DNA Binding and Gel Electrophoresis Studies of a Copper (II) Complex Containing Mixed Aliphatic and Aromatic Dinitrogen Ligands

Abstract

The interaction of a novel mixed ligand copper (II) complex, [Cu(N-N)(L)(EtOH)](NO₃)₂ · 2H₂O, in which N-N indicates 2,9-dimethyl-1,10-phenanthroline and L indicates N,N-dimethyltrimethylenediamine with calf thymus DNA was investigated by absorption, circular dichroism, voltammetric, and viscosimetric techniques. The absorption spectra of the complex with calf thymus DNA showed a marked hypochromism in the π → π* and metal to ligand charge transfer (MLCT) transitions, with no obvious red shift attributed to a partial intercalation. The intrinsic binding constant (K _b) was determined as 2 × 10⁵ M⁻¹. There was slight to appreciable changes in the relative viscosity of DNA, which is consistent with enhanced hydrophobic interaction of the methyl-substituted phen ring and partial intercalation mode of binding. Electrochemical studies showed a decrease in the peak current, which is ascribed to the strong binding between Cu (II) complex and DNA. The fluorescence spectral characteristics showed that the Cu (II) complex is able to displace the methylene blue bound to DNA, but not as complete as intercalative molecules. It is remarkable that this mixed ligand complex, in contrast to [Cu(2,9-dmp)₂]⁺ (2,9-dmp = 2,9-dimethyl-1,10-phenanthroline), which fails to cleave DNA, has ability to cleave the supercoiled plasmid DNA.

Introduction

T he interaction of transition metal polypyridyl and mixed ligand complexes with DNA has been extensively studied during the past several years, as their unusual binding properties, combined with their general photoactivity, make them suitable candidates as probes for DNA secondary structure, photocleavers, and antitumor drugs (Wang and Okabe, 2005; Ramakrishna Rao et al., 2009; Wang et al., 2009). The most important factor driving the overall binding of these molecules to DNA appears to be the match of shape and overlap between metal complex and the B-DNA helix. The application of mixed ligand complexes permits variation in the geometry, size, and hydrophobicity of the ligands and their substituents or in the metal, thereby providing an opportunity to determine and also understand how these factors contribute to the affinity in DNA binding and cleavage (Zhang et al., 2004; Ramakrishna Rao et al., 2007). Redox-active coordination complexes of either synthetic or natural origin that induce DNA cleavage have been studied as models for site-specific reactions and are useful tools in modern molecular biology and medicine. Thus, several copper (II) (Mahadevan and Palaniandavar, 1997; Santra et al., 2002) complexes have been interacted with DNA. Recently, there has been substantial interest in the design and study of DNA binding (Chikira et al., 2002) and cleavage properties of mixed ligand Cu (II) complexes (García et al., 2003; Reddy et al., 2004; Selvakumar et al., 2006; Barceló et al., 2007; Rajendiran et al., 2007) and development of the complexes as metallodrugs. Copper complexes have been extensively utilized in metal-mediated DNA cleavage for the generation of activated oxygen species (Nagane et al., 2000; Liu et al., 2003). It has been reported that tetraaza macrocyclic copper complexes have shown anti-HIV activities, and further, copper accumulates in tumors because of the selective permeability of cancer cell membranes to copper compounds. For this reason, a number of copper complexes were screened for anticancer activity and some of them were found active both in vivo and in vitro (Singh and Singh, 2002). Other complexes such as copper (II) phenanthroline l-threonine (Zhang et al., 2004) and copper (II) phenanthroline diisopropyl salicylato (Ranford et al., 1993) exhibit cytotoxicity and antiviral activity. Similarly, complexes of the types [Cu(phen)(edda)] (Ng et al., 2008) and [Cu(phen)(N-propylnorfloxacin) Cl] (Rossi et al., 2005; Katsarou et al., 2008) exhibit anticancer and antileukemic activities, respectively, indicating that the synergy between the metal and ligand results in enhancement of their antiproliferative properties.

Therefore, to design improved drugs that target cellular DNA, to understand the mechanism of action at the molecular level, and also to investigate the effect of mixed ligands on the structure and conformation of DNA, we used a new water-soluble copper (II) complex, [Cu(N-N)(L)(EtOH)](NO₃)₂ · 2H₂O, in which N-N indicates 2,9-dimethyl-1,10-phenanthroline and L indicates N,N-dimethyltrimethylenediamine (Shahabadi and Maghsudi, 2009), and binding studies of this potential drug complex, [Cu(N-N)(L)(EtOH)](NO₃)₂, with calf thymus DNA (CT-DNA) were carried out by electronic absorption spectroscopy, florescence spectroscopy, cyclic voltammetric, circular dichroic spectral, and viscosity measurements.

