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Studies of interaction between Safranine T and double helix DNA by spectral methods
Spectrochimica Acta Part A 54 (1998) 883 – 892
Studies of interaction between Safranine T and double helix DNA by spectral methods Ying Cao, Xi-wen He * Department of Chemistry, Nankai Uni6ersity, Tianjin 300071, China Received 11 August 1997; accepted 18 November 1997
Abstract In this paper, the DNA affinity properties of Safranine T (ST), which features a phenazinyl group, were studied. The studies indicated that ST could intercalate into the stack base pairs of DNA. Intrinsic binding constants obtained by different spectral methods were consistent within experimental errors. They were of the order of 104 M − 1 in DNA base pairs, and the binding site size was about 7 in DNA base pairs. Studies of fluorescence quenching by anionic quenchers and melting temperature of DNA all supported the intercalative binding of ST with DNA. The experiments also showed that electrostatic binding played an important role in the interaction of ST with DNA. This research offers a new intercalation functional group to DNA-targeted drug design. © 1998 Elsevier Science B.V. All rights reserved. Keywords: DNA; Intercalative binding; Safranine T; Phenazium dye; Fluorescence quenching
1. Introduction Several small molecules have been shown to interact with DNA at the molecular level by specific binding modes. These binding studies were driven partly by the need to understand the mechanism of anticancer drug action [1], to decipher the chemical basis for the carcinogenicity of environmental pollutants and toxic chemicals [2] and to serve as analogues in studies of protein-nucleic acid recognition [3]. A systematic investigation of the binding of metal complexes, porphyrins, natural antibiotics and other planar
heterocyclic cations have revealed several structural and electronic factors that control the DNA binding affinity and sequence specificity of small molecules [4]. The results of these various binding studies have been useful in designing new and promising anticancer agents for clinical use [5].
* Corresponding author. E-mail: [email protected]
1386-1425/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S1386-1425(97)00277-1
Scheme 1. Binding modes of small molecules with DNA.
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mechanism of bioactivity of phenazine derivatives on the biomolecular level. To date, most studied intercalators featured acridinyl and thiazinyl moieties, so the findings of intercalative binding of ST with DNA base pairs provide a new functional group for intercalators.
Scheme 2. Structure of safranine T (ST).
Small molecules are bound to DNA double helix by three binding modes: electrostatics binding, groove binding and intercalative binding (Scheme 1) [6]. The accepted point is that small molecules have some extent selectivity when binding with DNA except by the mode of electrostatic binding. The steric structure of small molecules and DNA determine the binding properties (such as affinity, binding site) of small molecules as well as the electronic state of small molecules. In general, planarity was suggested to be one of the important features needed for efficient intercalators [7]. A non-negative charge on the small molecules is considered another important feature, otherwise electrostatic repulsion between anionic DNA polymer and small negative molecules inhibit interaction efficiency. In this paper, we studied the DNA binding properties of a phenazium dye, Safranine T (ST), which features a planar phenazine ring and a positive charge (Scheme 2). Therefore, the planar hydrophobic phenazinyl moiety of ST is expected to facilitate intercalation of ST into the relatively nonpolar interior of the DNA helix. The strong absorption and fluorescence characteristics of ST provide a sensitive spectroscopic handle to study its interaction with DNA. The changes in the intensities of these spectra can be used to decipher the nature and the strength of the stacking interaction between the chromophore and the DNA base pairs. Phenazine derivatives are a kind of antibiotic. Studies showed that some phenazium dyes had antimalarial potency and selectivity, and inhibited many bacteria from growing [8]. What we report here on the interaction between ST and DNA may be relevant to our understanding of the
2. Experiments and methods
2.1. Chemicals The calf thymus (ct) DNA sample was purchased from Sino-American biological engineering company and purified by phenol extraction, as described in the literature [9]. The purity of the final DNA preparation was checked by monitoring the absorption spectrum and the ratio of the absorbance at 260 and 280 nm. The sample was dissolved in 0.05 M Tris buffer with HCl (pH 7.4). The DNA concentration per nucleotide (c(P)) was determined by absorption spectroscopy, using the molar extinction coefficients at 260 nm as 6600 M − 1 [10]. ST was purchased from Beijing No.2 Chemical plant, and used after recrystallized from water. Other reagents were at least analytical grade, and were used without further purification.
2.2. Spectral measurements The absorption spectra were recorded on a Shimadzu UV-240 spectrophotometer and the fluorescence spectra were recorded on a Shimadzu FL-510 spectrofluorophotometer. The absorption and fluorescence titrations with DNA were conducted by keeping the concentration of ST constant, and varying the nucleic acid concentrations. This was done by dissolving an appropriate amount of ST in the DNA stock solution and mixing various proportions of ST and the DNA stock solutions, while maintaining the total volume of the solution constant. This resulted in a series of solutions with varying concentrations of DNA but with a constant concentration of ST. Fluorescence quenching experiments were carried out by adding 10 ml aliquots of a 0.5 M quencher stock solution to samples each time and the volume effect could be ignored. The samples were
Y. Cao, X.-w. He / Spectrochimica Acta Part A 54 (1998) 883–892
excited at 520 nm and the fluorescence intensity was monitored at 568 nm. The intrinsic binding constants of ST with calf thymus DNA was determined by absorption and fluorescence titrations. In the case of the former, the absorbance at 520 nm was recorded after each addition of DNA. The intrinsic binding constant Ka was determined from Eq. (1) [11]: c/Doa =c/Do +1/DoKa
(1)
where c is the concentration of DNA determined by the absorbance at 260 nm, Doa =[oa −of], and Do = [ob −of]. oa, ob and of correspond to the apparent extinction coefficient of ST, the extinction coefficient of the bound form of ST and of the free ST, respectively. In fluorescence quenching experiments, the data were plotted according to the Stern–Volmer equation: I0/I =1+ Kq[Q]
(2)
885
3. Results and discussion
3.1. Absorption studies The electronic absorption spectra in the presence of increasing amounts of DNA of ST showed decreases (hypochromicity) and extensive red-shift at the peak intensities (Fig. 1). The absorbance of ST in the peak was decreased by about 20% and the maximum absorbance wavelength was shifted from 520 nm to 529 nm. The addition of further amounts of DNA to the dye solution did not cause any more absorption changes, due to the binding saturation of the dye with DNA. Two isobestic points were observed at 432 nm and 538 nm, respectively. Hypochromism was suggested to be due to strong interactions between the electronic states of the intercalating chromophore and that of the DNA base pairs [13]. The spectral changes that we observed (hypochromicity, red-
where I0 and I are the fluorescence intensities in the absence and in the presence of DNA. Kq is the Stern–Volmer fluorescence quenching constant, which is a measurement of the efficiency of quenching by quencher. [Q] is the concentration of quencher. Data from the fluorescence titrations were also used to determine the binding constant of ST with DNA by the modified Scatchard Eq. (3) given by McGhee and von Hippel [12]: r/cf =Ki(1−nr)[(1−nr)/[1 −(n − 1)r]]n − 1
(3)
where Ki is the intrinsic binding constant and n is the binding site size in base pairs, I0 and I are the same as in Eq. (2). A plot of r/cf vs. r was constructed, where r is equal to cb/[DNA]. cb, cf are concentrations of bound and free ST, respectively. The binding data were treated by leastsquares methods and values of Ki and n were obtained. The DNA melting studies were done by controlling the temperature of the sample cell with a Shimadzu circulating bath while monitoring the absorbance at 260 nm. The temperature of the solution was continuous monitored with a thermo-couple attached to the sample holder.
