Spectroscopic investigations of interactions between Hematoxylin–Ag+ complex and Herring-sperm DNA with the aid of the acridine orange probe

Journal of Molecular Structure 1010 (2012) 73–78

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Spectroscopic investigations of interactions between Hematoxylin–Ag+ complex and Herring-sperm DNA with the aid of the acridine orange probe Jianhang Huang, Xingming Wang ⇑ School of Materials Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China

a r t i c l e

i n f o

Article history: Received 9 July 2011 Received in revised form 20 November 2011 Accepted 21 November 2011 Available online 1 December 2011 Keywords: Spectroscopy Hematoxylin Ag+ Herring sperm DNA Interaction

a b s t r a c t Investigations of the interaction between deoxyribonucleic acid (DNA) and other molecules have captivated scientists over the past years because it is fundamental for many intracellular processes and biotechnological relevance. In this study, Hematoxylin (HE)–Ag+ complex was synthesized, and it was characterized by IR, UV–vis spectroscopy. The binding constant for the HE–Ag+ complex and hsDNA is obtained by double reciprocal method at 303 K and 310 K, respectively, and the corresponding thermodynamic parameters shown the interaction process is driven mainly by entropy. The subsequent experiments indicate that the binding mode between HE–Ag+ complex and hsDNA is a mixed binding which contains partial intercalation and electrostatic binding. Study on the influence of basic group to the complex suggests that the binding interaction has high selectivity. The intercalation mainly occurs between A:T base pair. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Numerous biological experiments have demonstrated that DNA is the primary intracellular target of anticancer drug, the interaction between DNA and other molecules is an important fundamental issue on life sciences [1–5]. Many investigations on the interaction of drug molecules with DNA have been carried out since the early 1960s. The impetus of these investigations are to understand the mechanism of drug action at molecular level and to understand the structure and function of DNA [6,7]. In pharmacological research, drug design based on metal complexes is particular fascinating. It is known that a combination of metal ion and agents can improve drug activity and decrease their toxicity [8]. Since Rosenberg et al. [9] reported anti-tumor activity of cis-platinum, the interaction between noble metals and DNA has attracted increasing attention. Both Ag+ and silver compounds present obvious bactericidal effect, AgNO3 is used to dye DNA and protein as well, so silver is a kind of bio-affinity metal. Hematoxylin (HE) (Fig. 1) is a derivative of catechol, which is one of the most bioactive flavonoids [10,11], it has strong coordinating qualities. It is well known that flavonoids have a variety of biological effects in numerous mammalian cell systems such as antiviral, anti-allergic, anti-platelet, anti-inflammatory and anti-tumor activities, and possibly even protective effects against chronic diseases [12]. Due to the existence of planar ring system, a number of polycyclic aromatic hydrocarbons and their derivatives are known ⇑ Corresponding author. Tel.: +86 13547133962; fax: +86 816 2419201. E-mail address: [email protected] (X. Wang). 0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2011.11.031

to intercalate into DNA, resulting in the anticancer activity [13]. They have potential to disrupt the normal function of cellular DNA and exhibit mutagenesis, carcinogenesis and even lead to cell death. Studies in the last few years indicate that hematoxylin has the remarkable inhibitory action to the stomach cancer [14,15]. So the binding studies between HE–Ag+ complex and DNA are useful for the understanding of the reaction mechanism and provide guidance for the application and design of new and more efficient drugs targeted to DNA. In this paper, we synthesize the HE–Ag+ complex, DNA binding mode of the complex was investigated by electronic absorption spectroscopy, fluorescence spectroscopy and viscosity measurement. Experimental evidences indicate that the complex can strongly bind to hsDNA, the binding modes of the complex binding to hsDNA can be used as models for the DNA binding of anticancer drugs [16]. 2. Methods 2.1. Materials hsDNA was purchased from Sigma Biological Co. and used as supplied. The stock solution of hsDNA was prepared by dissolving appropriate solid hsDNA in doubly distilled water and stored at 277 K. hsDNA purity estimation was done by recording the UV– visible absorbance at 260 nm and 280 nm. The ratio of absorbance (A260/A280) was found >1.8, which indicates that the hsDNA was free from any contamination [17]. HE was purchased from Sichuan Chengdu China Kelong chemical plant (A.R.). AgNO3 was purchased from Beijing Beihua Fine Chemicals Co., Ltd. acridine orange (AO)