The biological importances of this system are as follows: (i) the existence of copper (II) ion, which is supposed to play an important role in biological systems, such as toxicity against microorganisms, especially to protozoa, fungi, and algae, and also has antiseptic properties (Malik et al., 1979); (ii) the existence of 2,9-dimethyl-1,10-phenanthroline ligand, whose complexes possess interesting anticancer properties (Papadia et al., 2005); and (iii) this system containing mixed ligands and this kind of complexes play key roles in some biological processes (Sigel, 1975).

Materials and Methods

Materials

N,N-Dimethyltrimethylenediamine (L), ethylenediaminetetraacetic acid (EDTA), Tris-HCl, NaOH, sodium dodecyl sulphate, potassium acetate, chloroform, isoamylalcohol, methylene blue (MB), dimethyl sulfoxide (DMSO), KI, MeOH, sucrose, and agarose were purchased from Merck (Darmstadt, Germany). 2,9-Dimethyl-1,10-phenanthroline was purchased from Riedel-deHaën (Hanover, Germany). RNase A was purchased from Roche (Bielefeld, Germany). Doubly distilled deionized water was used throughout the experiment. Highly polymerized CT-DNA and HEPES buffer were purchased from Sigma. Plasmid DNA (pUC18) was extracted from Escherichia coli.

Experiments were carried out in HEPES buffer at pH 7.2. Solutions of CT-DNA gave an UV absorbance ratio (260 over 280 nm) of more than 1.8, indicating that the DNA was sufficiently free of protein. The stock solution of CT-DNA was prepared by dissolving DNA in 10 mM HEPES buffer at pH 7.2. The DNA concentration (monomer units) of the stock solution (1 × 10⁻² M per nucleotide) was determined by UV spectrophotometry in properly diluted samples using a molar absorption coefficient of 6600 M⁻¹ cm⁻¹ at 258 nm (Kennedy and Bryant, 1986). The stock solutions were stored at 4°C and used within 4 days.

Synthesis of copper (II) complex

The Cu (II) complex [Cu(N-N)(L)(EtOH)](NO₃)₂ · 2H₂O, in which N-N indicates 2,9-dimethyl-1,10-phenanthroline and L indicates N,N-dimethyltrimethylenediamine, was synthesized by a previously reported procedure (Shahabadi and Maghsudi, 2009). (Fig. 1)

FIG. 1.

Structure of the [Cu(2,9-dimethyl-phenanthroline)(N,N-dimethyltrimethylene diamine)(EtOH)](NO₃)₂ · 2H₂O complex.

Analytical data (%) for CuC₂₁H₃₅N₆O₉, found (calculated): C, 44.1 (43.56); H, 6.5 (6.11); N, 14.9 (14.51). ¹H NMR (200 MHz, CDCl₃): aromatic dinitrogen ligand: 8.48 (d, 2H), 8.01 (s, 2H), 7.75 (d, 2H), 2.39 (s, 6H); aliphatic diamine ligand: 2.46 (s, 6H), 0.87 (t, 2H), 1.21 (t, 2H), 1.28 (q, 2H), 4.17 (br NH2). ¹³C NMR (200 MHz, CDCl₃): aromatic dinitrogen ligand: 125.61, 127.64, 128.79, 130.95, 143.03, 157.62 (six carbon atoms of phen ligand), 25.88 (Me groups of phen ligand); aliphatic dinitrogen ligand: 22.96, 29.68, 25.88, 14.06 (three carbon atoms and Me groups, respectively).

Isolation of plasmid DNA and gel electrophoresis

A pure culture of E. coli plasmid DNA was incubated at 37°C for 12 h in a nutrient broth containing ampicillin. The broth was harvested after 12 h, centrifuged for 5 min at 4000 rpm, and decanted, and the entire medium was drained. The pellet was resuspended in 1 mL SET (sucrose, EDTA, Tris-HCl) buffer by vortexing. A 2 mL solution of NaOH and sodium dodecyl sulphate buffer was added, mixed well, kept on ice for 5 min, and then 1.5 mL of potassium acetate solution (5 M) was added and mixed immediately. After 5 min, 4.5 mL chloroform:isoamylalcohol (24:1) was added and centrifuged for 10 min at 8000 rpm at 4°C. The supernatant was collected in a new tube, 10 mL of 100% EtOH was added, and centrifuged for 5 min at 10,000 rpm. Then the pellet was washed with 5 mL of 70% EtOH, centrifuged for 5 min at 5000 rpm, and air dried. The pellet was dissolved in Tris-HCl–EDTA (TE) buffer (near 200 μL), 50 μL RNase A (1 mg/mL in TE) was added, and then incubated at 37°C for 1–2 h. The homogeneity of plasmid DNA was checked by gel electrophoresis and stored at −20°C until use. The DNA complex and the solution of plasmid DNA (control) was loaded in 0.8% agarose gel with the addition of 3 μL of loading dye (bromophenol blue). It was allowed to run for 1 h at 80 V. After electrophoresis, the gel was stained with ethidium bromide and photographed under UV light.