Fig. 1. Titration electronic absorption spectra of ST by DNA: a. 0 mM; b. 14.2 mM; c. 28.5 mM; d. 42.7 mM; e. 56.9 mM; f. 71.1 mM; g. 85.4 mM.
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Fig. 2. Half-reciprocal plot of ST (1.00 × 10 − 5 M) with DNA as determined from the electronic absorption spectra.
shift and isobestic point) were consistent with the intercalation of the chromophore into the stack DNA base pairs. Therefore, the phenazine dye, ST, was considered as an intercalator of DNA. Absorbance data of ST at 520 nm with different concentrations of DNA were used to obtain the intrinsic binding constant by Eq. (1) (Fig. 2). The best fit linear regression equation was: c/Do = 6.52× 10 − 9 +1.0 ×10 − 4c
there was DNA in the solution or not, and the quenching efficiency of ST fluorescence by DNA decreased (Fig. 4). That is, the fluorescence quenching of ST by DNA was fit to the static quenching model [14], i.e. a complex was formed between the DNA base pairs and the phenazinyl of ST. Data in Fig. 3 fitted poorly to the Stern– Volmer equation (Eq. (2)): The quenching curve tended toward the x-axis in the case of high concentration of DNA, and the curvature was reduced when the temperature rose (Fig. 4). These results indicated that there were two kinds of fluorophores in solution [14]. Considering the state of solution, the two kind of fluorophores should be phenazinyl of ST and the complex formed between ST and the DNA base pairs. The changes of fluorescence spectra of ST after adding DNA also indicated that there were strong interactions between the chromophore of ST and the base pairs of DNA. Combined with the changes of absorption spectra, the strong interaction
(r =0.9976)
Then, the extinction coefficient of the bound form of ST (ob =of −Do) of 1.85 × 104 M − 1 cm − 1 and the intrinsic binding constant of ST with DNA (Ka) of 1.53×104 M − 1 were deduced.
3.2. Fluorescence studies The binding of ST to DNA was further studied by fluorescence spectroscopy. Binding of ST to DNA was found to quench the fluorescence of ST (Fig. 3). The binding did not show saturation of the ST fluorescence spectra even when large amounts of DNA were added, which would have already saturated in the electronic absorption spectra. This indicated that only a fraction of the binding sites changed the electronic state of chromophore of ST. If the experimental temperature was raised, the fluorescence intensity of ST increased whether
Fig. 3. Fluorescence spectra of ST (1.00×10 − 5 M) with DNA (a to l: 0, 14.2, 28.5, 42.7, 56.9, 71.1, 85.4, 99.6, 113.8, 128.1, 142.3, 156.5 mM) (lex =520 nm).
Y. Cao, X.-w. He / Spectrochimica Acta Part A 54 (1998) 883–892
Fig. 4. Plots of fluorescence quenching of ST (1.00 ×10 − 5 M) by DNA (7.11 × 10 − 5 M) at different temperatures (lex = 520 nm, lem = 568 nm).
should be considered as the intercalative binding of ST with DNA. The interaction was weakened when the temperature rose. Thus the quenching efficiency of DNA to ST was reduced when the experimental temperature rose. Because it was the static quenching of DNA to ST, the quenching constant was considered as the formation constant of ST and DNA [14], i.e. the binding constant of ST with DNA. In order to obtain the quenching constant of ST by DNA, Eq. (3) was modified as [15]: 1 −1 −1 (I0 −I) − 1 =I − 0 +K %qI 0 [Q]
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Fig. 5. Double-reciprocal plot of fluorescence quenching of ST (1.00 ×10 − 5 M) by DNA (lex =520 nm, lem =568 nm).
obtained. As we discussed before, an intrinsic binding constant (Ka) of 1.53×104 M − 1 resulted from the electronic absorption titration data, and a binding constant (Kq) of 1.00× 104 M − 1 was deduced from the modified Stern–Volmer equation (Eq. (4)). The binding constants obtained by the three methods were consistent within the experimental error. This increased the credibility of these three measurements. The large binding constant for ST indicated that the phenazinyl chromophore had high affinity for DNA base pairs. The estimated site size of about 7 for this aromatic cation was consistent with the neighboring
(4)
where I0, I and Q were the same as formerly, the −1 binding constant was K ). The doubleq ( = (Kq) reciprocal plot was made by (I0 −I) − 1 vs. [Q] − 1 (Fig. 5) and the binding constant (Kq) of 1.00× 104 M − 1 was obtained. Compared with the intrinsic binding constant obtained by the electronic absorption spectra, the binding constant from the modified Stern–Volmer equation (Eq. (2)) was almost the same. The fluorescence titration data were also used to estimate the binding constant and the binding size of ST with DNA by the modified Scatchard equation (Eq. (3)). The plot of r/cf vs. r is shown in Fig. 6. From the best fit of the data to Eq. (3), an intrinsic binding constant (Ki) of 6.11 × 104 M − 1, and a site size of 6.7 base pairs were
Fig. 6. Scatchard plot of the ST fluorescence titration by DNA (lex =520 nm, lem =568 nm).
