Spectroscopic studies on the interaction between anthragallol and DNA using of ethidium bromide as a fluorescence probe

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 141 (2015) 239–243

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

Spectroscopic studies on the interaction between anthragallol and DNA using of ethidium bromide as a fluorescence probe Yan Gao, Junsheng Li ⇑, Guoxia Huang, Liujuan Yan, Zhen Dong Guangxi University of Science and Technology, Biological and Chemical Engineering, Liu Zhou 545006, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 We developed a new method to

evaluate ability of molecules embedded in DNA.  The bonding methods were investigated by various experiments.  We first calculated binding anthragallol saturation value at 3.75.

a r t i c l e

i n f o

Article history: Received 15 May 2014 Received in revised form 15 January 2015 Accepted 22 January 2015 Available online 3 February 2015 Keywords: Anthragallol Ethidium bromide DNA Action mode Intercalation Saturation value

a b s t r a c t The interaction of DNA with anthragallol (Ant) was investigated using ethidium bromide (EB) as a fluorescence probe, and the binding mechanism of Ant with DNA was researched via viscosity measurements. The results indicate that there is a complex of Ant and DNA, as confirmed by Ultraviolet visible absorption spectroscopy (UV–vis), Fluorescent and Resonance Light Scattering spectrum (RLS) and viscosity measurements. Ant molecules could intercalate with the base pairs of DNA as evidenced by the hyperchromic effect of absorption spectra, the relative viscosity of DNA and significant increases in the melting temperature. The binding constants of Ant and DNA were obtained by the fluorescence quenching technique. Furthermore, the binding mechanisms of the reaction of Ant with DNA were also investigated. The RLS assay successfully evaluated the saturated value and measured the potential toxicity of Ant. Adriamycin, chrysophanol, rhein, and alizarin can be used as references to build a method based on the mechanism of interactions with DNA and the DNA-saturation binding value to rapidly evaluate the potential toxicity of Ant. Ó 2015 Elsevier B.V. All rights reserved.

Introduction DNA is an important genetic substance in an organism. The regions of DNA that are involved vital processes, such as gene transcription and expression, are also related to mutagenesis and carcinogenesis [1]. Naturally occurring anthraquinones have been used as colorants in foods, drugs, cosmetics, hair dyes and textiles [2]. Madder (Rubia tinctorum L.) extract has been used for the ⇑ Corresponding author. E-mail address: [email protected] (J. Li). http://dx.doi.org/10.1016/j.saa.2015.01.049 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.

casing of ham and sausage as a food pigment in Japan. Although natural colors that are extracted from insects or plants are used as food colorants in large quantities, few have been studied for potential toxicity. Ant (1,2,3-trihydroxy-9,10-anthraquinone) can lead to direct frameshift mutagenesis to Salmonella Typhimurium [3] and is highly mutagenic in Salmonella [4]. In 1961, Lerman [5] first found that flat aromatic molecules have the ability to intercalate into DNA, and this model has been confirmed by a number of aromatic molecules. Ant has the characteristics of a flat-rigid structure, and a small molecule volume. The spatial dimensions of Ant fix the width of the DNA double helix well, so it very easy to embed

240

Y. Gao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 141 (2015) 239–243

into the double helix structure of DNA and participates in the formation of a typical mode of intercalation. In recent years, there have been many studies of the interaction mechanism between reagents with DNA and effective DNA florescent probes. DNA can enhance the fluorescence intensity of ethidium bromide (EB). In this paper, EB was selected as a probe to investigate the interaction of Ant with DNA by fluorescence methods. Based on the phenomenon by which small molecules combine with DNA to enhance RLS, the saturation value of Ant and DNA can be estimated. Experimental Material and reagents A stock solution of DNA (2.66  103 mol/L) was prepared by dissolving commercially purchased herring sperm DNA (Sigma Chem. Co., America) in 250 ml of double-distilled water overnight. The DNA stock solution was stored at 4 °C in the dark. The DNA concentration was calculated according to the absorbance at 260 nm with e DNA = 6600 L/mol cm (Bouguer–Lambert–Beer law). The DNA purity was checked by monitoring the ratio of >1.8 at A260/A280, indicating that DNA was sufficiently free from protein. Ant (Alfa Aesar, Tianjin) (Fig. 1) was prepared as 1  103 mol/L stock solutions. A stock solution of EB was prepared as 2  104 mol/L. BR buffer solution (pH 7.41) was used to control the pH. All of the chemicals that were used were of analytical reagent grade, and double-distilled water was used throughout the experiment. Apparatus The fluorescence spectra were measured using an F-280 fluorophotometer (Tianjin, China) with a quartz cell with a 1-cm path length to measure the fluorescence spectra. The UV spectra were measure using a Cary 60 UV-Spectrometer (Agilent Technology) and an 1835 Ubbelohde viscometer (Shanghai, China). Procedures UV–vis spectra When DNA with increasing concentration was added to the solution containing Ant, the UV–vis spectra were determined. Fluorescence spectra Fluorescence measurements were carried out by adding various concentrations of Ant to 5-mL test tubes containing a fixed concentration of EB–DNA. The mixture was diluted with double-distilled water and mixed thoroughly. The test tubes were placed in the thermostat water bath for 10 min for equilibrium. The above solution was transferred into the quartz cell, and fluorescence measurement was performed.

