. 10. Melting curves of DNA–EB in the absence of anthraquinones(a1, b1 and c1) and in the presence of chrysophanol (a2), physcion (b2), and 1,8-dihydroxy anthraquinone (c2). DNA and EB concentrations are 25.30 and 3.18 mol l−1 ; chrysophanol, physcion and 1,8-dihydroxy anthraquinone concentrations are 6.00, 6.00 and 0.60 mol l−1 , respectively.
[29]
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Spectrochimica Acta Part A 70 (2008) 136–143
Study of interactions of anthraquinones with DNA using ethidium bromide as a fluorescence probe Chunyu Qiao a , Shuyun Bi b , Ying Sun a , Daqian Song a , Hanqi Zhang a , Weihong Zhou a,∗ a
College of Chemistry, Jilin University, Changchun 130012, PR China b Changchun Normal College, Changchun 130000, PR China Received 26 May 2007; accepted 12 July 2007
Abstract The interactions of fish sperm deoxyribonucleic acid (DNA) with anthraquinones, such as chrysophanol, physcion and 1,8-dihydroxy anthraquinone, were investigated by using ethidium bromide (EB) as fluorescence probe. The binding constants of anthraquinones and DNA were obtained by the fluorescence quenching technique. Further, the binding mechanisms on the reaction of the three anthraquinones with DNA and effect of ionic strength on the fluorescence property of the system have also been investigated. The results of the assay indicate that the binding modes of chrysophanol, physcion and 1,8-dihydroxy anthraquinone with DNA were evaluated to be groove binding. And the binding constants of chrysophanol, physcion and 1,8-dihydroxy anthraquinone with DNA–EB complex were 1.64 × 104 , 3.04 × 104 and 2.88 × 105 l mol−1 , respectively. © 2007 Elsevier B.V. All rights reserved. Keywords: Chrysophanol; Physcion; 1,8-Dihydroxy anthraquinone; DNA; Ethidium bromide; Fluorescence quenching
1. Introduction Deoxyribonucleic acid (DNA) is an important genetic substance in the organism. The regions of DNA involved vital processes, such as gene expression, gene transcription, mutagenesis and carcinogenesis [1]. Since DNA is an important cellular receptor, many chemicals through binding to DNA exert their antitumor effects. Therefore, the changing of DNA replication and the inhibiting growth of the tumor cells, which is the basis of designing new and more efficient antitumor drugs and their effectiveness depend on the mode and affinity of the binding [2]. Therefore, the qualitative and quantitative analysis of nucleic acids as the material base of genetic inheritance is becoming more and more important. Especially in the quantitative analysis, their natural fluorescence intensity is so weak that their fluorescence spectra have scarcely been used for studying their biological properties, and usually some fluorescent probes have been employed for the investigation [3].
∗
Corresponding author. Tel.: +86 431 85095144; fax: +86 431 85095144. E-mail address: [email protected] (W. Zhou).
1386-1425/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2007.07.038
In recent years, there have been many reports on the study of the interaction mechanism between drugs with DNA and the investigation of new effective DNA fluorescent probes [4–6]. Different probes react with DNA in different ways. Among these probes, the fluorescence intensity of some probes is enhanced by DNA, such as ethidium bromide [7–9], acridine orange (AO) [10], bisbenzimidazole (Hoechst 33258) [11], thiazole orange homodimer (TOTO) [12], and berberine [13]. While, the fluorescence of some other probes is quenched by DNA, such as 9,10-anthraquinone-2-sulfonate [14], phosphin 3R [15] and nile blue [16]. In this paper, EB (Fig. 1) was selected as a probe to investigate the interactions of chrysophanol, physcion and 1,8-dihydroxy anthraquinone with DNA by spectral methods. The structures of three analytes used in this work are presented in Fig. 2. Chrysophanol, physcion and 1,8-dihydroxy anthraquinone are the effective components in traditional herbal used in China for treating various ailments. They possess antibacterial, laxative, and antitumor functions. The accepted point is that small molecules are bound to DNA double helix by three binding modes: electrostatic binding, intercalation binding and groove binding [17]. Generally, small molecules have some extent selectivity except
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DNA by using EB as fluorescence probe were the first reported. Consequently, binding studies of anthraquinones with DNA are valuable for the understanding of the reaction mechanism. The results of various binding studies may be contributive in designing new and promising drugs for a variety of medical conditions in clinic. 2. Experimental 2.1. Reagents Fig. 1. The structure of EB.