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Table 1 Binding constants of some intercalators and DNA Intercalators
Condition
Ethidium bromide
pH 7, 5 mM phosphate buffer and 5 mM Na2SO4 Acridine orange pH 7, 5 mM phosphate buffer and 5 mM Na2SO4 6-Chloro-2-methoxy-9-acdidine pH 6.50, 50 mM NaCl, in water-DMSO (9-Anthrylmethyl) ammonium 5 mM Tris–HCl buffer chloride Safranine T 50 mM Tris–HCl buffer
exclusion model, but it was much larger than the reported values for some typical intercalators such as ethidium bromide and acridine derivatives (Table 1). This may be due to the big size of the benezyl subsitituent, which is much larger than the other typical intercalators. The intercalative binding of ST was hindered in the wider neighboring region of bound site than other intercalators. But the size of substituent had no influence on the affinity of chromophore with DNA base pairs. Table 1 shows the binding constants of some intercalators with DNA. Though the methods and conditions are different, it still can be concluded that the affnity of ST with DNA is not weaker than the others.
3.3. Fluorescence quenching studies If small molecules intercalate into DNA base pairs, the double helix of DNA would protect them from some quenchers, e.g. iodide and bromide, owing to the base pairs above and below the intercalator. Otherwise, the fluorescence quenching of ST would have no observed changes after addition of DNA. In aqueous solutions, potassium iodide and potassium ferrocyanide quench the fluorescence of ST very efficiently, so we used these two anionic quenchers to determine the relative accessibilities of the free and bound ST. After adding KI to solution, the fluorescence quenching was much smaller in the presence of DNA than in the absence of DNA. The quenching data were plotted according to the Stern – Volmer equation (Eq. (2)), and treated by the
Binding constant, K Site size (n) (M−1)
Reference
2.6×106
2
[16]
4×105
2
[16]
6.6×104 7.8×104
2.2 4.8
[17] [18]
6.1×104
6.7
—
linear least-squares method. The quenching constants of KI to ST were 13 and 28 M − 1 with and without DNA, respectively (Fig. 7). The quenching of the dye fluorescence was decreased by a factor greater than two when ST was bound to DNA. This was due to the intercalative binding of ST fluorophores with helix of DNA, which prevented ST fluorescence quenching from the anionic quenchers. The experimental results of ST fluorescence quenching by potassium ferrocyanide was similar to those with KI. The quenching constants obtained by the Stern–Volmer equation were 18 and 30 M − 1 in the presence and in the absence of DNA (Fig. 8). In order to determine the role of ionic strength, we examined the changes of ST fluorescence by adding the strong electrolyte, sodium chloride, instead of KI and potassium ferrocyanide to solution with and without DNA. For NaCl, which is not an anionic quencher, its influence on the ST fluorescence intensity comes only from the ionic strength [19]. We found that the ionic strength had little or no effect on the ST fluorescence intensity no matter if there was DNA in the solution (Fig. 8). So the changes of fluorescence intensity after adding quenchers had little correlation with the ionic strength. The decreases of the quenching constants of KI and potassium ferrocyanide on ST after adding DNA resulted from the intercalative binding of ST with the DNA double helix, which prevented fluorescence quenching of ST from anionic quenchers because of the sandwich structure formed by one chromophore of ST and two DNA base pairs as well as by the polyphosphate anionic skeleton of DNA.
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Fig. 7. Fluorescence quenching of ST (1.00 × 10 − 5 M) by KI in the presence of DNA (7.11 ×10 − 5 M) and in the absence of DNA (lex = 520 nm, lem = 568 nm).
3.4. Salt effect on binding Because the intercalator of ST carries a positive charge and DNA has a negative polyphosphate skeleton, We considered the electrostatic interactions between the intercalator and DNA. In order to test if there was electrostatic interaction between ST and DNA, the strong electrolyte, sodium chloride (NaCl), was used. The electrostatic interaction would be weakened by the additional counter ion because of the conversion of the electrostatic atmosphere of DNA periphery. Some reports showed that very high concentration of NaCl would hinder small molecules from binding with DNA [19]. If there did exist electrostatic interaction between ST and DNA, the following phenomenon should happen by increasing the ionic strength of solutions of ST and DNA: The absorbance of solutions would increase when NaCl was added, but it would never exceed the absorbance of the ST solution. The experimental results confirmed this (Table 2). This indicated
that electrostatic binding was a part of the interaction of ST and DNA. But the effect was not as great as expected. However, if the adding order of DNA and NaCl was changed, the intrinsic binding constants were decreased greatly from 1.53× 104 M − 1 to 0.79 × 104 M − 1 when the concentration of NaCl (i.e. ionic strength) increased from 0.05 to 0.12 M (Table 2). The binding constant was decreased by 50% when the ionic strength increased. The ionic strength had great effect on the binding of ST with DNA, and the different adding sequence of DNA and NaCl had different effect on the binding of ST with DNA (Table 2). If NaCl was added before DNA, there existed a significant salt effect on binding, otherwise there was little salt effect. But they were the same at one point: the influence of ionic strength would because smaller and smaller with increasing amounts of NaCl. This indicated that there was competition interaction between ST and NaCl with DNA. The first additional cations assembled near the anionic DNA, and hindered
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Fig. 8. Fluorescence quenching of ST (1.00 × 10 − 5 M) by potassium ferrocyanide and NaCl in the presence of DNA (7.11 ×10 − 5 M), and in the absence of DNA (lex = 520 nm, lem = 568 nm).
the interaction of other cations added afterwards with DNA. So the probable intercalation process was that ST was attracted to the periphery of DNA by electrostatic attraction, and then intercalated into the stack base pairs. If other cations occupied the effective range of electrostatic interaction, the affinity of ST with DNA would be greatly weakened. However, the salt effect could not inhibit the binding of ST with DNA completely.