Instrumentation

¹H NMR spectra were recorded using a Bruker Avance DPX 200 MHz (4.7 tesla) spectrometer (Bruker Rheinstetten, Germany) with CDCl₃ as the solvent. The elemental analysis was performed using a Heraeus CHN elemental analyzer. Absorbance spectra were recorded using an HP spectrophotometer (Agilent 8453; Agilent, Santa Clara, CA) equipped with a thermostated bath (Huber Polystat cc1, Huber, Offenburg, Germany). Absorption titration experiments were conducted by keeping the concentration of Cu (II) complex constant (5 × 10⁻⁵ M) while varying the DNA concentration from 0 to 3.5 × 10⁻⁴ M (r _i = [DNA]/[Cu (II) complex] = 0–7). Absorbance values were recorded after each successive addition of DNA solution, followed by an equilibration period.

CD measurements were recorded on a JASCO (J-810) spectropolarimeter (JASCO, Osaka, Japan) by keeping the concentration of DNA constant (8 × 10⁻⁵ M) while varying the Cu (II) complex concentration from 0 to 8 × 10⁻⁶ M (r _i = [Cu (II) complex]/[DNA] = 0.0, 0.005, 0.03, 0.07, and 0.1).

Viscosity measurements were made using a viscosimeter (SCHOTT AVS 450, SCHOTT, Mainz, Germany), which was maintained at 25°C ± 0.5°C using a constant temperature bath. The DNA concentration was fixed at 5 × 10⁻⁵ M, and flow time was measured with a digital stopwatch. The mean values of three replicated measurements were used to evaluate the viscosity η of the samples. The values for relative specific viscosity (η/η ₀)^1/3, where η ₀ and η are the specific viscosity contributions of DNA in the absence (η ₀) and in the presence of the Cu (II) complex (η), respectively, were plotted against r _i (r _i = [DNA]/[Cu (II) complex]).

The cyclic voltammetry, linear sweep voltammetry, and differential pulse voltammetry (DPV) measurements were performed using an AUTOLAB model (PGSTAT C; Filderstadt, Germany), with a three-electrode system: a 0.10-cm-diameter glassy carbon disk as working electrode, an Ag/AgCl electrode as reference electrode, and a Pt wire as counter electrode. Electrochemical experiments were carried out in a 25-mL voltammetric cell at RT. All potentials are referred to the Ag/AgCl reference. Their surfaces were freshly polished with 0.05-mm alumina prior to each experiment and were rinsed using double-distilled water between each polishing step. The supporting electrolyte was 10 mM HEPES buffer solution (pH 7.2), which was prepared with double-distilled water. Cyclic voltametry (CV) and DPV measurements were recorded by keeping the concentration of Cu (II) complex constant (5 × 10⁻⁴ M) while varying the DNA concentration from 0 to 7.86 × 10⁻⁵ in CV measurements and from 0 to 7.88 × 10⁻⁵ M in DPV measurements. The current potential curves and experimental data were recorded on the software GPES (Ni et al., 2006; Kashanian et al., 2008).

Fluorescence measurements were carried out with a JASCO spectrofluorimeter (FP 6200). DNA (5 × 10⁻⁵ M) was pretreated with MB (5 × 10⁻⁶ M) for 30 min. Then Cu (II) complex was added to this mixture (from 5 × 10⁻⁵ to 1.5 × 10⁻⁴ M) and diluted to the volume with HEPES buffer, and after the incubation time the effect on emission intensity was measured. The samples were excited at 630 nm and emission was observed between 630 and 680 nm.

Reactions of Cu (II) complex with pUC18 (0.33 mg mL⁻¹) were performed in TE buffer (pH 8) and the contents were incubated for 2 h at 37°C. Concentration dependence studies were performed using Cu (II) complex ratios (r _i = [Cu(II) complex]/[DNA] = 0.00, 0.01, 0.03, 0.04 and 0.05). Applying 80 V for 1 h in Tris, Borate, EDTA (TBE) buffer solution with agarose gels (0.8% agarose), the DNA reactions were analyzed. Gel was stained with ethidium bromide and photographed using UV illumination. In control experiments, DMSO, KI, and MeOH were added to reaction mixtures.

Results and Discussion

Electronic spectral studies

The binding of intercalative drugs to DNA helix has been characterized classically through absorption spectral titrations, as a function of added DNA. Complex binding with DNA through intercalation usually results in hypochromism and bathchromism (red shift) due to the intercalative mode involving a strong stacking interaction between an aromatic chromophore and the base pairs of DNA. The extent of the hypochromism commonly parallels the intercalative binding strength.

The absorption spectra of the Cu (II) complex (Fig. 2) displays an intense intraligand π → π* absorption at λ = 274 nm and metal to ligand charge transfer (MLCT) band at λ = 455 nm (Shahabadi and Maghsudi, 2009).

FIG. 2.

Electronic absorption spectra for the titration of 5.0 × 10⁻⁵ M Cu (II) complex with DNA (r _i = 0.00, 1.5, 2, 3, 3.5, 5, and 7). Arrow shows the changes in absorbance after DNA addition.