for its binding with DNA by the mode of electrostatic binding. Electrostatic interaction is along the external DNA double helix. In this work, the binding mechanism on the reaction of the anthraquinones with DNA was investigated by UV–vis spectrophotometry and fluorescence quenching technique. And the effects of the melting temperatures and ionic strength were also obtained. These studies suggest that the groove-binding mode appears to be more acceptable. Here, the interactions of chrysophanol, physcion and 1,8-dihydroxy anthraquinone with
Deoxyribonucleic acid (DNA) was purchased from Sigma Chem. Co. Ethidium bromide (EB, 5 ml mg−1 ) was purchased from Changchun Baotaike Company. Chrysophanol, physcion and 1,8-dihydroxy anthraquinone were obtained from China Drug Biological Products Qualifying Institute. DNA used in this work was double stranded DNA (dsDNA) unless specified. All the chemicals used were of analytical reagent grade and doubly distilled water was used throughout the experiment. A stock solution of DNA was prepared by dissolving an appropriate amount of DNA in doubly distilled water overnight and hoard at 4 ◦ C in the dark for about a week. The concentration of DNA solution was determined from UV absorption at 260 nm using a molar absorption coefficient ε260 = 6600 mol−1 cm−1 . Concentration of DNA in the stock solution was 4.22 × 10−4 mol l−1 in the experiment. Purity of the DNA was checked by monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of A260/A280 > 1.8, indicating that DNA was sufficiently free from protein [18,19]. A stock solution of EB was prepared by adding 0.4 ml liquid EB into flasks of 50 ml and diluted to mark with doubly distilled water and mixed thoroughly. Then the concentration of EB was determined by UV–vis spectrophotometer based on ε480 = 5450 mol−1 cm−1 . The concentration of EB stock solution was 7.94 × 10−5 mol l−1 and the solution was stored in cool and dark place. Chrysophanol, physcion and 1,8-dihydroxy anthraquinone were prepared into 1.00 × 10−3 mol−1 l−1 stock solutions by dissolving an appropriate amount of the drugs in boil ethanol. Britton–Robinson (B–R) buffer (pH 7.4, 2.66 ml 85% H3 PO4 , 2.36 ml acetic acid, 2.4700 g boric acid were dissolved in 1000 ml doubly distilled water) was used to control the pH of the reaction system. 2.2. Apparatus
Fig. 2. Molecular structure of chrysophanol (a), physcion (b) and 1,8-dihydroxy anthraquinone (c).
All fluorescence measurements were conducted by using a Shimadzu RF-5301PC fluorophotometer (Kyoto, Japan) equipped with a xenon lamp source and quartz cells of 1 cm path length. All absorption spectra were measured on a GBC Cintra 10e UV–vis spectrophotometer (Australia) equipped with quartz cells. pH measurement was carried out with a pHS3C digital pH-meter (Hangzhou Dongxing Instrument Works, Hangzhou, China) with a combined glass–calomel electrode. An electronic thermostat water-bath (Tianjin Taisite Instrument Company, Tianjin, China) was used for controlling the temperature.
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2.3. Experimental procedures 2.3.1. The binding of EB with DNA The EB fluorescence spectra in the absence and presence DNA was measured by fluorophotometer. All fluorescence measurements were under a pH 7.4 Britton–Robinson buffer. Under the conditions, the excitation and emission wavelength were at 525 nm at 595 nm, respectively, and the slits width of the excitation and emission were all 5 nm. Then the change of absorption spectra was measured when DNA with increasing concentration were added. The slit width was 1.5 nm, the scan rate was 442 nm min−1 and limits of scan wavelength were from 220 nm to 600 nm. At room temperature, UV–vis absorption spectras were measured against the blank solution, which consists of a pH 7.4 Britton–Robinson buffer. 2.3.2. Binding of anthraquinones with DNA in the presence of EB When DNA with increasing concentration was added into the solution containing three anthraquinones, the UV–vis spectra were determined. All fluorescence measurements were carried out in a buffer by adding a various concentrations of chrysophanol, physcion and 1,8-dihydroxy anthraquinone into the 5 ml test tubes, which containing fixed concentration of DNA–EB. The mixture was diluted to mark with buffer and mixed thoroughly. The test tubes were placed in the thermostat water-bath for 5 min for equilibrium. Then the above solution was transferred into the quartz cell and the fluorescence measurement was performed. 2.3.3. Effect of ionic strength on the fluorescence properties The fluorescence measurements were performed with a series of admixture solutions containing constant concentration of DNA–EB and NaCl, and various concentrations of anthraquinones. Then when the concentration of NaCl in the solutions was changed and the concentration of other reagents was fixed, the fluorescence intensity was determined and was recorded at selected experimental conditions.