800 nm with Dk = 0 nm. The Ant–DNA system was measured at the maximum wavelength 467 nm. Results and discussion UV–vis spectra Absorption spectra studies The non-covalent interactions of small molecules with DNA involve three binding modes: groove binding, long-range assembly on the molecular surfaces of nucleic acids and intercalation [6]. Ultraviolet visible absorption spectroscopy (UV–vis spectra) is generally used as an effective method for DNA-binding research [7], fixing the concentration of DNA and changing the concentration of Ant to measure the absorbance between 200 nm and 300 nm. As seen in Fig. 2, the absorption peak of DNA at approximately 260 nm showed a gradual increase after the addition of different amounts of Ant. Across a range of concentrations, one isosbestic point at 241 nm appeared in the family of absorption curves. The hyperchromic effect and isosbestic point are special spectral properties of DNA that are closely related to the double helix structure of DNA [8]. For instance, EB is a typical intercalating protein that binds with DNA, resulting in a bathochromic shift (red shift) and hypochromic effect. Compared with the spectra of EB–DNA binding [9], a red shift and hypochromic effect are observed for the binding of DNA and Ant, fixing the concentration of Ant and changing the concentration of DNA to measure the absorbance between 200 nm and 600 nm. UV–vis spectra are shown in Fig. 3; the absorption peaks of Ant are at 375 nm. After DNA was added to the solution containing the Ant, the absorption peaks of Ant at 375 nm gradually decreased along with the increasing concentration of DNA, and the absorption peak at 375 nm of Ant has 2-nm red shifts, and an isosbestic point appeared at 312 nm. Moreover, the increase in the region from 200 nm to 300 nm is due to the increasing DNA concentration. Therefore, this analysis of UV–vis spectra suggests that intercalation binding is the major binding mode of Ant with DNA. Ant inserts into DNA double helix bases while packing p electronics, strengthening the interaction of base pair electrons and reducing ultraviolet absorption. From the above observations, we surmised that intercalation occurred upon the binding of Ant to DNA. Viscosity experiments Measuring viscosity is an important method to identify the binding mode, which plays a critical role in various studies. Depending on this theory, some small molecules intercalate the

1.0 0.55 t

0.50

a 0.45 250

0.6

255

260

265

270

λ ⁄ nm

A

RLS spectra Appropriate amounts of DNA and Ant were added to a cuvette. The RLS spectrum was obtained by scanning from 200 nm to

A

0.8

a

t

0.4

O

OH

t

OH

200

OH O Fig. 1. Structure of anthragallol (Ant).

a

0.2 220

240

260

280

300

λ /nm Fig. 2. The absorption spectra of DNA in the absence of Ant (a) and in the presence of Ant (b)–(t). Ant concentrations are 5  105 mol/L (10 lL per scan, b–t: 10– 200 lL). The concentration of DNA is 6.65  105 mol/L.

241

Y. Gao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 141 (2015) 239–243

4

1.0

DNA(1.5×10-4mol/L) DNA+ant(1.25×10-6mol/L)

0.6

0.8

0.5

A

a

0.4

a

0.3

A

2

0.6

t 360 390 wavelength(nm)

fss

h

3

420

0.4

a 1

0.2

h

0 200

300

400 500 wavelength(nm)

0.0 30

600

40

50

60

70

80

90

100 110

T/°C

Fig. 5. Melting curves of DNA in the absence of Ant and in the presence of Ant. Fig. 3. The absorption spectra of Ant in the absence of DNA (a) and in the presence of DNA (b)–(t). DNA concentrations are 5  103 mol/L (10 lL per scan, b–t: 10–200 lL). The concentration of Ant is 1  104 mol/L.