In the presence of increasing concentrations of CT-DNA, a marked hypochromism in the π → π* and MLCT transitions is apparent with no obvious red shift. The extent of hypochromism in the spectrum at λ = 455 nm was measured using the following equation (% H = 22.97%): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}{\rm H } \% = \frac {A_ {\rm Free} - A_{\rm Bounded}} {A_{\rm Free}} \times 100 \end{align*} \end{document}

A large degree of hypochromism in the spectrum in the presence of double-helical DNA is a typical characteristic of interactions between DNA and Cu (II) complex, and it arises from the strong stacking interactions between the aromatic chromophore and the base pairs. Strong intercalative binding of small molecules to the DNA are known to cause large bathochromic shifts on the spectral bands (Nair et al., 1998). Therefore, comparing the hypochromism and bathochromism of the titled complex with other complexes (Tan et al., 2005; Tan and Chao, 2007), it was found that it binds to DNA via partial intercalative mode.

To further investigate the intensity of the interaction between the Cu (II) complex and CT-DNA, the intrinsic binding constant, k _b, was calculated using the equation 1 (Wolfe et al., 1987) (Fig. 3). \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} [{\rm DNA}] / ( \varepsilon_{\rm a} - \varepsilon_{\rm f}) = [{\rm DNA}] / (\varepsilon_{\rm b} - \varepsilon_{\rm f}) + 1 / K_{\rm b} (\varepsilon_{\rm b} - \varepsilon_{\rm f} ) \tag{1} \end{align*} \end{document}

FIG. 3.

Spectrophotometric titration of Cu (II) complex at 455 nm with calf thymus DNA in HEPES buffer.

where ɛ _a, ɛ _b, and ɛ _f are the apparent, bound, and free complex extinction coefficients, respectively. In particular, ɛ _f was determined by a calibration curve of the isolated Cu (II) complex in aqueous solution, following Beer's law. ɛ _a was determined as the ratio between the measured absorbance and the Cu (II) complex concentration, A _obs/[Cu (II) complex]. A plot of [DNA]/(ɛ _a − ɛ _f) vs. [DNA] gives a slope of 1/(ɛ _b − ɛ _f) and a y-intercept equal to 1/K _b (ɛ _b − ɛ _f); K _b is the ratio of the slope to the y-intercept (Fig. 3).

The K _b value obtained was 2.9 × 10⁴ M⁻¹. In comparing the intrinsic binding constant (K _b) of the Cu (II) complex with some DNA partial intercalative, such as [Cu(dpq)₃](ClO₄)₂ (7.5 × 10⁴ M⁻¹) (Ramakrishnan and Palaniandavar, 2008), [Cu(phen)₃](ClO₄)₂ (9.6 × 10³ M⁻¹) (Ramakrishnan and Palaniandavar, 2008), homodinuclear macrocyclic complex of copper(II) (4.25 × 10⁴ M⁻¹) (Arjmand and Aziz 2009), the hexaaza macrocyclic copper(II) complex (Cu(II)l-1,8-dihydroxyethyl-1,3,6,8,10,13-hexaazacyclotetradecane copper(II) perchlorate monohydrate) (2.4 × 10⁴ M⁻¹) (Kang et al. 2006), and [Ru(phen)₂NMPIP]²⁺ (2.1 × 10⁴ M⁻¹) (Tan et al., 2005), we can deduce that Cu (II) complex binds to CT-DNA via a partial intercalation mechanism.

Viscosity measurements

In the absence of crystallographic structural data, hydrodynamic measurements, which are sensitive to length changes, are the least ambiguous and most critical tests for confirming a classical intercalation model in solution. A classical intercalative mode causes a significant increase in viscosity for the DNA solution because of the increase in separation of base pairs at intercalation sites and hence an increase in overall DNA length. By contrast, complexes that binds exclusively in the DNA grooves by partial and/or nonclassical intercalation, under the same conditions, typically cause less pronounced (positive or negative) or no change in DNA solution viscosity (Kelly et al., 1985; Meadows et al., 1993). To further explore the binding mode of Cu (II) complex, viscosity measurements were carried out on CT-DNA bound to varying concentrations of Cu (II) complex. The value of the relative specific viscosity (η/η ₀)^1/3 versus r _i (r _i = [Cu (II) complex]/[DNA]) was plotted in the absence and presence of Cu (II) complex (Fig. 4). The results showed that it exerts essentially no effect on DNA viscosity, which is indicative of a mode of binding involving partial intercalation.

FIG. 4.

Effect of increasing amounts of Cu (II) complex on the viscosity of calf thymus DNA (5 × 10⁻⁵ M) in 10 mM HEPES buffer (r _i = 0, 0.05, 0.2, 0.3, and 0.4).

In addition, some partial intercalators, such as Cu (II) tris(2,2′-bipyridine) (Arias et al., 2009), mixed ligand copper (II) maltolate complexes (Barve et al., 2009) were shown to give the same results like Cu (II) complex.