Fig. 3. The fluorescence spectrum of EB (a) and DNA–EB (b). The concentration of EB and DNA is 3.18 and 25.30 mol l−1 , respectively.
intensity of the system markedly increased when EB is added into the DNA solution (Fig. 3). Thus it is a sensitive reagent to determine diversified character of DNA. The absorption spectrum of EB and DNA are shown in Fig. 4. It can be seen from Fig. 4 that the absorption peaks of EB are at 284.48 and 479.36 nm. After DNA was added into the solution containing the EB, the absorption peaks of EB at 284.48 and 479.36 nm gradually decrease along with the increasing concentration of DNA, and the absorption peak at 479.36 nm of EB has a red-shifts. Generally, bathochromic shift and hypochromic effects are the spectral effect when the small molecules intercalate with DNA [20]. The experimental outcomes validate again that EB intercalates into the base pairs in a DNA double helix and the complex formed is stabilized by the stacking interaction between EB and the DNA bases. The optimal ratio of DNA to EB in the system was determined by a fluorescence method. Result is shown in Fig. 5. It is seen from Fig. 5 that the fluorescence intensity of EB was increased along with the increasing concentration of DNA.
2.3.4. DNA melting studies When the solution was in the absence and presence of anthraquinones, the fluorescence intensity was separately measured in the temperature range from 20 to 100 ◦ C. The data was recorded every other 2–5 ◦ C. 3. Results and discussion 3.1. Binding properties of EB with DNA In recent years, many literatures reported that EB was one of the most frequently used probe molecule in the binding studies of small molecules to DNA. It is well known that the binding of EB to DNA is representative intercalation binding, and it can insert between two adjacent base pairs in a DNA double helix. EB has the characteristics of high sensitivity and selectivity. Fluorescence intensity of EB is so weak, while the fluorescence
Fig. 4. The effect of DNA on the absorption spectrum of EB. DNA concentrations are 0, 16.87, 33.74, 50.61, 67.48, 84.35, 101.22 and 118.09 mol l−1 for absorption spectra (a)–(h), respectively. EB concentration is 15.87 mol l−1 .
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Fig. 5. Fluorescence intensity of EB with increasing concentration of DNA. EB concentration is 3.18 mol l−1 .
And for 3.18 × 10−6 mol l−1 EB, when the DNA concentration was higher than 33.76 × 10−6 mol l−1 , the fluorescence intensity of the system was not enhanced any more. This indicates that binding of EB to DNA near upon saturation. Based on this curve, for 3.18 × 10−6 mol l−1 EB, the DNA concentration in the experiment was selected as 25.30 × 10−6 mol l−1 . It is said that for 5.00 ml solution, 200 l of EB and 300 l of DNA were added. 3.2. Binding characteristics of anthraquinones with DNA in the presence of EB 3.2.1. UV–vis spectra UV–vis spectra are shown in Fig. 6, which is the results of chrysophanol, physcion and 1,8-dihydroxy anthraquinone binding with DNA. Compared with spectra of DNA–EB binding, bathochromic shift and hypochromic effect are not observed for the bining of DNA and anthraquinones. It is say that anthraquinones not intercalate with DNA. When increasing concentration of DNA was continuously added to the solution, maximum absorption wavelength of chrysophanol at 410 nm, physcion at 410 nm and 1,8-dihydroxy anthraquinone at 431 nm almost did not change. Therefore, this analysis of UV–vis spectra suggests that the binding modes of chrysophanol, physcion and 1,8-dihydroxy anthraquinone with DNA are not the classical intercalation binding. 3.2.2. Fluorescence studies When chrysophanol, physcion and 1,8-dihydroxy anthraquinone were added into the DNA–EB solution, the shape and position of the fluorescence peak did not change. While their fluorescence intensity decreased regularly with the increasing concentration of the three anthraquinones. The results are shown in Fig. 7. This indicated that the interaction occurred between the drugs and system of DNA–EB. There are three imaginable reasons for such phenomenon. First, the petition of anthraquinones compound compete binding to DNA on binding sites with EB. And along with concen-
Fig. 6. The absorption spectra of chrysophanol (1), physcion (2) and 1,8dihydroxy anthraquinone (3) in the absence of DNA (a) and in the presence of DNA (b)–(f). DNA concentrations are 16.87, 33.74, 50.61, 67.48 and 84.35 mol l−1 for spectrum (b)–(f), respectively. Concentrations of chrysophanol and physcion are 20.00 mol l−1 and 1,8-dihydroxy anthraquinone is 2.00 mol l−1 .