interspace between the base pairs, which lengthens the intercalated DNA helix. Using Ubbelohde viscometer for the viscosity experiments, the immersing thermostated water bath was maintained at 30 ± 0.1 °C. The data were presented as (g/go)1/3 versus the ratio of the concentration of Ant to that of the Herring Sperm DNA, where g and go are the viscosities of DNA solutions in the presence and absence of Ant. The viscosity values were calculated from the observed flow time of DNA-containing solutions (t) that were corrected for that of BR buffer alone (t0), g = t  t0. From Fig. 4, with the intercalation of Ant, the viscosity of the Ant–DNA increases. Therefore, we can conclude that Ant molecules were intercalated into the base pairs of DNA. DNA thermal denatured When the solutions of DNA were close to the extremes of pH or temperature, the double helical structure of DNA experienced a transition into a randomly single-stranded form at the melting temperature (Tm). The intercalation of some small molecules into the double helix could increase the DNA melting temperature, during which the double helix denatures into single-stranded DNA [10]. The Tm of Ant–DNA was determined by the absorption degree. When the temperature ranged from 30 °C to 100 °C, the value of absorption was noted, and from the curve on the dependence of fss = (A  A0)/(Af  A0) vs. T, the Tm was obtained. The results are shown in Fig. 5. The transition midpoint of the melting curve is the Tm of the mixed solution. After Ant was added to the DNA solution, the Tm of the system increased from 71 °C to 91 °C. The results demonstrate that the binding of Ant and DNA is mainly intercalated. It can be seen from the increase in Tm that, due to the binding of Ant with DNA, the conformation of DNA changed to some degree, and the stabilization of the systems increased.

Fluorescence spectra Fluorospectrophotometry When Ant was added to the EB–DNA solution, the shape and position of the fluorescence peak slightly changed, while the fluorescence intensity decreased regularly with the increasing concentration of Ant. The results are shown in Fig. 6 and indicate that the interaction occurred between the Ant and the system of EB–DNA. According to the theory of fluorescence quenching [11], regardless of the dynamic quenching or static quenching, F0/F vs. [Q] is always a straight line or can be considered a mixed mode. As seen in Fig. 7, the fluorescence quenching curve of Ant to EB–DNA is not a straight line; therefore, the quenching mode is mixed. It was hypothesized that the quenching of the Ant compound to EB–DNA was dynamic and was caused by molecule collision. According to the equation of Stern–Volmer [12],

F 0 =F ¼ 1 þ K q C0 ½Q  ¼ 1 þ K SV ½Q 

ð1Þ

where F0 is the fluorescence intensity in the absence of the Ant in this paper, F is that after adding the quenching reagent, Kq is the quenching rate constant of the biomacromolecule (DNA), and C0 is the average lifetime of the fluorescence molecule in the absence of the quenching regent, whose value is approximately 108 s [13]. [Q] is the concentration of the quenching reagent, which is the concentration of Ant in this paper; KSV is the Stern–Volmer dynamic quenching constant; and KSV = KqC0 under standard atmospheric pressure. Fig. 8 was obtained based on the Stern–Volmer equation. The Stern–Volmer plots of Ant binding to EB–DNA were obtained at 25 °C, 35 °C, 45 °C, and 55 °C. The value of KSV did not decrease with the increasing temperature. Their change is irregular. If the value of KSV decreases with the temperature increase, the quenching of Ant 2000

2.5 1500 a

η0 )1/3

F

2.0 1000

j

(η ⁄

1.5 500

1.0 0 550

0

100

200

300

400

600

650

700

750

wavelength(nm)

[Ant]/[DNA]

Fig. 4. Effect of increasing amounts of the complex on the viscosities of DNA at pH 7.41. [DNA] = 2.66  104 mol/L.

Fig. 6. Fluorescence spectra of EB–DNA in the presence of Ant. The total concentrations of Ant were 1  103 mol/L (10 lL per scan, a–j: 0–80 lL); the EB and DNA concentrations were 3  106 mol/L and 2.5  105 mol/L, respectively.

242

Y. Gao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 141 (2015) 239–243

1.5

70

1.4

60 a

rc-1/(mol/l)

F0/F

1.3

1.2

50 b

40 c

1.1

30 d

1.0

0.0001

0

5

10

15

20

25

30

0.0002

0.0003

35

c(anthragallol)/(10 -6 mol/L)

Fig. 10. Scatchard plot of the interaction between Ant with EB–DNA. CAnt = 1  104 mol/L; CDNA = 2.5  103 mol/L; CEB = 2  104 mol/L (10 lL per scan); Rt = CAnt/CDNA; a, Rt = 0.00; b, Rt = 0.04; c, Rt = 0.08; d, Rt = 0.12.