Electrochemical studies

Recently, electrochemical techniques were used as a simple and rapid method to study DNA interaction with different compounds (Kashanian and Ezzati Nazhad Dolatabadi, 2009, Mahadevan and Palaniandavar, 1998). The cyclic voltammogram of 5 × 10⁻⁴ M Cu (II) complex in the absence and presence of CT-DNA is shown in Figure 5. The cathodic peak potential (E _pc) and the anodic peak potential (E _pa) in the absence of DNA are 0.398 and 0.542 V, respectively. The formal potential (E _1/2), taken as the average of E _pc and E _pa, is 0.470 V in the absence of DNA. When CT-DNA was added to a solution of Cu (II) complex, both the anodic and cathodic peak current heights of the Cu (II) complex decreased (Fig. 5). This suggested that there existed interaction between Cu (II) complex and DNA (Liu et al., 2002; Jiao et al., 2005). The decrease in current may be attributed to the diffusion of the complexes bound to the large, slowly diffusing DNA molecule. The decrease in the peak current is ascribed to the strong binding between Cu (II) complex and DNA (Xu et al., 2008).

FIG. 5.

Cyclic voltammograms for Cu (II) complex in the presence of DNA at different concentrations. C _{Cu (II) complex} = 5 × 10⁻⁴ M; C _DNA = 0.00, 4.45 × 10⁻⁶, 2.67 × 10⁻⁵, 3.99 × 10⁻⁵, 7.01 × 10⁻⁵, and 7.86 × 10⁻⁵ for curves 1–5, respectively. Arrows show the changes in the cathodic and anodic peak current heights of Cu (II) complex after DNA addition.

Also the Cu (II) complex interaction with DNA at the glassy carbon electrode was followed by DPV (Fig. 6). The voltammograms indicate that the peak current of Cu (II) complex decreased as DNA was added, which was consistent with the results of cyclic voltammetry.

FIG. 6.

Differential pulse voltammograms for Cu (II) complex (C _{Cu (II) complex} = 5 × 10⁻⁴ M) in the presence of different concentrations of DNA; C _DNA = 0.00, 2.23 × 10⁻⁵, 3.55 × 10⁻⁵, 4.88 × 10⁻⁵, 6.5 × 10⁻⁵, and 7.88 × 10⁻⁵ M for curves 1–6, respectively.

Circular dichroic spectral studies

A solution of CT-DNA exhibits a positive band (275 nm) from base stacking interactions and a negative band (245 nm) from the right-handed helicity of DNA (Lincoln et al., 1997). Classical intercalation reactions tend to enhance the intensities of both bands because of strong base stacking interactions and stable DNA conformations (right-handed B conformation of CT-DNA), whereas simple groove binding and electrostatic interactions with small molecules show less of a perturbation or no perturbation whatsoever on the base stacking and helicity bands (Norden and Tjerneld, 1982).

The interaction between the Cu (II) complex and CT-DNA was studied using circular dichroism (CD) spectroscopy (Fig. 7). This complex showed changes in both the positive and negative bands, which are characteristic of partial intercalative interaction of the heterocyclic rings of the complex. The observed increase and decrease, respectively, in the positive and negative bands is consistent with a B to A conformational change (Rajendiran et al., 2007).

FIG. 7.

Circular dichroism spectra of DNA (8.0 × 10⁻⁵ M) in 10 mM HEPES buffer in the presence of increasing amounts of Cu (II) complex (r _i = [Cu (II) complex]/[DNA] = 0.0, 0.005, 0.03, 0.07, and 0.1). Arrows show the changes after Cu (II) complex addition.

Fluorescence studies (competitive binding studies)

To further clarify the nature of the interaction between Cu (II) complex and DNA, competitive MB displacement assay studies were undertaken. The experiment involves the addition of the present complex to CT-DNA pretreated with MB as a fluorescence probe ([DNA]/[MB] = 10). Interestingly, the emission intensity of MB is quenched on adding CT-DNA. This emission quenching phenomenon reflects the change in the excited state structure in consequence of the electronic interaction in the MB–DNA complexes (Long and Barton, 1990). The emission quenching phenomenon and the hypochromic and red shift effects in the absorption spectra fit the intercalative mode of MB to DNA.

The emission spectra of the MB–DNA solutions in the presence of increasing amounts of Cu (II) concentrations (r _i = 1–3) is shown in Figure 8, which clearly reveals an increase in the fluorescence intensity of the probe molecule on adding the Cu (II) complex. In the case of the highest concentration of Cu (II) complex, the emission intensity of the MB–DNA complex could not approach that of pure MB. The increase in fluorescence intensity should be due to a greater amount of free MB molecules in solution. Considering that MB molecules have been already intercalated into the DNA helix, these results indicate that some MB molecules are released from the DNA nucleobases after addition of the Cu (II) complex. That is, the formation of metal complex–DNA prevents the binding of MB. The increase in fluorescence intensity and the extent of MB release are applied to compare the strength of binding. Figure 8 shows that our complex could not release MB completely. Therefore, this experiment confirms our previous theories and reconfirms our prior evidence-based conclusions.