tration of anthraquinones ceaseless increases, EB was crushed continuously from the DNA double helix, following with the decreases of the system fluorescence intensity. Secondly, probe molecule and anthraquinones compound bound, which made
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concentration of EB binding with DNA reduces, and fluorescence intensity of system decreases, consequently. Thirdly, after anthraquinones compound binding to DNA–EB to form a new shape of non-fluorescence complex DNA–EB–anthraquinones, and following with the decrease of the fluorescence intensity of DNA–EB. In the UV–vis experiment, it has been already indicated that the binding mode of these anthraquinones compound to DNA was not classical intercalation binding. So, there was no competition between anthraquinones compound and EB. Also the first reason is not true of interpreting the fluorescence intensity decrease of DNA–EB. When anthraquinones compound were added into the EB solution, the change of EB fluorescence intensity did not observed in the investigation. This indicates that there were no reaction between anthraquinones compound and EB. Therefore, the second reason also seems not the real reason. Summarize, the third reason seems to be more reasonable, that the binding of anthraquinones compound to DNA–EB conduces fluorescence quenching phenomenon. And the binding modes of DNA and anthraquinones compound have already proven not the intercalation binding by UV–vis experimentation. The experiment result concludes that groove binding is more primarily binding mode rather than intercalation binding. The addition of small molecule to biomacromolecule solution can cause quenching which was divided into static quenching and dynamic quenching. It was assumed that the quenching of anthraquinones compound to DNA–EB were dynamic quenching, which was caused by molecule collision. According to the equation of Stern–Volmer [21,22]: F0 = 1 + Kq τ0 [Q] = 1 + Ksv [Q] (1) F where F0 is the fluorescence intensity in the absence of the quenching reagent, F is the one after adding the quenching reagent, Kq the quenching rate constant of biomacromolecule, τ 0 the average lifetime of the fluorescence molecule in the absence of quenching reagent and its value is about 10−8 s [23]. [Q] is the concentration of quenching reagent, Ksv is the Stern–Volmer dynamic quenching constant, and Ksv = Kq τ 0 . Primary data were transferred to the ORIGIN graphic program for plotting and analysis. For correcting anthraquinones compound absorption influence at excitation wavelength (525 nm) and emission wavelength (595 nm), all fluorescence data was corrected by following formula [24]: Fc = F0 e(A1 +A2 )/2 Fig. 7. Fluorescence spectra of DNA–EB in the presence of chrysophanol (1), physcion (2) and 1,8-dihydroxy anthraquinone (3). The total concentrations of chrysophanol are 0.00 (a), 2.00 (b), 4.00 (c), 6.00 (d), 8.00 (e), 10.00 (f), 12.00 (g) and 14.00 mol l−1 (h); the total concentrations of physcion are 0.00 (a), 2.00 (b), 4.00 (c), 6.00 (d), 8.00 (e), 10.00 (f), 12.00 (g) and 14.00 mol l−1 (h); the total concentrations of 1,8-dihydroxy anthraquinone are 0.00 (a), 0.20 (b), 0.40 (c), 0.60 (d), 0.80 (e), 1.00 (f), 1.20 (g) and 1.40 mol l−1 (h). EB and DNA concentrations are 3.18 and 25.30 mol l−1 , respectively.
(2)
where Fc and F0 are the corrected and measured fluorescence intensity, respectively. A1 and A2 are absorbance of anthraquinones compound at excitation wavelength and emission wavelength, respectively. Fig. 8 was obtained based on Stern–Volmer equation. The Stern–Volmer plots of three anthraquinones binding to DNA–EB were obtained at 15, 25, 35 and 45 ◦ C, respectively. The value of Ksv decreases along with the temperature increases. This indicated that the quenching of anthraquinones to DNA–EB is not controlled by diffusion. Also, the quenching rate constant Kq for chrysophanol, physcion and 1,8-dihydroxy anthraquinone were
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Fig. 9. The plots of 1 /(F0 − F) vs. [Q]−1 for chrysophanol (a), physcion (b) and 1,8-dihydroxy anthraquinone (c).