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J. Huang, X. Wang / Journal of Molecular Structure 1010 (2012) 73–78

Fig. 1. Structure of Hematoxylin.

was purchased from Shanghai—China medicine chemical plant (A.R.). Other reagents used were of analytical grade, and were used as supplied. 2.2. Instruments The absorption spectra were recorded on a Shimadzu UV-3150 spectrophotometer. The fluorescence spectra were recorded on a Hitachi FL-4500 spectrofluorophotometer. The infrared spectra were recorded on a PE Spectrum One FTIR. In fluorescence mode, emission bandwidths were set at 5 nm, excitation wavelength was set at 441 nm, but in competitive binding experience, excitation wavelength was set at 320 nm in order to suppress inner-filter effect. 2.3. Spectral measurements Spectral measurements were performed by the spectrophotometric titration method using a quartz cuvette of 1 cm path length. The absorption spectra were plotted with fixed concentration of the HE–Ag+ complex while gradually increasing the concentration of hsDNA. Each injection is 10 lL, the volume effect was so small that could be ignored. Analogously, the fluorescence spectra were measured with fixed the concentration of DNA–AO while gradually increasing the concentration of the HE–Ag+ complex. All solutions were shaken thoroughly and allowed to equilibrate for 10 min before spectral measurements were made at room temperature. Scatchard method was performed as follows: hsDNA was incubated with HE–Ag+ complex on different Rt (Rt = [HE–Ag+ complex]/[DNA]. Rt = 0.00, 0.80, 1.60 respectively) for 10 min so that they could react completely, then the samples were titrated by AO solution and recorded the fluorescence intensity. 2.4. Viscosity measurements Viscosity measurements were carried out using a viscometer, which was immersed in a thermostat water-bath at room temperature. The hsDNA concentration was kept constant while increasing the concentration of the HE–Ag+ complex. The flow times of the samples were repeatedly measured with an accuracy of ±0.2 s by using a digital stopwatch. Flow time was recorded three times for each sample and took the average flow time. The value of relative viscosity (g/g0) were plotted against [HE–Ag+ complex], where g and g0 are the relative viscosities for hsDNA in the presence and absence of the HE–Ag+ complex, respectively. 3. Results and discussion 3.1. Formation of HE–Ag+ complex The accession of Ag+ made significant effects on absorption spectral of HE, indicating the formation of a complex between Ag+ and HE. The absorption spectra of HE in absence and presence of different amounts of Ag+ was given in Fig. 2. In the ultraviolet region spectra, we could see that with increasing concentration

Fig. 2. Absorption spectra of HE in different concentrations of Ag+; [HE] = 2.00  104 mol L1 (3.0 mL), [Ag+] = 0.00, 0.30, 0.60, 0.90, 1.20  104 mol L1. Inset: Mole ratio plots of HE–Ag+.