FIG. 8.

Emission spectra of MB–DNA complexes in the presence of increasing amounts of Cu (II) complex (r _i = [complex]/[DNA] = 0.0 (MB + DNA), 1, 1.5, 1.7, 2.0, 2.5, and 3). MB, methylene blue. Arrows show the emission spectrum of MB solution and the changes in the DNA emission spectrum after Cu (II) complex addition.

Cleavage of pUC18 DNA Cu (II) complex

Transition metal-mediated radical production may result in efficient DNA cleavage. DNA cleavage was controlled by relaxation of the supercoiled circular form of pUC18 into the nicked circular and linear forms. When circular plasmid DNA is run on horizontal gel using electrophoresis, the fastest migration will be observed for the supercoiled form (Form I). If one strand is cleaved, the supercoils will relax to produce a slower-moving open circular form (Form II). If both strands are cleaved, a linear form (Form III) will be generated, which migrates in between.

The ability of Cu (II) complex to perform DNA cleavage is generally monitored by agarose gel electrophoresis and the pUC18 plasmid DNA is involved. Agarose gel electrophoresis is shown in Figure 9. As shown in this figure, two clear bands were observed for the control in which the Cu (II) complex was absent (lane 0). The relatively fast migration is the intact supercoil form (Form I) and the slower moving migration is the open circular form (Form II), which was generated from the supercoiled form when scission occurred on its one strand (Patra et al., 2005; Gao et al., 2006). The Cu (II) complex at different concentrations is able to perform cleavage of pUC18 plasmid DNA (lanes 1, 2, 3, and 4; Fig. 9). The amount of Form I diminished gradually and partly converted to Form II, whereas the intensity of the Form II band increased as the concentration of the Cu (II) complex increased. It is obvious that the Cu (II) complex has ability to cleave the supercoiled plasmid DNA and this cleavage system does not require addition of any external agents. In control experiments, radical scavengers such as DMSO, KI, and MeOH were added to the reaction mixtures, which did not show any apparent effect on the DNA cleavage activity. These observations suggested that DNA cleavage reaction did not proceed via radical mechanism and indicated the hydrolytic nature of the cleavage. Further studies on the reaction mechanism are in progress.

FIG. 9.

Cleavage of pUC18 DNA in the presence of increasing amounts of Cu (II) complex (r _i = [Cu(II) complex]/[DNA] = 0.00, 0.01, 0.03, 0.04, and 0.05): lane 0 was DNA alone (r _i = 0.00), lane 1 (r _i = 0.01), lane 2 (r _i = 0.03), lane 3 (r _i = 0.04), and lane 4 (r _i = 0.05).

Conclusion

We examined the interaction of a water-soluble Cu (II) complex to CT-DNA and found sufficient evidence for its binding mode. We view this as a significant finding because of the ubiquitous medical roles found for phenanthroline ligands. 2,9-Dimethyl substitution strongly interferes with the partial intercalation of the complex and the results of this study prove it again. The main finding is that this mixed ligand complex, in contrast to [Cu(2,9-dmp)₂]⁺ (2,9-dmp = 2,9-dimethyl-1,10-phenanthroline), which fails to cleave DNA (Sigman et al., 1979), has ability to cleave the supercoiled plasmid DNA and it could be useful in molecular biology and drug design.

Footnotes

Acknowledgment

The financial support from Razi University Research Center is gratefully acknowledged.

Disclosure Statement

No competing financial interests exist.

References

Arias

M.S.

, González-Álvarez

, Fernández

M.J.

, Lorente

, Alzuet

, Borrás

2009. Synthesis and copper-mediated nuclease activity of a tetracationic tris(2,2′-bipyridine) ligand. J Inorg Biochem, 103:1067–1073.

Arjmand

, Aziz

2009. Synthesis and characterization of dinuclear macrocyclic cobalt(II), copper(II) and zinc(II) complexes derived from 2,2,2',2'-S,S[bis (bis-N, N-2-thiobenzimidazolyloxalato-1,2-ethane)]: DNA binding and cleavage studies. Eur J Med Chem, 44:834–844.

Barceló-Oliver

, García-Raso

Ä.

, Terrón

A.Ä.

, Molins

, Prieto

M.J.

, Moreno

, Martínez

, Lladó

, López

, Gutiérrez

, Escribá

P.V.

2007. Synthesis and mass spectroscopy kinetics of a novel ternary copper(II) complex with cytotoxic activity against cancer cells. J Inorg Biochem, 101:649–659.

Barve

, Kumbhar

, Bhat

, Joshi

, Butcher

, Sonawane

, Joshi

2009. Mixed-ligand copper(II) maltolate complexes: synthesis, characterization, DNA binding and cleavage, and cytotoxicity. Inorg Chem, 48:9120–9132.