3.5. Melt studies Other strong evidence for the intercalative binding of ST into the double helix DNA was obtained from DNA melting studies. Intercalation of small molecules into the double helix is known to increase the DNA melting temperature (Tm) at which the double helix denatures into single-
stranded DNA, owing to the increased stability of the helix in the presence of an intercalator [20]. The extinction coefficient of DNA bases at 260 nm in the double-helical form is much less than in the single-stranded form; hence, melting of the helix leads to an increase in the absorption at 260 nm [21]. Thus, the helix-to-coil transition temperature can be determined by monitoring the absorbance of DNA at 260 nm as a function of temperature. The DNA melting studies were carried out with DNA in the absence and in the presence of ST (1:5 ratio of ST to DNA-c(P)). The plot of the DNA absorbance at 260 nm as a function of temperature (Fig. 9) clearly showed that the value of Tm of DNA was increased from 70°C (in the absorbance ST) to 74°C (in the presence of ST). These various DNA melting experiments strongly supported the intercalation of ST into the double helix DNA.
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Table 2 Influence of ionic strength on the binding of ST (1.00×10−5 M) with DNA (7.11×10−5 M) Ionic strength (NaCl) (M)
0.05 0.057 0.079 0.120 a
Adding DNA after NaCl
Adding DNA before NaCl
Binding constant (M−1)
Decreased (%)
A/A a0
Decreased (%)
1.53×104 1.32×104 0.83×104 0.79×104
— 13.7 45.8 48.4
1 0.977 0.969 0.960
— 2.3 3.1 4.0
A and A0 are the absorbance of ST with and without DNA at 520 nm, respectively.
4. Conclusions ST binds to the double helical DNA with a high affinity, and the binding constant obtained by spectral methods was on the order of 104 M − 1 in DNA base pairs. The strong hypochromism, extensive red-shift and isobestic points of ST absorption spectra after adding DNA supported the intercalative binding of ST chromophore with DNA stack base pairs. From fluorescence titration data, the binding site sizes of about 7 were obtained. The intercalative binding was proven by melting studies and fluorescence quenching experiment by anionic quenchers. The binding of ST to DNA increased the melting temperature about 4°C, and the fluorescence of ST was protected from quenching by anionic quenchers after adding
DNA. Different adding sequences of DNA and NaCl had different salt effect on binding of ST with DNA. The binding constant of ST with DNA was decreased greatly because of a salt effects. That is, there existed electrostatic binding between ST and DNA. Presumably, it is because of being attracted by anionic DNA that ST was intercalated into the interior of the DNA double helix. In conclusion, ST binds to DNA by intercalative binding and electrostatic binding. As a dye, ST with characteristics of high affinity with DNA, may be a photoactivater to DNA. Current studies indicate that it is possible to design molecular systems based on the phenazinyl chromophore that bind to DNA avidly.
Acknowledgements This work was supported by National Natural Science Foundation of China (NNFSC).
References
Fig. 9. Absorbance of ST (1.00 × 10 − 5 M) and of DNA (7.11 ×10 − 5 M) with and without ST (1.00 × 10 − 5 M) as a function of temperature.
[1] M.J. Waring, in: G.C.K. Roberts (Ed.), Drug Action at the Molecular Level, Macmillan, London, 1977, p. 167. [2] M.A. Warpehoskj, L.H. Hurley, Chem. Res. Toxicol. 1 (1988) 313. [3] D. Porschke, in: W. Guschlballer, W. Saenger (Eds.) DNA – Ligand Interactions Specificity and Dynamics of Protein – Nucleic Acid Interactions, Plenum, New York, 1986, p. 85. [4] J.K. Barton, J. Biomol. Struct. Dyn. 1 (1983) 621. [5] M.P. Singh, T. Joseph, S. Kumar, J.W. Lown, Chem. Res. Toxicol. 5 (1992) 597. [6] Y. Chen, Y.-Z. Li, W.-B. Chang, Y.-X. Ci, J. Anal. Sci. (Chinese) 10 (1994) 67.
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[7] L.S. Lerman, J. Mol. Biol. 3 (1961) 18. [8] J.L. Vennerstrom, M.T. Makler, Antimicrob. Agents Chemother. 39 (1995) 2671; Chem. Abstr. 124 (1996) 25431h. [9] T. Mamaus, E.F. Fritsch, J. Sambrook, Molecular Colning. A Laboratory Manual, Coid Soring Harbor Laboratory, New York, 1982, p. 458. [10] J.K. Barton, J.M. Goldberg, C.V. Kumar, J. Am. Chem. Soc. 108 (1986) 2081. [11] A. Wolfe Jr., G.H. Shimer, T. Meeham, Biochemistry 26 (1987) 6392. [12] J.D. McGhee, D.H. von Hippel, J. Mol. Biol. 86 (1974) 496. [13] (a) E.C. Long, J.K. Barton, Acc. Chem. Res. 23 (1990) 271; (b) Q.-G. Li, H.-M. Wang, A.-Z. Li, Molecular Biophysical Chemistry (Chinese), Higher Education Publisher, Beijing, 1992, p. 276.
.
[14] P. Yang, Introduction of Bioinorganic Chemistry (Chinese), Xi’An JiaoTong University Publisher, Xi An, 1991, p. 152. [15] G.-Z. Chen, X.-Z. Huang, J.-G. Xu, Z.-Z Zheng, Z.-B. Wang, The Analytical Method of Fluorescence (Chinese), Science Publisher, Beijing, 1990, p. 118. [16] A.M. Brun, A. Harriman, J. Am. Chem. Soc. 114 (1992) 3656. [17] M. Asakawa, K. Endo, H. Tol, Y. Aoyama, Bull. Chem. Soc. Jpn. 65 (1992) 2050. [18] C.V. Kumar, E.H. Asuncion, J. Am. Chem. Soc. 115 (1993) 8574. [19] G. Prativel, J. Bermadou, B. Meunier, Angew. Chem. Int. Ed. Engl. 34 (1995) 746. [20] D.J. Patel, Acc. Chem. Res. 12 (1979) 118. [21] G.L. Zubay, Biochemistry, 2nd edn., Macmillan, New York, 1988, p. 236.
