Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 1–6

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Interaction of a copper (II) complex containing an artificial sweetener (aspartame) with calf thymus DNA Nahid Shahabadi a,⇑, Mohammad Mehdi Khodaei b,⇑, Soheila Kashanian b, Fahimeh Kheirdoosh c a

Department of Inorganic Chemistry, Faculty of Chemistry, Razi University, Kermanshah, Islamic Republic of Iran Department of Chemistry, Sensor and Biosensor Research Center (SBRC) & Nanoscience and Nanotechnology Research Center (NNRC), Faculty of Chemistry, Razi University, Kermanshah, Islamic Republic of Iran c Department of Applied Chemistry, Faculty of Chemistry, Razi University, Kermanshah, Islamic Republic of Iran b

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

 Copper is an essential trace element

A copper (II) complex containing aspartame (APM) as ligand, Cu(APM)2Cl22H2O, was synthesize and characterized. In vitro binding interaction of this complex with native calf thymus DNA (CT-DNA) was studied at physiological pH.

in plants and animals which binds to aspartame and makes stable complex.  Aspartam rapidly picks up toxic metals and carries into the body.  All the results showed that Cu(APM)2Cl22H2O complex was a DNA groove binder.

a r t i c l e

i n f o

Article history: Received 31 May 2013 Received in revised form 14 August 2013 Accepted 2 October 2013 Available online 12 October 2013 Keywords: Aspartame Cu(APM)2Cl22H2O Calf thymus DNA (CT-DNA) Spectroscopy Groove binding

a b s t r a c t A copper (II) complex containing aspartame (APM) as ligand, Cu(APM)2Cl22H2O, was synthesized and characterized. In vitro binding interaction of this complex with native calf thymus DNA (CT-DNA) was studied at physiological pH. The interaction was studied using different methods: spectrophotometric, spectrofluorometric, competition experiment, circular dichroism (CD) and viscosimetric techniques. Hyperchromicity was observed in UV absorption band of Cu(APM)2Cl22H2O. A strong fluorescence quenching reaction of DNA to Cu(APM)2Cl22H2O was observed and the binding constants (Kf) and corresponding numbers of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy change (DH) and entropy change (DS) were calculated to be +89.3 kJ mol1 and +379.3 J mol1 K1 according to Van’t Hoff equation which indicated that reaction is predominantly entropically driven. Experimental results from spectroscopic methods were comparable and further supported by viscosity measurements. We suggest that Cu(APM)2Cl22H2O interacts with calf thymus DNA via a groove interaction mode with an intrinsic binding constant of 8  10+4 M1. Binding of this copper complex to DNA was found to be stronger compared to aspartame which was studied recently. Ó 2013 Elsevier B.V. All rights reserved.

Introduction ⇑ Corresponding authors. Tel./fax: +98 831 8360795. E-mail addresses: [email protected] (N. Shahabadi), mm_khodaei@ yahoo.com (M.M. Khodaei). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.10.008

Aspartame (C14H18N2O5) (Fig. 1a) is the methyl ester of the dipeptide of the natural amino acids L-aspartic acid and L-phenyl-