of Ag+, the intensity of the maximal peak of HE (at 289 nm) reduce gradually. The absorption peak at 289 nm is attributed to the n–p⁄ transition energy of the oxygen in the Hematoxylin [18], so the change of absorbance at 289 nm is caused by the interaction of Ag+ with oxygen heteroatom. In the visible region spectra, the complex presents a strong absorbance peak (at 444 nm), which is due to metal-to-ligand charge transfer interactions. From the detail view in Fig. 2, we find two isosbestic points at 282 nm and 300 nm, respectively. They also indicate that HE has been bound with Ag+. In order to determine stoichiometry of the HE–Ag+ complex in solution, a mole-ratio study was performed. Ligand HE was titrated by Ag+ solution and the absorbance was measured at 289 nm. In the mole-ratio plots two lines were obtained, the intersection which determines the stoichiometry of the complex in buffer solution (inset in Fig. 2) suggests that the stoichiometry of the complex in solution for metal to ligand is 1:2. The complex was also characterized with the infrared spectra. The infrared spectra of free HE in KBr pellets show stretching vibration band at 1643 cm1 (mCO). After HE binding to Ag+, the complex exhibits band at 1604 cm1 (mCO), which indicates that the coordination of Ag+ to the oxygen of hydroxy. This is further supported by the appearance of a new medium intensity band in the region 470–480 cm1 assignable to (M–O) vibration. 3.2. Absorption spectra of interaction between HE–Ag+ complex and hsDNA Titration with UV absorption spectroscopy is an effective method to examine the binding mode of DNA with metal complexes, so the interaction of HE–Ag+ complex with hsDNA has been studied with UV spectroscopy in order to investigate the possible binding mode and calculate the binding constants. Absorption titration experiment was performed by maintaining the HE–Ag+ complex concentration and varying the concentration of hsDNA (Fig. 3). In the absence of hsDNA, the spectrum of the HE–Ag+ complex (Curve 1) is characterized by the strong absorption peaks at 444 nm. With the addition of hsDNA, the intensity of the 444 nm band reduces significantly. Generally, large hypochromism of an aromatic dye in presence of double helical DNA is characteristic of intercalation into DNA base-pairs for the dye, due to the strong stacking interaction between the aromatic chromophore and the base-pairs [19], so the observed hypochromism reflect the intercalating mode between HE–Ag+ complex and hsDNA. On the other hand, a shoulder peak at ca. 510 nm is observed in the presence of hsDNA, the small

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Fig. 3. Absorption spectra of HE–Ag+ complex in different concentrations of DNA; [HE–Ag+ complex] = 5.00  105 mol L1 (3.0 mL), [DNA] = 0.00, 0.30, 0.60, 0.90, 1.20  105 mol L1.

hyperchromic effect suggests that the impetus of the binding process include external contact [20]. The change in the absorbance of the HE–Ag+ complex band with increasing amounts of hsDNA is used to derive the intrinsic binding constants (K). From the absorption data, K was determined using the following equation by plotting 1/(A0  A) versus 1/[DNA] (Fig. 4) [21,22]:

1=ðA0  AÞ ¼ 1=A0 þ 1=ðK  A0  ½DNAÞ where A0 and A are the absorbencies of HE–Ag+ complex in the absence and presence of DNA, respectively, K is the binding constant between HE–Ag+ complex and hsDNA, [DNA] is the concentration of DNA. The data were fitted to the above equation with a slope equal 1/(K  A0) and y-intercept equal to 1/A0, than K was obtained from the ratio of the intercept to the slop. The values of K at two 1 4 H temperatures (303, 310 K): K H 303K ¼ 3:13  10 L mol , K 310K ¼ 1 4 4:63  10 L mol . K values are lower than those typical classical intercalators such as ethidium bromide, the result indicate that the affinity between the title complex and DNA is weaker than that between classical intercalators and DNA [23]. 3.3. Thermodynamics studies The thermodynamic parameters dependent on temperatures were analyzed in order to further characterize the interaction

Fig. 5. Emission spectra of DNA–AO admixture in different concentration of HE–Ag+ complex; [DNA–AO] = 1.00  106 mol L1 (3.0 mL); [HE–Ag+ complex] = 0.00, 0.33, 0.67, 1.0, 1.33, 1.67, 2.0, 2.33, 2.67, 3.0, 3.33  106 mol L1.

forces between HE–Ag+ complex and hsDNA. The interaction forces between a small molecule and biomolecules may include hydrophobic force, electrostatic interactions, van der Waals interactions, hydrogen bonds, etc. [24]. If temperature changes slightly, the enthalpy change (DrHmH) could be considered to be a constant. Then the value of DrHmH and entropy change (DrSmH) can be determined from the van’t Hoff equation:

lgK H ¼ Dr HmH =ð2:303RTÞ þ Dr SmH =ð2:303RÞ where K and R are binding constant and gas constant, respectively. 1 1 Then Dr HmH ¼ 4:37  104 J mol , Dr SmH ¼ 230:31 J mol was deduced. The free energy change (DrGmH) was estimated from the following relationship:

Dr GmH ¼ RTlnK H ¼ Dr HmH  T Dr SmH 1

4 Then Dr GmH was deduced. 303K ¼ 2:61  10 J mol The negative value DrGmH reveals the interaction process is spontaneous, the both positive DrHmH and DrSmH values indicate that the entropy is favorable for the interaction process, namely, it is more likely that hydrophobic interaction is involved in the binding process [25].