Double reciprocal chart were obtained using the equation of the static quenching [26]: (F0 − F )−1 = F0−1 + KD F0−1 [Q]−1
(3)
where KD is the dissociation constants for the reaction of quenching reagent and fluorophore. It is defined as the ratio of the slope KD F0 −1 and the intercept F0 −1 . Then the binding constants KA (KA = 1/KD ) can be obtained. Fig. 9 is the curve of 1/(F0 − F) versus [Q]−1 at 25 ◦ C. The binding constants KA of chrysophanol, physcion and 1,8-dihydroxy anthraquinone to DNA–EB at various temperatures are listed in Table 1. In the assay, the binding of three anthraquinones and single stranded DNA (ssDNA) was investigated. The ssDNA was obtained by heating the dsDNA in a boiling water bath for 30 min and quickly cooling in an ice-water bath for 10 min. The binding constants of anthraquinone compounds with dsDNA and ssDNA under the same conditions are also listed in Table 2. The results indicate that binding constants of anthraquinones with dsDNA are much higher than those of anthraquinones with ssDNA. Because the probability of ssDNA binding to anthraquinones is much lower than that of dsDNA, it is reasonable that the anthraquinones binding to DNA is groove binding. 3.3. Effect of ionic strength on the fluorescence properties
Fig. 8. Stern–Volmer curves at 15 (a), 25 (b), 35 (c) and 45 ◦ C (d) of chrysophanol–DNA–EB (A), physcion–DNA–EB (B) and 1,8-dihydroxy anthraquinone–DNA–EB (C), respectively.
1.69 × 1012 , 1.36 × 1012 and 2.01 × 1013 l (mol s)−1 at 25 ◦ C, which was much more than the maximum collision quenching rate constants 2.0 × 1010 l (mol s)−1 [25]. These indicated the process was static quenching rather than the dynamic collision quenching.
Under different concentrations of NaCl, the effect of ionic environment on system of anthraquinones compound and DNA–EB was studied. When the concentration of NaCl ranged from 0 to 0.3 mol l−1 , the fluorescence intensity of system continuously decreased and binding constants of DNA–EB–anthraquinones increased with the concentration of NaCl (Table 3). Because NaCl is not an anionic quencher of DNA [27], the influence comes mostly from ionic strength. When three anthraquinone compounds is absent from the solution of DNA–EB, it was discovered that concentration of NaCl had no effect on the system. Due to the protection of base pairs above and below, EB is not sensitive to surrounding change, when EB insert between two adjacent base pairs in a DNA double helix
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Table 1 The binding constants (KA ) of the anthraquinones with DNA–EB at various temperatures Drug
15 ◦ C
25 ◦ C
KA (l mol−1 ) Chrysophanol Physcion 1,8-Dihydroxy anthraquinone
6.11 × 103 1.96 × 104 1.61 × 105
r
KA (l mol−1 )
0.9987 0.9987 0.9974
1.64 × 104 3.04 × 104 2.88 × 105
Table 2 Binding constants of anthraquinones and DNA–EB at 25 ◦ C Drug
dsDNA KA (l mol−1 )
ssDNA KA (l mol−1 )
Chrysophanol Physcion 1,8-Dihydroxy anthraquinone
1.64 × 104 3.04 × 104 2.88 × 105
4.26 × 103 1.96 × 104 1.42 × 105
[28]. Nevertheless, small molecule binds DNA in the mode of groove binding, and it was exposed in the solution much more than it does in the mode of the intercalation [28,29]. So, the groove binder is much more sensitive to the surrounding change. DNA is an anionic polyelectrolyte, so Na+ ions can exhibit a tendency to coordinate with the phosphate groups of DNA by electrostatic interaction. It was much more in favor of compacting with DNA–EB configuration, narrowing the minor groove and forming the van der Waals, hydrogen bond and hydrophobic force. Accordingly, binding constants were increased. 3.4. Studies of DNA melting When solutions of DNA are exposed to extremes of pH or heat, the double helical structure of DNA undergoes a transition into a randomly single-stranded form at the melting temperature (Tm ). The intercalation of small molecules into the double helix is known to increase the DNA melting temperature, at which the double helix denatures into single stranded DNA [30]. Intercalation binding can stabilize the double helix structure and Tm increases by about 5–8 ◦ C, but the non-intercalation binding causes no obvious increase in Tm [28]. Tm of DNA–EB, chrysophanol–DNA–EB, physcion– DNA–EB and 1,8-dihydroxy anthraquinone–DNA–EB were determined by fluorescence method. When the temperature ranged from 25 to 100 ◦ C, the maximum fluorescence intensity of the systems was noted, and by the curve on the dependence of F25◦ C /F versus T was obtained. The results were shown in Fig. 10. Transition midpoint of the melting curve was Table 3 The effect of NaCl concentrations on the binding constants (KA ) of the anthraquinones with DNA (25 ◦ C) Drug