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alanine [1]. The mentioned molecule is an artificial sweetener and widely used in many fields such as medicine and food [2]. There are many reports concerning aspartame (APM) and its harmful effects ranging from brain damage to pre-term delivery [3–5]. APM has carboxylate and amino groups suitable for binding metal ions. Aspartame also chelates the toxic heavy metals from the ingested food and drink, as well as from the very containers from which it is served in, and carries them directly into the body as their highly absorbed chelated heavy metal forms. Copper is an essential trace element. It is essential to all living organisms and is a universally important cofactor for many hundreds of metalloenzynes. Copper deficiency is widespread and appears in many forms. Copper is required in many physiological functions (RNA, DNA, lysil oxidase cofactor, melanin production (hair and skin pigment), electron transfer of oxygen subcellular respiration, tensile strength of elastic fibers in blood vessels, skin, vertebral discs, etc.) [6,7]. Deficiencies of copper can result in hernias, aneurysms, and blood vessel breakage manifesting as bruising or nosebleeds. The diet must be as low as possible in sweets, fruits and sugars as possible. These foods, along with all stimulants, stress the adrenal glands and tend to make copper imbalance worse. Stimulants include sugars, caffeine and food additives such as aspartame and other excitotoxins in the diet. Literature review proves the interaction between copper complexes and DNA [8]. There have also been recent developments in the genetic and non-genetic abnormalities of copper [9]. The interaction of aspartame with copper as a biologically significant metal has been studied in [10] where the emphasize is on the pathological mechanism of aspartame interactions. Deoxyribonucleic acid (DNA) plays an important role in living organisms, such as gen expression, gen transcription, mutagenesis and carcinogenesis. Studies toward the interactions between small molecules and DNA will be valuable for preventing and caring disease. DNA is known to be a major target for drugs and some harmful chemicals to be attacked. The studies of complex – DNA interactions are of current general interest and significance [11,12], particularly, for the designing of new DNA-targeted complex and the experience of these in vitro. During recent years, the interest for metal complexes containing planar aromatic ligands has increased tremendously, mainly for their usage as probes capable to utilize the nucleic acid structures and as DNAmolecular light switches [13]. The above mentioned studies motivated us to investigate the interaction of a copper (II) complex containing APM as ligand, Cu(APM)2Cl22H2O, with DNA. The main goal is to study DNA dam-

COO-

age and understand the toxic effect of aspartame in the presence of copper. In this study, different spectroscopic methods including: fluorimetry, competition experiment, circular dichroism (CD), UV absorption and viscometric techniques were considered and the results are compared thoroughly.

Experimental Material and methods Aspartame and CT-DNA were purchased from Sigma, TrisAHCl buffer was purchased from Merck. Cu(APM)2Cl22H2O was prepared according to the method published in [14]. Experiments were performed in TrisAHCl buffer at pH = 7.4. Stock solution of CT-DNA was prepared by dissolving approximately 1–2 mg of CTDNA fibers in 2 mL TrisAHCl buffer (10 mM), shaken gently and stored at 4 °C for 24 h. Concentration of DNA solution was expressed in monomer units, determined by spectrophotometry to be 260 nm using an extinction coefficient (ep) of 6600 M1cm1. DNA solutions were used after 4 days at most. A solution of CTDNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm of 1.8–1.9:1, indicating that the DNA was sufficiently protein-free. Stock solution of Cu(APM)2Cl22H2O was prepared by dissolving sufficient amount of the complex in 2.0 mL of TrisAHCl buffer (10 mM) (final concentration = 103 M). Aliquots of the DNA solution were treated with the complex at several input molar ratios (ri) (ri = [DNA]/[complex]) with a constant complex concentration. The final volume of samples was made with TrisAHCl (10 mM).

Synthesis of the Cu(APM)2Cl22H2O The complex (Fig. 1b) was prepared as a mononuclear complex. A solution of CuCl22H2O (85.24 mg, 0.5 mmol) in 20 mL methanol was added to a solution containing APM (290.4 mg, 1 mmol) in 30 mL methanol, and the mixture was stirred at room temperature (RT) for 4 h and then left at RT for 6 h. Resulting solution was stirred at RT for 24 h. The solution was evaporated to dryness. A crude green solid, [Cu(APM)2Cl22H2O] was obtained. Elemental Analysis for C28H40CuN4O13Cl2 Calc C, 43.28; H, 5.19; N, 7.21. Found: C, 42.45; H, 5.19; N, 6.8%. IRmmax: 3600–2400 Cm1 (hydrogen bond HAN/O); 1738– 1420 Cm1 (symmetric and anti symmetric C@O stretching);

O H N

+H N 3

OCH 3

O

(a)

(b)

Fig. 1. (a) Molecular structure of APM and (b) molecular structure of Cu(APM)2Cl22H2O.

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(UV–Vis) (nm): MeOH solution (274 MLCT CuACl shifted to 265 MLCT in complex,208, d–d transition).