3.4. Competitive binding experiments using AO as probe

Fig. 4. Double reciprocal 5.00  105 mol L1.

plots

of

HE–Ag+–DNA;

[HE–Ag+

complex] =

Although DNA has a natural fluorescence, the intensity is so weak that the direct use of the fluorescence emission of DNA is limited to study its properties. Ethidium bromide, acridine orange, methylene blue and similar fluorescent compounds are normally used to probe DNA structure in drug-DNA and protein-DNA interactions. In this work, AO was chosen for probe, because of its spectral and self-aggregation characteristics, widely use for fluorescence chromophore marker of DNA, it can offers lower toxicity and more convenience of use than Ethidium bromide. AO displays a dramatic enhancement of DNA fluorescence efficiency when intercalated into DNA [26]. Like AO, if HE–Ag+ complex intercalates into the helix of DNA, it would compete with AO for the intercalation sites in DNA and remove it to outside, provide a quenched AO fluorescence intensity. The spectrum of HE–Ag+ complex shows intense absorption band at 440 nm. Given the inner-filter effect, excitation wavelength was set at 320 nm, the influence on emission spectra of HE–Ag+ complex to DNA–AO system was shown in Fig. 5, a significant decrease in fluorescence intensity was

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J. Huang, X. Wang / Journal of Molecular Structure 1010 (2012) 73–78 Table 2 Data of Scatchard equation of the interaction between HE–Ag+ complex and DNA. Curve

Rt

NaCl (mol L1)

Scatchard

K (La mol1)

n

a

0.00

0.50

2.40  105– 6.91  106r 3.01  105– 3.96  106r

6.91  106

0.035

6

0.076

1.79  105– 5.40  106r 1.00  105– 1.55  106r

5.40  106

0.033

1.55  106

0.065

4.47  106

0.029

6

0.056

0 b

0.80

0.50 0

c

1.60

0.50 0

Fig. 6. The Stern–Volmer plots for the quenching of DNA–AO by HE–Ag+ complex [DNA–AO] = 1.00  106 mol L1 (3.0 mL); [HE–Ag+ complex] = 0.00, 0.33, 0.67, 1.0, 1.33, 1.67, 2.0, 2.33, 2.67, 3.0, 3.33  106 mol L1.

Table 1 KSV and Kq at different temperatures. Curve

T (K)

KSV (104 mol L1)

Kq (1011–13 L s1 mol1)

r

a b

298 293

6.09 4.27

6.09 4.27

0.9978 0.9982

observed. As we known there are two quenching processes: static and dynamic quenching. Fluorescence quenching can be dynamic, resulting from the collisional encounters between the fluorophore and quencher, or static, resulting from the formation of a groundstate complex between the fluorophore and quencher [27]. The Stern–Volmer constant KSV is often used to evaluate the quenching efficiency for a compound, it is given by the equation [28]:

F 0 =F ¼ 1 þ K SV ½complex ¼ 1 þ K q s0 ½complex where F0 and F are the emission intensities in the absence and the presence of the complex, respectively. Kq is the DNA–AO quenching rate constant. s0 is the average lifetime of DNA–AO in the absence of the complex and its value is 109–107 s [27]. [Complex] is concentration of the HE–Ag+ complex. KSV is the quenching constant and equals Kq multiplied by s 0. Each static and dynamic quenching is