Chikira

, Tomizava

, Fukita

, Sugizaki

, Sugawara

, Yamazaki

, Sasano

, Shindo

, Palaniandavar

, Anthroline

W.E.J.

2002. DNA-fiber EPR study of the orientation of Cu(II) complexes of 1,10-phenanthroline and its derivatives bound to DNA: mono(phenanthroline)-copper(II) and its ternary complexes with amino acids. Inorg Biochem, 89:163–173.

Gao

, Sun

, Liu

, Duan

2006. An anticancer metallobenzylmalonate:crystal structure and anticancer activity of a palladium complex of 2,20-bipyridine and benzylmalonate. J Coord Chem, 59:1295–1300.

García-Raso

AÄ.

, Fiol

J.J.

, Adrover

, Moreno

, Mata

, Espinso

, Molins

2003. Synthesis, structure and nuclease properties of several ternary copper(II) peptide complexes with 1,10-phenanthr. J Inorg Biochem, 95:77–86.

Jiao

, Wang

Q.X.

, Sun

, Jian

F.F.

2005. Synthesis, characterization and DNA-binding properties of a new cobalt(II) complex: Co(bbt)₂Cl₂ . J Inorg Biochem, 99:1369–1375.

Kang

, Dong

, Lu

, Su

, Wu

, Sun

2006. Study on DNA cleavage by the hexaaza macrocyclic copper(II) complex. J Macromol Sci Part A: Pure Appl Chem, 43:279–288.

10.

Kashanian

, Ezzati Nazhad Dolatabadi

2009. DNA binding studies of 2-tert-butylhydroquinone (TBHQ) food additive. Food Chem, 116:743–747.

11.

Kashanian

, Gholivand

M.B.

, Ahmadi

, Ravan

2008. Interaction of diazinon with DNA and the protective role of selenium in DNA damage. DNA Cell Biol, 27:325–332.

12.

Katsarou

, Efthimiadou

, Psomas

, Karaliota

, Vourloumis

2008. Novel copper(II) complex of N-propyl-norfloxacin and 1,10-phenanthroline with enhanced antileukemic and DNA nuclease activities. J Med Chem, 51:470–478.

13.

Kelly

T.M.

, Tossi

A.B.

, McConnell

D.J.

, OhUigin

1985. A study of the interactions of some polypyridylruthenium(II) complexes with DNA using fluorescence spectroscopy, topoisomerization and thermal denaturation. Nucleic Acids Res, 13:6017–6034.

14.

Kennedy

S.D.

, Bryant

R.G.

1986. Manganese-deoxyribonucleic acid binding modes. Nuclear magnetic relaxation dispersion results. Biophys J, 50:669–676.

15.

Lincoln

, Tuite

, Norden

1997. Hybrid electrochemical/chemical synthesis of supported, luminescent semiconductor nanocrystallites with size selectivity: copper(I) iodide. J Am Chem Soc, 119:1454–1455.

16.

Liu

, Zhang

, Chen

, Deng

, Lu

, Ji

2003. Interaction of macrocyclic copper(II) complexes with calf thymus DNA: effects of the side chains of the ligands on the DNA-binding behaviors. Dalton Trans, 1:114–119.

17.

Liu

, Zhang

, Lu

, Qu

, Zhou

, Zhang

, Ji

2002. DNA-binding and cleavage studies of macrocyclic copper(II) complexes. J Inorg Biochem, 91:269–276.

18.

Long

E.C.

, Barton

J.K.

1990. On demonstrating DNA interaction. Acc Chem Res, 23:271–273.

19.

Mahadevan

, Palaniandavar

1997. Electrochemical study of the enantioselective interaction of Tris(phen)Ru (II) with calf thymus DNA. Inorg Chim Acta, 254:291–302.

20.

Mahadevan

, Palaniandavar

1998. Spectroscopic and voltammetric studies on copper complexes of 2,9-dimethyl-1,10-phenanthrolines bound to calf thymus DNA. Inorg Chem, 37:693–700.

21.

Malik

G.S.

, Singh

S.P.

, Tandon

J.P.

1979. Mixed ligand complexes involving ligands of biological importance [Cu(II)-1,10-phenanthroline or 2,2′-bipyridinl-α-Aminoacids] Moatshefte für Chemie, 110:149–155.

22.

Meadows

K.A.

, Liu

, Sou

, Hudson

P.B.

, McMillin

D.R.

1993. Spectroscopic and photophysical studies of the binding interactions between copper phenanthroline complexes and RNA. Inorg Chem, 32:2919–2923.

23.

Nagane

, Chikira

, Oumi

, Shindo

, Antholine

W.E.

2000. How amino acids control the binding of Cu(II) ions to DNA: part III. A novel interaction of a histidine complex with DNA. J Inorg Biochem, 78:243–249.

24.

Nair

R.B.

, Teng

E.S.

, Kirkland

S.L.

, Murphy

C.J.

1998. Synthesis and DNA-binding properties of [Ru(NH₃)₄dppz]²⁺ Inorg Chem, 37:139–141.

25.