Studies of interaction between Safranine T and double helix DNA by spectral methods Ying Cao, Xi-wen He * Department of Chemistry, Nankai Uni6ersity, Tianjin 300071, China Received 11 August 1997; accepted 18 November 1997
Abstract In this paper, the DNA affinity properties of Safranine T (ST), which features a phenazinyl group, were studied. The studies indicated that ST could intercalate into the stack base pairs of DNA. Intrinsic binding constants obtained by different spectral methods were consistent within experimental errors. They were of the order of 104 M − 1 in DNA base pairs, and the binding site size was about 7 in DNA base pairs. Studies of fluorescence quenching by anionic quenchers and melting temperature of DNA all supported the intercalative binding of ST with DNA. The experiments also showed that electrostatic binding played an important role in the interaction of ST with DNA. This research offers a new intercalation functional group to DNA-targeted drug design. © 1998 Elsevier Science B.V. All rights reserved. Keywords: DNA; Intercalative binding; Safranine T; Phenazium dye; Fluorescence quenching
1. Introduction Several small molecules have been shown to interact with DNA at the molecular level by specific binding modes. These binding studies were driven partly by the need to understand the mechanism of anticancer drug action [1], to decipher the chemical basis for the carcinogenicity of environmental pollutants and toxic chemicals [2] and to serve as analogues in studies of protein-nucleic acid recognition [3]. A systematic investigation of the binding of metal complexes, porphyrins, natural antibiotics and other planar
heterocyclic cations have revealed several structural and electronic factors that control the DNA binding affinity and sequence specificity of small molecules [4]. The results of these various binding studies have been useful in designing new and promising anticancer agents for clinical use [5].
* Corresponding author. E-mail: [email protected]
1386-1425/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S1386-1425(97)00277-1
Scheme 1. Binding modes of small molecules with DNA.
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Y. Cao, X.-w. He / Spectrochimica Acta Part A 54 (1998) 883–892
mechanism of bioactivity of phenazine derivatives on the biomolecular level. To date, most studied intercalators featured acridinyl and thiazinyl moieties, so the findings of intercalative binding of ST with DNA base pairs provide a new functional group for intercalators.
Scheme 2. Structure of safranine T (ST).
Small molecules are bound to DNA double helix by three binding modes: electrostatics binding, groove binding and intercalative binding (Scheme 1) [6]. The accepted point is that small molecules have some extent selectivity when binding with DNA except by the mode of electrostatic binding. The steric structure of small molecules and DNA determine the binding properties (such as affinity, binding site) of small molecules as well as the electronic state of small molecules. In general, planarity was suggested to be one of the important features needed for efficient intercalators [7]. A non-negative charge on the small molecules is considered another important feature, otherwise electrostatic repulsion between anionic DNA polymer and small negative molecules inhibit interaction efficiency. In this paper, we studied the DNA binding properties of a phenazium dye, Safranine T (ST), which features a planar phenazine ring and a positive charge (Scheme 2). Therefore, the planar hydrophobic phenazinyl moiety of ST is expected to facilitate intercalation of ST into the relatively nonpolar interior of the DNA helix. The strong absorption and fluorescence characteristics of ST provide a sensitive spectroscopic handle to study its interaction with DNA. The changes in the intensities of these spectra can be used to decipher the nature and the strength of the stacking interaction between the chromophore and the DNA base pairs. Phenazine derivatives are a kind of antibiotic. Studies showed that some phenazium dyes had antimalarial potency and selectivity, and inhibited many bacteria from growing [8]. What we report here on the interaction between ST and DNA may be relevant to our understanding of the
2. Experiments and methods
2.1. Chemicals The calf thymus (ct) DNA sample was purchased from Sino-American biological engineering company and purified by phenol extraction, as described in the literature [9]. The purity of the final DNA preparation was checked by monitoring the absorption spectrum and the ratio of the absorbance at 260 and 280 nm. The sample was dissolved in 0.05 M Tris buffer with HCl (pH 7.4). The DNA concentration per nucleotide (c(P)) was determined by absorption spectroscopy, using the molar extinction coefficients at 260 nm as 6600 M − 1 [10]. ST was purchased from Beijing No.2 Chemical plant, and used after recrystallized from water. Other reagents were at least analytical grade, and were used without further purification.
2.2. Spectral measurements The absorption spectra were recorded on a Shimadzu UV-240 spectrophotometer and the fluorescence spectra were recorded on a Shimadzu FL-510 spectrofluorophotometer. The absorption and fluorescence titrations with DNA were conducted by keeping the concentration of ST constant, and varying the nucleic acid concentrations. This was done by dissolving an appropriate amount of ST in the DNA stock solution and mixing various proportions of ST and the DNA stock solutions, while maintaining the total volume of the solution constant. This resulted in a series of solutions with varying concentrations of DNA but with a constant concentration of ST. Fluorescence quenching experiments were carried out by adding 10 ml aliquots of a 0.5 M quencher stock solution to samples each time and the volume effect could be ignored. The samples were
Y. Cao, X.-w. He / Spectrochimica Acta Part A 54 (1998) 883–892
excited at 520 nm and the fluorescence intensity was monitored at 568 nm. The intrinsic binding constants of ST with calf thymus DNA was determined by absorption and fluorescence titrations. In the case of the former, the absorbance at 520 nm was recorded after each addition of DNA. The intrinsic binding constant Ka was determined from Eq. (1) [11]: c/Doa =c/Do +1/DoKa
(1)
where c is the concentration of DNA determined by the absorbance at 260 nm, Doa =[oa −of], and Do = [ob −of]. oa, ob and of correspond to the apparent extinction coefficient of ST, the extinction coefficient of the bound form of ST and of the free ST, respectively. In fluorescence quenching experiments, the data were plotted according to the Stern–Volmer equation: I0/I =1+ Kq[Q]
(2)
885
3. Results and discussion
3.1. Absorption studies The electronic absorption spectra in the presence of increasing amounts of DNA of ST showed decreases (hypochromicity) and extensive red-shift at the peak intensities (Fig. 1). The absorbance of ST in the peak was decreased by about 20% and the maximum absorbance wavelength was shifted from 520 nm to 529 nm. The addition of further amounts of DNA to the dye solution did not cause any more absorption changes, due to the binding saturation of the dye with DNA. Two isobestic points were observed at 432 nm and 538 nm, respectively. Hypochromism was suggested to be due to strong interactions between the electronic states of the intercalating chromophore and that of the DNA base pairs [13]. The spectral changes that we observed (hypochromicity, red-
where I0 and I are the fluorescence intensities in the absence and in the presence of DNA. Kq is the Stern–Volmer fluorescence quenching constant, which is a measurement of the efficiency of quenching by quencher. [Q] is the concentration of quencher. Data from the fluorescence titrations were also used to determine the binding constant of ST with DNA by the modified Scatchard Eq. (3) given by McGhee and von Hippel [12]: r/cf =Ki(1−nr)[(1−nr)/[1 −(n − 1)r]]n − 1
(3)
where Ki is the intrinsic binding constant and n is the binding site size in base pairs, I0 and I are the same as in Eq. (2). A plot of r/cf vs. r was constructed, where r is equal to cb/[DNA]. cb, cf are concentrations of bound and free ST, respectively. The binding data were treated by leastsquares methods and values of Ki and n were obtained. The DNA melting studies were done by controlling the temperature of the sample cell with a Shimadzu circulating bath while monitoring the absorbance at 260 nm. The temperature of the solution was continuous monitored with a thermo-couple attached to the sample holder.