1.29  105– 4.47  106r 0.92  105– 1.65  106r

3.96  10

1.65  10

in agreement with the linear Stern–Volmer equation, if there exist two kinds of quenching mode at the same time, the Stern–Volmer curve would loose its linearity. Beside above quenching modes, inner-filter effect can also induce the fluorescence quenching, so the inner-filter effect must be evaluated. The corrected fluorescence intensity is given by Fcorr = Fobs e(Aex+Aem)/2 [29], where Fcorr and Fobs are the corrected and observed fluorescence intensities, and Aex and Aem are the absorbance values of the title complex at the excitation and emission wavelengths. HE–Ag+ complex was added into the DNA–AO solution with fixed concentration at 293 K and 298 K respectively, in order to infer the nature of the quenching process. From the corrected equation, we calculated the value of e(Aex+Aem)/2, it was about 1.0 when the concentration of the title complex was less than 1.00  106 mol L1. Then the Stern–Volmer curves were plotted (Fig. 6). As we can see in Fig. 6, the behaviors of the first four points are linear. However, with the further increasing concentration of the complex, the curves shown a small upward concave curvature toward the y-axis, it reflects that there exists not only one type of quenching process. First, from the Stern–Volmer curves, the corresponding KSV and Kq were obtained (Table 1). The values of Kq is much greater than 2.0  1010 L s1 mol1, the maximum diffusion collision quenching rate constant of various quenchers with biopolymers [30]. It proves that the quenching process was static quenching. Secondly, dynamic quenching depends upon diffusion, so an increase in temperature increases the quenching and the quenching constant is expected to increase. In Fig. 6, the degree

Fig. 7. Scatchard plots of HE–Ag+–DNA in different concentration of AO. [DNA] = 1.00  106 mol L1; [AO] = 3.00  105 mol L1; Rt = [HE–Ag+ complex]/[DNA]; (a) Rt = 0.00; (b) Rt = 0.80; (c) Rt = 1.60.

J. Huang, X. Wang / Journal of Molecular Structure 1010 (2012) 73–78

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HE–Ag+ complex. The characteristics of AO bind to DNA can be expressed by Scatchard equation [31,32]:

rAO =½AO ¼ Kðn  r AO Þ

Fig. 8. Influence on DNA viscosity with different concentrations of HE–Ag+ complex. [DNA] = 1.00  105 mol L1.

of quenching at 298 K is obviously higher than which at 293 K, it indicates the occurrence of dynamic quenching. 3.5. Scatchard analysis Scatchard analysis was performed in order to further understand the mechanisms of the interaction between hsDNA and

where rAO is the proportion of bound AO per nucleic acid phosphate, [AO] is the concentrations of free AO, n is the number of binding sites per nucleic acid phosphate, K is the intrinsic association constant to a site. The Scatchard plots belong to the influence of AO on hsDNA in the absence and presence of drugs were obtained by using rAO/[AO] as a function of rAO (Fig. 7). When a drug binds to DNA by intercalation mode, the K value in the Scatchard plot would change but the n value would not. When a drug binds to DNA by non-intercalation mode, the n value would decrease but the K value would not change, when a drug binds to DNA by both intercalation and nonintercalation modes (a mixed type), both K and n value would change [32]. According to Scatchard equation, the values of n and K were listed in Table 2. As shown in Table 2, with the increasing of Rt value, both the values of parameter n and K change, it suggests that the interaction sites of HE–Ag+ complex and AO are not identical, there exists mixed binding mode between hsDNA and the complex. Besides, in order to test whether there exists electrostatic binding between HE–Ag+ complex and hsDNA, the strong electrolyte sodium chloride (concentration of 0.10 mol L1 NaCl) was used in Scatchard studies. Because Na+ can combine with the negative polyphosphate skeleton of hsDNA, if there is electrostatic interaction between HE–Ag+ complex and hsDNA, the existence of Na+

Fig. 9. Emission spectra of HE–Ag+ complex in different concentrations of basic group. [HE–Ag+ complex] = 1.00  104 moL L1 (3.0 mL); [basic group] = 0.00, 0.17, 0.33, 0.50, 0.67, 0.83, 1.00, 1.17, 1.33  104 moL L1.