½DNA=ðea  ef Þ ¼ ½DNA=ðeb  ef Þ þ 1=K b ðeb  ef Þ

DNA interaction studies

where [DNA] is the concentration of DNA in base pairs. ea is the extinction coefficient for compound absorption band at a given DNA concentration, ef is extinction coefficient of free compound, eb the extinction coefficient of compound when fully bound to DNA (it is assumed when further addition of DNA does not change the absorbance). In particular, ef was determined by a calibration curve of the isolated compound in aqueous solution, following Beer’s law. ea was determined as the ratio between the measured absorbance and the compound concentration. Plot of [DNA]/(ea–ef) versus [DNA] gives a slope of 1/(eb–ef) and a y-intercept equal to 1/Kb (eb–ef); Kb is the ratio of the slope to the y-intercept (Fig. 2 inset). The binding constant, Kb, for this complex was 8  104 L mol1. The binding constant of APM ligand [18] was 5  104 M1 which is smaller than that of its Cu (II) complex, showing that the DNA binding affinity of the complex is stronger than the ligand. However, they are much lower than the potential intercalators like ethidium bromide (Kb = 106–107 M1) [19], but the intrinsic binding constant (Kb) of Cu(APM)2Cl22H2O was consistent with previously reported DNA groove binders such as isatin, Kb = 7.32  104 M1 [20] and Tartrazine = 3.75  104 M1 [21], therefore, we deduce that complex binds to CT-DNA via groove binding mode [22,23].

Physical measurements and Instrumentation Absorbance spectra were recorded using an HP spectrophotometer (Agilent 8453) equipped with a thermostated bath (Huber polysat cc1). The absorbance measurements were performed by keeping the Cu(APM)2Cl22H2O concentration constant (5  105 M) while varying the DNA concentration from 0 to 2.5  104 M (ri = [DNA]/ [complex] = 0.0–5). The spectra were recorded in the range of 230–300 nm. Fluorescence measurements were carried out with a JASCO spectrofluorimeter (FP 6200) by keeping the concentration of the complex Cu(APM)2Cl22H2O constant (5  105 M) while varying the DNA concentration from 0 to 2.25  104 M, (ri = [DNA]/[complex] = 0.0–4.5) at three different temperatures (298, 310 and 318 K). The emission spectra were recorded from 260–330 nm. Viscosity measurements were made using a viscometer (SCHOT AVS 450) which was maintained temperature at 25 ± 0.5 °C using a constant temperature bath. The DNA concentration was fixed at 5  105 M and flow time was measured with a digital stopwatch; the mean values of three replicated measurements were used to evaluate the viscosity (g) of the samples. The data were reported as (g/g°)1/3 versus the [complex]/[DNA] ratio, where g° is the viscosity of the DNA solution alone [15]. CD measurements were recorded on a JASCO (J-810) spectropolarimeter by keeping the concentration of DNA constant (8  105 M) while varying the Cu(APM)2Cl22H2O concentration from 0 to 8.0  105 M (ri = [Complex]/[DNA] = 0.0–1.0). In the competitive binding studies, concentrations of DNA and Hoechst 33258 were kept constant while varying the complex concentration. Results and discussion UV/vis spectroscopic studies Electronic absorption spectroscopy is usually used to determine the binding strength and the mode of DNA binding with small molecules [16]. The absorption spectra of Cu(APM)2Cl22H2O with increasing concentrations of CT-DNA are increasing (Fig. 2). It means resulting in hyperchromicity. The intrinsic binding constant was calculated according to this equation [17]:

Fig. 2. Electronic absorption spectra for the titration of 5.0  105 M Cu(APM)2Cl22H2O with DNA (ri = 0.0, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0).