Fig. 1. Titration electronic absorption spectra of ST by DNA: a. 0 mM; b. 14.2 mM; c. 28.5 mM; d. 42.7 mM; e. 56.9 mM; f. 71.1 mM; g. 85.4 mM.
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Fig. 2. Half-reciprocal plot of ST (1.00 × 10 − 5 M) with DNA as determined from the electronic absorption spectra.
shift and isobestic point) were consistent with the intercalation of the chromophore into the stack DNA base pairs. Therefore, the phenazine dye, ST, was considered as an intercalator of DNA. Absorbance data of ST at 520 nm with different concentrations of DNA were used to obtain the intrinsic binding constant by Eq. (1) (Fig. 2). The best fit linear regression equation was: c/Do = 6.52× 10 − 9 +1.0 ×10 − 4c
there was DNA in the solution or not, and the quenching efficiency of ST fluorescence by DNA decreased (Fig. 4). That is, the fluorescence quenching of ST by DNA was fit to the static quenching model [14], i.e. a complex was formed between the DNA base pairs and the phenazinyl of ST. Data in Fig. 3 fitted poorly to the Stern– Volmer equation (Eq. (2)): The quenching curve tended toward the x-axis in the case of high concentration of DNA, and the curvature was reduced when the temperature rose (Fig. 4). These results indicated that there were two kinds of fluorophores in solution [14]. Considering the state of solution, the two kind of fluorophores should be phenazinyl of ST and the complex formed between ST and the DNA base pairs. The changes of fluorescence spectra of ST after adding DNA also indicated that there were strong interactions between the chromophore of ST and the base pairs of DNA. Combined with the changes of absorption spectra, the strong interaction
(r =0.9976)
Then, the extinction coefficient of the bound form of ST (ob =of −Do) of 1.85 × 104 M − 1 cm − 1 and the intrinsic binding constant of ST with DNA (Ka) of 1.53×104 M − 1 were deduced.
3.2. Fluorescence studies The binding of ST to DNA was further studied by fluorescence spectroscopy. Binding of ST to DNA was found to quench the fluorescence of ST (Fig. 3). The binding did not show saturation of the ST fluorescence spectra even when large amounts of DNA were added, which would have already saturated in the electronic absorption spectra. This indicated that only a fraction of the binding sites changed the electronic state of chromophore of ST. If the experimental temperature was raised, the fluorescence intensity of ST increased whether
Fig. 3. Fluorescence spectra of ST (1.00×10 − 5 M) with DNA (a to l: 0, 14.2, 28.5, 42.7, 56.9, 71.1, 85.4, 99.6, 113.8, 128.1, 142.3, 156.5 mM) (lex =520 nm).
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Fig. 4. Plots of fluorescence quenching of ST (1.00 ×10 − 5 M) by DNA (7.11 × 10 − 5 M) at different temperatures (lex = 520 nm, lem = 568 nm).
should be considered as the intercalative binding of ST with DNA. The interaction was weakened when the temperature rose. Thus the quenching efficiency of DNA to ST was reduced when the experimental temperature rose. Because it was the static quenching of DNA to ST, the quenching constant was considered as the formation constant of ST and DNA [14], i.e. the binding constant of ST with DNA. In order to obtain the quenching constant of ST by DNA, Eq. (3) was modified as [15]: 1 −1 −1 (I0 −I) − 1 =I − 0 +K %qI 0 [Q]
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Fig. 5. Double-reciprocal plot of fluorescence quenching of ST (1.00 ×10 − 5 M) by DNA (lex =520 nm, lem =568 nm).
obtained. As we discussed before, an intrinsic binding constant (Ka) of 1.53×104 M − 1 resulted from the electronic absorption titration data, and a binding constant (Kq) of 1.00× 104 M − 1 was deduced from the modified Stern–Volmer equation (Eq. (4)). The binding constants obtained by the three methods were consistent within the experimental error. This increased the credibility of these three measurements. The large binding constant for ST indicated that the phenazinyl chromophore had high affinity for DNA base pairs. The estimated site size of about 7 for this aromatic cation was consistent with the neighboring
(4)
where I0, I and Q were the same as formerly, the −1 binding constant was K ). The doubleq ( = (Kq) reciprocal plot was made by (I0 −I) − 1 vs. [Q] − 1 (Fig. 5) and the binding constant (Kq) of 1.00× 104 M − 1 was obtained. Compared with the intrinsic binding constant obtained by the electronic absorption spectra, the binding constant from the modified Stern–Volmer equation (Eq. (2)) was almost the same. The fluorescence titration data were also used to estimate the binding constant and the binding size of ST with DNA by the modified Scatchard equation (Eq. (3)). The plot of r/cf vs. r is shown in Fig. 6. From the best fit of the data to Eq. (3), an intrinsic binding constant (Ki) of 6.11 × 104 M − 1, and a site size of 6.7 base pairs were
Fig. 6. Scatchard plot of the ST fluorescence titration by DNA (lex =520 nm, lem =568 nm).