Chrysophanol Physcion 1,8-Dihydroxy anthraquinone
35 ◦ C
CNaCl (mol l−1 ) 0.1
0.2
0.3
2.35 × 104 3.93 × 104 3.99 × 105
3.05 × 104 4.14 × 104 4.11 × 105
4.05 × 104 5.41 × 104 4.99 × 105
r
KA (l mol−1 )
0.9976 0.9993 0.9995
5.93 × 104 4.19 × 104 3.51 × 105
45 ◦ C r
KA (l mol−1 )
r
0.9960 0.9995 0.9972
1.29 × 105
0.9990 0.9979 0.9997
8.09 × 104 3.62 × 105
the Tm of the mixed solution. After three anthraquinones was added into the solution containing the DNA–EB, Tm of the system was no increases. The results once more proved that the binding modes of these anthraquinones with DNA were nonintercalated. It can be seen from the decrease of Tm , that due to the binding of anthraquinones with DNA, the conformation of DNA changed in some degree and the stabilization of systems decreased. 3.5. Determination of the thermodynamics parameter The binding constants of three anthraquinones with DNA were determined at 25, 35, and 45 ◦ C. It is seen from Table 1 that the binding constants of three anthraquinones increase along with temperature. This result indicates that the binding action of anthraquinones and DNA is endothermal reaction. There are several acting force between small molecular and biomacromolecule, such as hydrogen bond, van der Waals, electrostatic force, hydrophobic force, etc. When there is little change of temperature, the enthalpy change can be seen as a constant. The formulas which reflect the relationship to the change of enthalpy, free energy and entropy are as follows: ln
K2 (1/T1 − 1/T2 ) = H K1 R
(4)
G = H − TS = −RT ln K
(5)
with KA value at different temperatures, H, G and S between three anthraquinones and DNA–EB were obtained at 25 ◦ C, respectively (Table 4). It can be seen from the results obtained, that for the binding systems of chrysophanol, physcion and 1,8-dihydroxy anthraquinone to DNA–EB, H > 0, binding action is endothermal. When H < 0 or H ≈ 0, S > 0, the mainly acting force is electrostatic force; when H < 0, S < 0, the mainly acting force is van der Waals or hydrogen bond and when H > 0, S > 0, the mainly force is hydrophobic [31]. For all the binding system of chrysophanol, physcion and 1,8-dihydroxy with DNA–EB, H > 0, S > 0. Therefore, the hydrophobic forces Table 4 Thermodynamic parameters of the binding reactions of the anthraquinones with DNA at 25 ◦ C Drug
H (kJ mol−1 )
G (kJ mol−1 )
S (J mol−1 k−1 )
Chrysophanol Physcion 1,8-Dihydroxy anthraquinone
98.08 31.64 19.51
−24.04 −25.57 −31.14
409.82 191.99 169.97
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are the main acting force in the binding of three anthraquinones and DNA–EB. 4. Conclusion In this work, the binding of chrysophanol, physcion and 1,8dihydroxy anthraquinone with DNA was in detail investigated by fluorescence quenching and UV–vis spectrophotometry techniques using ethidium bromide as a DNA probe. The binding constants of the three anthraquinones with DNA in the presence of EB were obtained. The effects of the melting temperatures and ionic strength on the binding systems were also studied. All the results revealed that the binding of anthraquinones and DNA–EB are mainly groove binding mode. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] Fig. 10. Melting curves of DNA–EB in the absence of anthraquinones(a1, b1 and c1) and in the presence of chrysophanol (a2), physcion (b2), and 1,8-dihydroxy anthraquinone (c2). DNA and EB concentrations are 25.30 and 3.18 mol l−1 ; chrysophanol, physcion and 1,8-dihydroxy anthraquinone concentrations are 6.00, 6.00 and 0.60 mol l−1 , respectively.
[29]
[30] [31]
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