ð1Þ

Fluorescence quenching studies Since luminescence was observed for the Cu(APM)2Cl22H2O solution, it is possible to monitor the interaction of the complex with DNA by employing direct fluorescence emission method. In order to investigate the interaction mode between Cu(APM)2Cl22H2O and CT-DNA, the fluorescence titration experiments were performed. The complex can emit luminescence with maximum wavelength of about 282 nm in TrisAHCl buffer. Fig. 3 shows the emission spectra of the complex in the absence and presence of different concentrations of CT-DNA. The emission intensity of Cu(APM)2Cl22H2O decreases in the presence of increasing amounts of DNA. In previous study, the emission intensity of APM decreased in the presence of increasing amounts of DNA [18] as well.

Fig. 3. Fluorescence spectra of the Cu(APM)2Cl22H2O in the absence and presence of increasing amounts of DNA in 0.01 M TrisAHCl buffer (pH 7.4) at 298 K (ri = [DNA]/[complex] = 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5).

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Temperature effect on quenching efficiency The titration data obtained from the interaction study of DNA and Cu(APM)2Cl22H2O at different temperatures were fitted into the Stern–Volmer equation (Eq. (2)) [24]:

F 0 =F ¼ 1 þ K q s½DNA ¼ 1 þ K sv ½DNA

ð2Þ

where F0 and F are the fluorescence intensities in the absence and presence of DNA, respectively, Kq is the bimolecular quenching constant, s is the lifetime of the fluorophore and KSV is the Stern–Volmer quenching constant which can be considered as a measure for efficiency of fluorescence quenching by DNA. In fact, two quenching processes are known: static and dynamic. Dynamic quenching or collisional quenching requires contact between the excited lumophore and the quenching specie, the quencher. The rate of quenching is diffusion controlled and depends on the temperature and viscosity of the solution. The quencher concentration must be high enough so that the probability of collision between the analyte and quencher is significant during the lifetime of the excited species. The other form of quenching, as mentioned above, is static quenching in which the quencher and the fluorophore in ground state form a stable complex [25]. Dynamic and static quenching can also be discerned by their differing dependence on temperature [26]. Dynamic quenching depends upon diffusion, since higher temperatures lead to larger diffusion coefficients, the Ksv can be increased by rising the temperature. In contrast, increasing temperature is likely the result of decrease in complex stability resulting in lower values of the static quenching constants. The values of Ksv at different temperatures (298, 310 and 318 K) were obtained and the results are shown in Fig. 4 and Table 1. These results show that DNA can quench Cu(APM)2Cl22H2O fluorescence in a dynamic quenching procedure, because the Ksv increased due to the temperature rising [20,27]. According to the previous study [18] the results indicate that the quenching mechanisms of the complex in comparison with APM are different. Equilibrium binding titration Fluorescence titration data were used to determine the binding constant (Kf) and the number of binding sites (n) for the complex formation of Cu(APM)2Cl22H2O with CT-DNA. It can be seen that the fluorescence intensity at 282 nm decreases in the presence of CT-DNA. This change in fluorescence intensity was used to estimate Kf and n for the binding of Cu(APM)2Cl22H2O to CT-DNA from the following equation [28]:

log ðF 0  F=FÞ ¼ log K f þ n log ½DNA

Table 1 The Ksv values of Cu(APM)2Cl22H2O – DNA at different temperatures. R2

Ksv (L mol1)

Temperature (K)

+3

298 310 318

8.0  10 8.8  10+3 1.0  10+4

0.98 0.99 0.98

Here, F0 and F are the fluorescence intensities of the fluorophore in the absence and presence of different concentrations of CT-DNA, respectively. The linear equations of log (F0–F/F) versus log [DNA] at different temperatures are shown in Table 2. The values of Kf clearly indicate the remarkably high affinity of Cu(APM)2Cl22H2O for DNA. Thermodynamic studies In order to better understand the thermodynamic behavior of the complexation reaction between Cu(APM)2Cl22H2O and DNA, contributions of enthalpy and entropy should be determined in the reaction. Thermodynamic parameters describing the binding reactions can be divided into three contributions. The first contributions are due to hydrogen bonding and hydrophobic interactions between the compound and DNA binding sites. The next contribution arises from the conformational changes in either the nucleic acid or the compound upon binding. The third contributions are provided by coupled processes such as ion release, proton transfer, or changes in the hydration water [29]. Evaluation of the formation constant for the Cu(APM)2Cl22H2O – DNA complex at three different temperatures (298, 310, and 318 K) allows us to determine Table 2 Linear equations of log (F–F0/F) versus log [DNA], Kf and n of Cu(APM)2Cl22H2O with DNA at different temperatures. Temperature (°K)