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Table 1 Binding constants of some intercalators and DNA Intercalators
Condition
Ethidium bromide
pH 7, 5 mM phosphate buffer and 5 mM Na2SO4 Acridine orange pH 7, 5 mM phosphate buffer and 5 mM Na2SO4 6-Chloro-2-methoxy-9-acdidine pH 6.50, 50 mM NaCl, in water-DMSO (9-Anthrylmethyl) ammonium 5 mM Tris–HCl buffer chloride Safranine T 50 mM Tris–HCl buffer
exclusion model, but it was much larger than the reported values for some typical intercalators such as ethidium bromide and acridine derivatives (Table 1). This may be due to the big size of the benezyl subsitituent, which is much larger than the other typical intercalators. The intercalative binding of ST was hindered in the wider neighboring region of bound site than other intercalators. But the size of substituent had no influence on the affinity of chromophore with DNA base pairs. Table 1 shows the binding constants of some intercalators with DNA. Though the methods and conditions are different, it still can be concluded that the affnity of ST with DNA is not weaker than the others.
3.3. Fluorescence quenching studies If small molecules intercalate into DNA base pairs, the double helix of DNA would protect them from some quenchers, e.g. iodide and bromide, owing to the base pairs above and below the intercalator. Otherwise, the fluorescence quenching of ST would have no observed changes after addition of DNA. In aqueous solutions, potassium iodide and potassium ferrocyanide quench the fluorescence of ST very efficiently, so we used these two anionic quenchers to determine the relative accessibilities of the free and bound ST. After adding KI to solution, the fluorescence quenching was much smaller in the presence of DNA than in the absence of DNA. The quenching data were plotted according to the Stern – Volmer equation (Eq. (2)), and treated by the
Binding constant, K Site size (n) (M−1)
Reference
2.6×106
2
[16]
4×105
2
[16]
6.6×104 7.8×104
2.2 4.8
[17] [18]
6.1×104
6.7
—
linear least-squares method. The quenching constants of KI to ST were 13 and 28 M − 1 with and without DNA, respectively (Fig. 7). The quenching of the dye fluorescence was decreased by a factor greater than two when ST was bound to DNA. This was due to the intercalative binding of ST fluorophores with helix of DNA, which prevented ST fluorescence quenching from the anionic quenchers. The experimental results of ST fluorescence quenching by potassium ferrocyanide was similar to those with KI. The quenching constants obtained by the Stern–Volmer equation were 18 and 30 M − 1 in the presence and in the absence of DNA (Fig. 8). In order to determine the role of ionic strength, we examined the changes of ST fluorescence by adding the strong electrolyte, sodium chloride, instead of KI and potassium ferrocyanide to solution with and without DNA. For NaCl, which is not an anionic quencher, its influence on the ST fluorescence intensity comes only from the ionic strength [19]. We found that the ionic strength had little or no effect on the ST fluorescence intensity no matter if there was DNA in the solution (Fig. 8). So the changes of fluorescence intensity after adding quenchers had little correlation with the ionic strength. The decreases of the quenching constants of KI and potassium ferrocyanide on ST after adding DNA resulted from the intercalative binding of ST with the DNA double helix, which prevented fluorescence quenching of ST from anionic quenchers because of the sandwich structure formed by one chromophore of ST and two DNA base pairs as well as by the polyphosphate anionic skeleton of DNA.
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Fig. 7. Fluorescence quenching of ST (1.00 × 10 − 5 M) by KI in the presence of DNA (7.11 ×10 − 5 M) and in the absence of DNA (lex = 520 nm, lem = 568 nm).
3.4. Salt effect on binding Because the intercalator of ST carries a positive charge and DNA has a negative polyphosphate skeleton, We considered the electrostatic interactions between the intercalator and DNA. In order to test if there was electrostatic interaction between ST and DNA, the strong electrolyte, sodium chloride (NaCl), was used. The electrostatic interaction would be weakened by the additional counter ion because of the conversion of the electrostatic atmosphere of DNA periphery. Some reports showed that very high concentration of NaCl would hinder small molecules from binding with DNA [19]. If there did exist electrostatic interaction between ST and DNA, the following phenomenon should happen by increasing the ionic strength of solutions of ST and DNA: The absorbance of solutions would increase when NaCl was added, but it would never exceed the absorbance of the ST solution. The experimental results confirmed this (Table 2). This indicated
that electrostatic binding was a part of the interaction of ST and DNA. But the effect was not as great as expected. However, if the adding order of DNA and NaCl was changed, the intrinsic binding constants were decreased greatly from 1.53× 104 M − 1 to 0.79 × 104 M − 1 when the concentration of NaCl (i.e. ionic strength) increased from 0.05 to 0.12 M (Table 2). The binding constant was decreased by 50% when the ionic strength increased. The ionic strength had great effect on the binding of ST with DNA, and the different adding sequence of DNA and NaCl had different effect on the binding of ST with DNA (Table 2). If NaCl was added before DNA, there existed a significant salt effect on binding, otherwise there was little salt effect. But they were the same at one point: the influence of ionic strength would because smaller and smaller with increasing amounts of NaCl. This indicated that there was competition interaction between ST and NaCl with DNA. The first additional cations assembled near the anionic DNA, and hindered
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Fig. 8. Fluorescence quenching of ST (1.00 × 10 − 5 M) by potassium ferrocyanide and NaCl in the presence of DNA (7.11 ×10 − 5 M), and in the absence of DNA (lex = 520 nm, lem = 568 nm).
the interaction of other cations added afterwards with DNA. So the probable intercalation process was that ST was attracted to the periphery of DNA by electrostatic attraction, and then intercalated into the stack base pairs. If other cations occupied the effective range of electrostatic interaction, the affinity of ST with DNA would be greatly weakened. However, the salt effect could not inhibit the binding of ST with DNA completely.