Fig. 7. The fluorescence quenching curve of Ant to EB–DNA. CDNA = 2.5  105 mol/L; CEB = 3  106 mol/L; CAnt = 1  103 mol/L (10 lL per scan); kex = 510 nm.

Table 2 Scatchard equation of the interaction between Ant and DNA.

a b c d

1.6

F0 /F

1.4

CAnt/CDNA

Scatchard equation

K/(L mol1)

n

a b c d

0 0.04 0.08 0.12

72.741–6.46  104r 51.275–4.61  104r 36.013–3.34  104r 37.119–6.33  104r

6.46  104 4.61  104 3.34  104 6.33  104

0.0011 0.0011 0.0011 0.0006

1.2 1 ðF 0  FÞ1 ¼ F 0  1 þ K D F 1 0 ½Q

1.0 0

10

20

ð2Þ

where KD is the dissociation constant for the reaction of the quencher and fluorophore. KA is the binding constant (KA = 1/KD). Fig. 9 is the curve of 1/(F0  F) vs. [Q]1 at 25 °C. The binding constants KA of Ant to EB–DNA at various temperatures are listed in Table 1.

30

[Q]/(10-5mol/L) Fig. 8. Stern–Volmer curves at 25 °C (a), 35 °C (b), 45 °C (c), 55 °C (d) of Ant–EB– DNA.

Scatchard method The Scatchard method is usually used to research the binding mode of DNA with some small molecules [16]. The Scatchard equation analysis demonstrates the bonding mode between DNA and EB in the Ant solution, whose concentration is gradually changing [17].

0.0030 0.0028 0.0026 1/(F0-F)

Curve

0.0024

r=c ¼ kðn  rÞ

ð3Þ

0.0022

where r is the number of molecules of EB, which binds with DNA; c is the free concentration of EB; k is the binding constant, which is an inherent single locus; and n is the binding site multiplicity per class of binding site. Usually, if the value of n in the absence of Ant is the same as that in the presence of Ant, the binding mode was intercalation. If the values of k and n are different from each other, the binding mode between Ant and DNA is mixed mode (non-intercalation and intercalation binding) [18]. In Fig. 10, the Scatchard plots of Ant–DNA are shown at different concentrations of EB. As shown in Table 2, the value of n is the same at lower concentrations and different at higher concentrations. Therefore, there exist an intercalation binding mode between Ant and DNA [19].

0.0020 0.0018 0.0016

0.03

0.04

0.05

0.06

0.07

0.08

[Q]-1/(105mol L-1)

Fig. 9. The plots of 1/(F0  F) vs. [Q]1 for Ant.

is not controlled by diffusion. However, the results of the Stern– Volmer plots are irregular; therefore, the quenching of Ant to EB– DNA may be controlled by diffusion. The quenching rate constant Kq for Ant was 1.75  106 L (mol s)1 at 25 °C, which was much less than the maximum collision quenching rate constants 2.0  1010 L (mol s)1 [14]. These results indicate that the process is dynamic collision rather than static collision quenching. A double reciprocal chart was obtained using the equation of the static quenching [15] as follows:

RLS spectrum The RLS spectra of DNA, Ant, and Ant–DNA (Ant–DNA) at pH 7.41 are shown in Fig. 11. All of the RLS peaks for DNA, Ant, Ant– DNA are at 467 nm. According to the RLS intensity, we know that

Table 1 The binding constants (KA) of the Ant with EB–DNA at various temperatures. Reagent

Anthragallol

25 °C

35 °C

45 °C

55 °C

KA (L mol1)

r

KA (L mol1)

r

KA (L mol1)

r

KA (L mol1)

r

3.32  102

0.9971

6.53  103

0.9936

5.31  102

0.9964

7.84  103

0.9958

Y. Gao et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 141 (2015) 239–243

we can see that the potential toxicity of Ant is lower than that of adriamycin and higher than that of chrysophanol, rhein and alizarin.

500 400 Intensity of RLS

243

300

Conclusion 6

200

1

100 0 200

300

400

500

600

700

800

λ(nm)

Fig. 11. Resonance light-scatting spectra of DNA and Ant. CAnt2–6: 1  106 mol/L; CDNA1–6: 1  107, 0, 1  107, 3  107, 2  107, 2.67  107 mol/L.

Table 3 Change in the rate of the saturation value binding with DNA. Reagent

Saturation value

Method

Ref.