n

298 310 318

R2

Kf +4

1.08 1.20 1.32

1.6  10 5  10+4 1.5  10+5

0.98 0.99 0.98

ð3Þ

Fig. 5. Van’t Hoff plot for Cu(APM)2Cl22H2O – DNA complex.

Table 3 Thermodynamic parameters and binding constants for binding of Cu(APM)2Cl22H2O to calf thymus DNA.

Fig. 4. Stern–Volmer plots of the quenching of fluorescence of Cu(APM)2Cl22H2O with DNA at different temperatures (298, 310 and 318 K).

Temperature (°K)

DH° (kJ mol1)

DS° (J mol1 k1)

DG° (kJ mol1)

298 310 318

+89.3 +89.3 +89.3

+379 +379 +379

23.6 28.2 31.2

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thermodynamic parameters of Cu(APM)2Cl22H2O – DNA formation via Van’t Hoff equation (Eq. (4)) [30] as:

Ln K f ¼ DH=RT þ DS=R

ð4Þ

By plotting ln Kf versus 1/T (Fig. 5), DH and DS were determined and then DG was calculated from the following standard equation [31]:

DG ¼ DH  T DS

ð5Þ

The results are shown in Table 3. The DH and DS values of the Cu(APM)2Cl22H2O – DNA complex were +89.3 kJ mol1 and +379.3 J mol1 K1, respectively. When 4H < 0 or 4H  0, 4S > 0, the main acting force is electrostatic; when 4H < 0, 4S < 0, the main acting forces are van der Waals or hydrogen bonds and when 4H > 0, 4S > 0, the main force is hydrophobic [32]. These results indicate that the Cu(APM)2Cl22H2O – DNA binding is entropically–favored but enthalpically-disfavored. The free energy changes (DG) for Cu(APM)2Cl22H2O – DNA interactions are negatively large due to their strong association. As stated above, the reaction proceeds with an unfavorable change in enthalpy. It can be deduced that the positive value of DH in the Cu(APM)2Cl22H2O – DNA interaction signifies the contribution of the positive DS resulting in a more negative DG favoring binding process. It appears that the major contributing factor in the Cu(APM)2Cl22H2O – DNA complex stability is entropic in origin. Consequently, the release of water molecules or counter-ions results in positive enthalpy and entropy values in Cu(APM)2Cl22H2O – DNA interactions [33]. It should also be added that groove-binding is predominantly entropically driven, while intercalation is enthalpically driven [34]. Competitive binding between Hoechst 33258 and Cu(APM)2Cl22H2O for DNA In order to investigate the mode of the DNA binding of Cu(APM)2Cl22H2O, the competitive binding experiments were carried out. Measurements were performed using the emission intensity of Hoechst 33258 bound to DNA as a probe. Hoechst 33258 binds strongly to the minor groove of double-stranded B-DNA with specificity for AT-rich sequences [35]. It is reported that the Hoechst 33258 fluorescence emission spectral is significantly enhanced by increasing the DNA concentration [36]. The competitive binding experiment in the presence of Cu(APM)2Cl22H2O and Hoechst 33258 indicated that Cu(APM)2Cl22H2O could interact as a groove binder. To explain this, it should be considered that although Hoechst interacts to DNA with a high affinity [37], but it can be replaced by Cu(APM)2Cl22H2O. This is proved by the decrease in Hoechst-DNA solution fluorescence band which

Fig. 6. Emission spectra of the Hoechst–DNA complex [ri = [Hoechst]/[DNA] = 0.05] in the presence of increasing Cu(APM)2Cl22H2O in aqueous solution at room temperature [Cu(APM)2Cl22H2O] = 5.5, 8.2, 19, and 24  106 M1.