3.5. Melt studies Other strong evidence for the intercalative binding of ST into the double helix DNA was obtained from DNA melting studies. Intercalation of small molecules into the double helix is known to increase the DNA melting temperature (Tm) at which the double helix denatures into single-
stranded DNA, owing to the increased stability of the helix in the presence of an intercalator [20]. The extinction coefficient of DNA bases at 260 nm in the double-helical form is much less than in the single-stranded form; hence, melting of the helix leads to an increase in the absorption at 260 nm [21]. Thus, the helix-to-coil transition temperature can be determined by monitoring the absorbance of DNA at 260 nm as a function of temperature. The DNA melting studies were carried out with DNA in the absence and in the presence of ST (1:5 ratio of ST to DNA-c(P)). The plot of the DNA absorbance at 260 nm as a function of temperature (Fig. 9) clearly showed that the value of Tm of DNA was increased from 70°C (in the absorbance ST) to 74°C (in the presence of ST). These various DNA melting experiments strongly supported the intercalation of ST into the double helix DNA.
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Table 2 Influence of ionic strength on the binding of ST (1.00×10−5 M) with DNA (7.11×10−5 M) Ionic strength (NaCl) (M)
0.05 0.057 0.079 0.120 a
Adding DNA after NaCl
Adding DNA before NaCl
Binding constant (M−1)
Decreased (%)
A/A a0
Decreased (%)
1.53×104 1.32×104 0.83×104 0.79×104
— 13.7 45.8 48.4
1 0.977 0.969 0.960
— 2.3 3.1 4.0
A and A0 are the absorbance of ST with and without DNA at 520 nm, respectively.
4. Conclusions ST binds to the double helical DNA with a high affinity, and the binding constant obtained by spectral methods was on the order of 104 M − 1 in DNA base pairs. The strong hypochromism, extensive red-shift and isobestic points of ST absorption spectra after adding DNA supported the intercalative binding of ST chromophore with DNA stack base pairs. From fluorescence titration data, the binding site sizes of about 7 were obtained. The intercalative binding was proven by melting studies and fluorescence quenching experiment by anionic quenchers. The binding of ST to DNA increased the melting temperature about 4°C, and the fluorescence of ST was protected from quenching by anionic quenchers after adding
DNA. Different adding sequences of DNA and NaCl had different salt effect on binding of ST with DNA. The binding constant of ST with DNA was decreased greatly because of a salt effects. That is, there existed electrostatic binding between ST and DNA. Presumably, it is because of being attracted by anionic DNA that ST was intercalated into the interior of the DNA double helix. In conclusion, ST binds to DNA by intercalative binding and electrostatic binding. As a dye, ST with characteristics of high affinity with DNA, may be a photoactivater to DNA. Current studies indicate that it is possible to design molecular systems based on the phenazinyl chromophore that bind to DNA avidly.
Acknowledgements This work was supported by National Natural Science Foundation of China (NNFSC).
References
Fig. 9. Absorbance of ST (1.00 × 10 − 5 M) and of DNA (7.11 ×10 − 5 M) with and without ST (1.00 × 10 − 5 M) as a function of temperature.
[1] M.J. Waring, in: G.C.K. Roberts (Ed.), Drug Action at the Molecular Level, Macmillan, London, 1977, p. 167. [2] M.A. Warpehoskj, L.H. Hurley, Chem. Res. Toxicol. 1 (1988) 313. [3] D. Porschke, in: W. Guschlballer, W. Saenger (Eds.) DNA – Ligand Interactions Specificity and Dynamics of Protein – Nucleic Acid Interactions, Plenum, New York, 1986, p. 85. [4] J.K. Barton, J. Biomol. Struct. Dyn. 1 (1983) 621. [5] M.P. Singh, T. Joseph, S. Kumar, J.W. Lown, Chem. Res. Toxicol. 5 (1992) 597. [6] Y. Chen, Y.-Z. Li, W.-B. Chang, Y.-X. Ci, J. Anal. Sci. (Chinese) 10 (1994) 67.
892
Y. Cao, X.-w. He / Spectrochimica Acta Part A 54 (1998) 883–892
[7] L.S. Lerman, J. Mol. Biol. 3 (1961) 18. [8] J.L. Vennerstrom, M.T. Makler, Antimicrob. Agents Chemother. 39 (1995) 2671; Chem. Abstr. 124 (1996) 25431h. [9] T. Mamaus, E.F. Fritsch, J. Sambrook, Molecular Colning. A Laboratory Manual, Coid Soring Harbor Laboratory, New York, 1982, p. 458. [10] J.K. Barton, J.M. Goldberg, C.V. Kumar, J. Am. Chem. Soc. 108 (1986) 2081. [11] A. Wolfe Jr., G.H. Shimer, T. Meeham, Biochemistry 26 (1987) 6392. [12] J.D. McGhee, D.H. von Hippel, J. Mol. Biol. 86 (1974) 496. [13] (a) E.C. Long, J.K. Barton, Acc. Chem. Res. 23 (1990) 271; (b) Q.-G. Li, H.-M. Wang, A.-Z. Li, Molecular Biophysical Chemistry (Chinese), Higher Education Publisher, Beijing, 1992, p. 276.
.
[14] P. Yang, Introduction of Bioinorganic Chemistry (Chinese), Xi’An JiaoTong University Publisher, Xi An, 1991, p. 152. [15] G.-Z. Chen, X.-Z. Huang, J.-G. Xu, Z.-Z Zheng, Z.-B. Wang, The Analytical Method of Fluorescence (Chinese), Science Publisher, Beijing, 1990, p. 118. [16] A.M. Brun, A. Harriman, J. Am. Chem. Soc. 114 (1992) 3656. [17] M. Asakawa, K. Endo, H. Tol, Y. Aoyama, Bull. Chem. Soc. Jpn. 65 (1992) 2050. [18] C.V. Kumar, E.H. Asuncion, J. Am. Chem. Soc. 115 (1993) 8574. [19] G. Prativel, J. Bermadou, B. Meunier, Angew. Chem. Int. Ed. Engl. 34 (1995) 746. [20] D.J. Patel, Acc. Chem. Res. 12 (1979) 118. [21] G.L. Zubay, Biochemistry, 2nd edn., Macmillan, New York, 1988, p. 236.