Gradient (100%)

Adriamycin Chrysophanol Rhein Alizarin Anthragallol

10.58 0.53 0.66 0.2 3.75

RLS RLS RLS RLS RLS

[20] [20] [20] [21] This work

0 95 93.8 98.1 64.6

In this paper, the intercalation binding of Ant to DNA was demonstrated in detail by the red shift and hyperchromic effect of absorption spectra, fluorescence quenching techniques using ethidium bromide as a DNA probe and significant increases in the melting temperature and relative viscosity of DNA. The binding constants of Ant with DNA in the presence of EB were also obtained. These results indicate that the binding mode of Ant and EB–DNA was intercalation. The saturation value of binding with DNA can be evaluated by RLS. Other anthraquinones were compared to evaluate the potential toxicity of Ant. Based on the present results, depending on the saturation value, a greater potential toxicity of anthraquinones can be immediately measured. This result would open up possibilities for future development, such molecules food science, food chemistry, biomedicine and clinical medicine. Acknowledgement This paper was supported by Natural Science Foundation of Guangxi Province (2010GXNSFA013134).

Ant–DNA emitted a stronger scattering light than did DNA. The concentration of planar rigid molecules and DNA are two main factors affecting the scattering intensity. We know that the RLS intensity is extremely sensitive to the changes in the form of the molecule. Therefore, the Ant–DNA emitted a stronger scattering light than did the emitted DNA. Yang [20] reported that the DNA binding saturation value = plane rigid structure food additive/the concentration of saturated DNA molecules. This formula will be represented as SV = CAnt/CDNA in this study, where CAnt and CDNA are the concentrations of Ant and DNA, respectively, and SV indicates the DNA binding saturation value. From Fig. 11, we can see that the strongest RLS signal belonged to Ant–DNA, whose concentration of DNA is 2.67  107 mol/L. However, when the DNA concentration was 2.67  107 mol/L, even if the DNA concentration increased, the resonance scattering signal no longer increased. At this time, the concentration of DNA was the saturation concentration of DNA, and the Ant and DNA binding saturation value was 3.75, suggesting that Ant could bind to DNA and form binary aggregation. The results of the combination of other reagents with DNA are listed in Table 3, showing that the saturation value that was developed in this work is very sensitive and convenient. From Table 3,

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]

E.C. Miller, J.A. Miller, Cancer 47 (1981) 1055. Y. Yasui, N. Takeda, Mutat. Res. 121 (1983) 185–190. J.P. Brown, R.J. Brown, Mutat Res 40 (1976) 203–224. E.R. Nestmann, D.J. Kowbel, J.A. Wheat, Carcinogenesis 2 (1981) 879–883. L.S. Lerman, Mol. Biol. 3 (1961) 18–30. M.L. Yola, N. Ozaltm, J. Electroanal. Chem. 653 (2011) 56. X.Y. Xu, D.D. Wang, X.J. Sun, et al., Thermochim. Acta 493 (2009) 30. P. Yang, C.Q. Zhou, Acta Chim. Sin. 61 (2003) 1455. C.Y. Qiao, S.Y. Bi, Y. Sun, et al., Spectrochim. Acta A 70 (2008) 136–143. D.J. Patel, Acc. Chem. Res. 12 (1979) 118. G.Z. Chen, X.Z. Huang, Z.X. Zheng, et al., Fluorescence Analytical Method, Science Press, Beijing, 1990. p. 263. G.Z. Chen, X.Z. Huang, J.G. Xu, et al., The Methods of Fluorescence Analysis, second ed., Science Press, Beijing, 1990, p. 112. J.R. Lakowica, G. Weber, Biochemistry 12 (1973) 4161. W.R. Ware, Phys. J. Chem. 66 (1962) 455. C.Q. Jiang, M.X. Gao, X.Z. Meng, Spectrochim. Acta A 59 (2003) 1605. H.M. Torshizi, R. Mital, T.S. Srivastava, J. Inorg. Biochem. 44 (1991) 239. J.B. Lepecq, C. Paoletti, Mol. Biol. 27 (1967) 87. Q.H. Zhou, P. Yang, Acta Chim. Sin. 1 (2005) 71. D.K. Jangir, S. Charak, R. Kundu, et al., J. Photochem. Photobiol. 105 (2011) 143. X.M. Yang, J.S. Li, Q.Q. Li, et al., Asian J. Chem. 23 (2011) 3631. C.C. Li, J.S. Li, G.X. Huang, et al., Asian J. Chem. 25 (2013) 1015.