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would weaken this electrostatic interaction, then the value of n would decrease. The values of n in the present of NaCl are basically lower than those in the absence of NaCl in Table 2, it suggests that there are electrostatic binding and intercalation binding between the complex and hsDNA. 3.6. Viscosity method Optical photophysical probe is necessary but not a sufficient condition for the judgment of binding mode, whereas hydrodynamic measurements is sensitive to the length change, it is regarded as the most critical tests of a binding model in solution [33]. Thus, in order to further clarify the interaction nature between the HE–Ag+ complex and hsDNA, viscosity measurements were carried out. Classical intercalator is found to increase the relative viscosity of DNA due to the lengthening of DNA double helix resulting from intercalation; the groove-binding ligand does not appreciably alter DNA viscosity; in contrast, a partial and non-classical intercalation ligand reduces the relative viscosity of DNA, and the behavior is explained by a binding mode that produces bend or kink in DNA helix [34]. As illustrated in Fig. 8, the relative viscosity of hsDNA steadily decreases with the addition of the HE–Ag+ complex, the reason is related to the aromatic ring of HE being partially inserted into the hsDNA base pairs and the hsDNA secondary structure is changed, therefore decreasing its effective length. 3.7. The influence of basic group to the HE–Ag+ complex system The influence of basic group on the complex system was studied in order to further realize the interaction mechanism between HE– Ag+ complex and hsDNA. Two possibilities could explain the change in emission spectrum of the complex: Firstly, HE–Ag+ complex interact with the base pair of DNA through the major groove or minor groove of DNA molecule and it is hardly selective, because in the major and minor grooves, the bases are exposed to solvent, namely, the complex can directly interaction with basic group; Second, p–p interaction and hydrophobic interaction had been occurred between the HE–Ag+ complex and the p system of base pair, which means that there are intercalation binding mode. As shows in Fig. 9, the emission intensity of complex increased steadily with the increasing concentration of A and T, but hardly changed for G and C. Similar phenomenon was reported by Zhao et al. [35]. The essence of groove binding is van der Waals interactions, hydrogen bonds, it is hardly selective. So the phenomenon indicates that there is intercalation binding: the empty p⁄-orbital of the complex could couple with the p⁄-orbital of the base pair of A:T, it indicates that the intercalation is selective and the location of HE–Ag+ complex action are mainly in the base pair of A:T. We think the selective intercalation is very useful to design the drug or probe which acts to DNA. 4. Conclusion The interaction between HE–Ag+ complex and hsDNA has been studied by spectral analysis and viscosity method. The findings demonstrated that the binding mode between HE–Ag+ complex and hsDNA are partially intercalation binding and electrostatic binding. The rigid plane of the complex can partially intercalate into A:T base pairs and induce bend of the hsDNA secondary structure. The highly