Fig. 7. Circular dichroism spectra of DNA (8  105 M) in 10 mM TrisAHCl buffer, in the presence of increasing amounts of Cu(APM)2Cl22H2O (ri = [complex]/ [DNA] = 0.0, 0.5, 0.9, and 1.2).

indicates Cu(APM)2Cl22H2O has ability to interact with DNA in the minor groove (Fig. 6). Circular dichroic spectral studies CD spectroscopic technique is applied to monitor the conformational variations of DNA in solution [38] and as a useful technique to analyze interactions between small molecules and DNA. Free helix DNA shows the well-known features of a so-called right-handed B form. CD spectrum: a positive band at 277 nm due to base stacking and a negative band at 245 nm due to helicity. Simple groove binding and electrostatic interaction of small molecules with DNA shows little or no perturbation of the two bands [39]. Incubation of DNA with Cu(APM)2Cl22H2O shows little perturbation of the two bands (Fig. 7), which is indicative of a non-intercalative interaction between Cu(APM)2Cl22H2O and DNA and offers support to its groove binding nature [40,41]. Viscosity measurements Viscosity experiment is an effective tool to determine the binding mode of small molecules and DNA. A classical intercalation binding requires the space of adjacent basepairs to be large enough to accommodate the bound small molecules and to elongate the

Fig. 8. Effect of increasing amounts of Cu(APM)2Cl22H2O on the viscosity of calf thymus DNA (5  105 M1) in 10 mM TrisAHCl buffer (pH 7.4) at 298 K (ri = [complex]/[DNA] = 0.0, 0.2, 0.5,0.7, 1.0, 1.3 and 1.5).

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double helix, resulting in an increase of DNA viscosity [42]. If the electrostatic or groove surface binding occurs in the binding process, the effect on the viscosity of DNA is negligible [43,44]. As a validation of the above statement, viscosity measurements were carried out. The effect of Cu(APM)2Cl22H2O on the viscosity of DNA at 25.0 °C is shown in Fig. 8. This result supports the nonintercalative binding mode of Cu(APM)2Cl22H2O with DNA. Conclusion In summary, we have studied the binding of CT-DNA with Cu(APM)2Cl22H2O complex. Spectrophotometric and spectrofluorometric methods were used to verify that the complex exhibits binding affinity for CT-DNA. The intrinsic binding constant observed (8  10+4 M1) was comparable to other groove dichroism (CD) and binder. Thermodynamic studies, 4H > 0 and 4S > 0, show that the main force acting is hydrophobic and the reaction is predominantly entropically driven, similar to the other groove binders [23] as well as the results of circular viscosimetric techniques. The hyperchromic changes observed in the UV–visible spectra are the result of DNA’s damaged double-helix structure. Therefore, we must thought about intercalation, but another result, especially competitive binding between Hoechst 33258 and Cu(APM)2Cl22H2O for DNA indicated that Cu(APM)2Cl22H2O could interact as a groove binder. In comparison with APM – DNA interaction (Kb = 5  10+4 M1) the Cu(APM)2Cl22H2O complex can bind to DNA at 1.6 order of magnitude stronger [18]. Disclosure statement No competing financial interests exist. Acknowledgment Financial support from Razi University Research Center is gratefully acknowledged. References [1] D. Eric Walters, Aspartame, A Sweet-Tasting Dipeptide. Dept. of Biochemistry and Molecular Biology Finch University of Health Sciences/The Chicago Medical School, 2001. [2] M. Soffritti, F. Belpoggi, D. DegliEsposti, L. Lambertini, Euro. J. Oncol. 10 (2005) 101–107. [3] P. Humphries, E. Pretorius, H. Naude, Euro. J. Clin. Nutr. 62 (2008) 451–462. [4] J. Donahue, The Poison In American-Made Chewing Gum, 2010, perdurabo 10 tripod.com.

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