selective interaction with A:T base pairs strongly supports the idea that HE–Ag+ complex could be useful for design of new and more efficient drugs targeted to DNA. The binding constants of HE–Ag+ 1 4 H complex to hsDNA are K H 303K ¼ 3:13  10 L mol , K 310K ¼ 4:63 1 104 L mol , and the corresponding thermodynamic parameters are DrHmH = 4.37  104 J mol1, DrSmH = 230.31 J mol1, DrGmH = 2.61  104 J mol1. In this interaction process, the entropy is the driven force. These data provide important biophysical information relate to structure–activity relationship, to help understand the nature of the complex binding to DNA. Acknowledgement This work was supported by Postgraduate Innovation Fund sponsored by Southwest University of Science and Technology. References [1] E.K. Efthimiadou, N. Katsaros, A. Karaliota, G. Psomas, Inorg. Chim. Acta 360 (2007) 4093–4102. [2] G.G. Song, M. Peng, Q.J. Yan, Luminescence 26 (2011) 17–22. [3] G.M. Zhang, Y.H. Pang, S.M. Shuang, C. Dong, M.M. Choi, D.S. Liu, J. Photochem. Photobiol. A 169 (2005) 153–158. [4] M.T. Hassan, I.M. Mahboube, D. Adeleh, A.A. Saboury, Bioorg. Med. Chem. 16 (2008) 9616–9625. [5] A. Radi, M.A. El Ries, S. Kandil, Anal. Chim. Acta 495 (2003) 61–67. [6] G.W. Zhang, J.B. Guo, N. Zhao, J.R. Wang, Sens. Actuators B 29 (2009) 243–246. [7] L.H. Guo, B. Qiu, G.N. Chen, Anal. Chim. Acta 588 (2007) 123–130. [8] M.N. Patel, M.R. Chhasatia, P.A. Dosi, H.S. Bariya, V.R. Thakkar, Polyhedron 29 (2010) 1918–1924. [9] B. Rosenberg, L. Vancamp, J. Trosko, V. Mansour, Nature 222 (1969) 385. [10] H.R. Zare, N. Nasirizadeh, M. Mazloum-Ardakani, N. Mamazian, Sens. Actuators B 120 (2006) 288–294. [11] H.R. Zare, N. Nasirizadeh, Sens. Actuators B 143 (2010) 666–672. [12] J.W. Kang, L. Zhuo, X.Q. Lu, H.D. Liu, M. Zhang, H.X. Wu, J. Inorg. Biochem. 98 (2004) 79–86. [13] A. Kamal, K. Sreekanth, P. Praveen Kumar, N. Shankaraiah, G. Balakishan, M. Janaki Ramaiah, S.N.C.V.L. Pushpavalli, P. Ray, M.P. Bhadra, Eur. J. Med. Chem. 45 (2010) 2173–2181. [14] L.S. Ren, J.G. Xu, J.Y. Ma, China J. Chin. Mater. Med. 15 (1990) 50–51. [15] Y.B. Zhou, F.Z. Yao, J.R. Han, Y. Yang, C.F. Zhang, Y.P. Shi, Chin. J. Integr. Trad. West Med. 23 (2003) 370–372. [16] F. Qu, N.Q. Li, Y.Y. Jiang, Talanta 45 (1998) 787–793. [17] J. Marmur, J. Mol. Biol. 3 (1961) 208–218. [18] Q.Y. Deng, L. Liu, H.M. Deng (Eds.), Spectral Analysis Course, Science Publishing, Beijing, 2007 (Chapter 1). [19] B.D. Wang, Z.Y. Yang, M.H. Lü, J. Hai, Q. Wang, Z.N. Chen, J. Organomet. Chem. 694 (2009) 4069–4075. [20] K.C. Skyrianou, C.P. Raptopoulou, V. Psycharis, D.P. Kessissoglou, G. Psomas, Polyhedron 28 (2009) 3265–3271. [21] A.A. Ouameur, R. Marty, H.A. Tajmir-Riahi, Biopolymers 77 (2005) 129–136. [22] Y. Wang, A.H. Zhou, J. Photochem. Photobiol. A 190 (2007) 121–127. [23] R. Vijayalakshmi, M. Kanthimathi, V. Subramanian, Biochim. Biophys. Acta Gen Subj. 1475 (2000) 157–162. [24] H. Ojha, B.M. Murari, S. Anand, M.I. Hassan, F. Ahmad, N.K. Chaudhury, Chem. Pharm. Bull. 57 (2009) 481–486. [25] Y.Q. Wang, H.M. Zhang, Q.H. Zhou, Eur. J. Med. Chem. 44 (2009) 2100– 2105. [26] S. Nafisi, A.A. Saboury, N. Keramat, J.F. Neault, H.A. Tajmir-Riahi, J. Mol. Struct. 827 (2007) 35–43. [27] Y.T. Sun, S.Y. Bi, D.Q. Song, C.Y. Qiao, D. Mu, H.Q. Zhang, Sens. Actuators B 129 (2008) 799–810. [28] C.Q. Cai, X.M. Chen, F. Ge, Spectrochim. Acta A 76 (2010) 202–206. [29] R.F. Steiner, L. Weinryb (Eds.), Excited State of Protein and Nucleic Acid, Plenum Press, New York, 1971, p. 40. [30] W.R. Ware, J. Phys. Chem. 66 (1962) 455–458. [31] J.B. Lepecq, C. Paoletti, J. Mol. Biol. 27 (1967) 87. [32] M.L. Guo, P. Yang, B.S. Yang, Z.G. Zhang, Chin. Sci. Bull. 41 (1996) 1098–1103. [33] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 31 (1992) 9319– 9324. [34] Z.G. Zhang, X.D. Dong, Biometals 22 (2009) 283–288. [35] Z. Zhao, X.M. Wang, H.Z. Pan, Y.M. Hu, L.S. Ding, Spectrochim. Acta A 75 (2010) 1435–1442.