C.H.

, Kong

K.C.

, Von

S.T.

, Balraj

, Jensen

, Thirthagiri

, Hamada

, Chikira

2008. Synthesis, characterization, DNA-binding study and anticancer properties of ternary metal(II) complexes of edda and an intercalating ligand. Dalton Trans, 4:447–454.

26.

, Lin

, Kokat

2006. Synchronous fluorescence, UV–visible spectrophotometric, and voltametric studies of the competitive interaction of bis(1,10-phenanthroline)copper(II) complex and neutral red with DNA. Anal Biochem, 352:231–242.

27.

Norden

, Tjerneld

1982. Structure of methylene-blue DNA complexes studied by linear and circular-dichroism spectroscopy. Biopolymers, 21:1713–1734.

28.

Papadia

, Margiotta

, Bergamo

, Sava

, Natile

2005. Platinum(II) complexes with antitumoral/antiviral aromatic heterocycles: effect of glutathione upon in vitro cell growth inhibition. J Med Chem, 48:3364–3371.

29.

Patra

A.K.

, Dhar

, Nethaji

, Chakravarty

A.R.

2005. Metal-assisted red light-induced DNA cleavage by ternary l-methionine copper(II) complexes of planar heterocyclic bases. Dalton Trans, 5:896–902.

30.

Rajendiran

, Karthik

, Palaniandavar

, Stoeckli-Evans , Periasamy

V.S.

, Akbarsha

M.A.

, Srinag

B.S.

, Krishnamurthy

2007. Mixed-Ligand copper(II)-phenolate complexes: effect of coligand on enhanced DNA and protein binding, DNA cleavage, and anticancer activity. Inorg Chem, 46:8208–8221.

31.

Ramakrishnan

, Palaniandavar

2008. Interaction of rac-[Cu(diimine)₃]²⁺ and rac-[Zn(diimine)₃]²⁺ complexes with CT DNA: effect of fluxional Cu(II) geometry on DNA binding, ligand-promoted exciton coupling and prominent DNA cleavage. Dalton Trans, 29:3866–3878.

32.

Ramakrishna Rao , Patra

A.P.

, Chetana

P.R.

2007. DNA binding and oxidative cleavage activity of ternary (l-proline)copper(II) complexes of heterocyclic bases. Polyhedron, 26:5331–5338.

33.

Ramakrishna Rao , Patra

A.P.

, Chetana

P.R.

2009. Synthesis, structure, DNA binding and oxidative cleavage activity of ternary (l-leucine/isoleucine) copper(II) complexes of heterocyclic bases. Polyhedron, 27:1343–1352.

34.

Ranford

J.D.

, Sadler

P.J.

, Tocher

D.A.

1993. Cytotoxicity and antiviral activity of transition-metal salicylato complexes and crystal structure of bis (diisopropylsalicylato) (1,10-phenanthroline) copper (II) J Chem Soc Dalton Trans, 22:3393–3399.

35.

Reddy

P.A. N.

, Nethaji

, Chakravarty

A.R.

2004. Hydrolytic cleavage of DNA by ternary amino acid Schiff base copper(II) complexes having planar heterocyclic ligands. Eur J Inorg Chem, 7:1440–1446.

36.

Rossi

L.M.

, Neves

, Bortoluzzi

A.J.

, Horner

, Szpoganicz

, Terenzi

, Mangrich

A.S.

, Pereira-Maia

, Castellano

E.E.

, Haase

2005. Synthesis, structure and properties of unsymmetrical μ-alkoxo-dicopper(II) complexes: biological relevance to phosphodiester and DNA cleavage and cytotoxic activity. Inorg Chim Acta, 358:1807–1822.

37.

Santra

B.K.

, Reddy

P.A.N.

, Neelakanta

, Mahadevan

, Nethaji

, Chakravarty

A.R.

2002. Oxidative cleavage of DNA by a dipyridoquinoxaline copper(II) complex in the presence of ascorbic acid. J Inorg Biochem, 89:191–196.

38.

Selvakumar

, Rajendiran

, Maheswari

P.U.

, Evans

H.S.

, Palaniandavar

2006. Structures, spectra, and DNA-binding properties of mixed ligand copper(II) complexes of iminodiacetic acid: The novel role of diimine co-ligands on DNA conformation and hydrolytic and oxidative double strand DNA cleavage. J Inorg Biochem, 100:316–330.

39.

Shahabadi

, Maghsudi

2009. Binding studies of a new copper (II) complex containing mixed aliphatic and aromatic dinitrogen ligands with bovine serum albumin using different instrumental methods. J Mol Struct, 929:193–199.

40.

Sigel

1975. Ternary Cu²⁺ complexes: stability, structure, and reactivity. Angew Chem Int Ed, 14:394–402.

41.

Sigman

D.S.

, Graham

D.R.

, Aurora

V.D.

, Stern

A.M.

1979. Oxygen-dependent cleavage of DNA by the 1,10-phenanthroline-cuprous complex. J Biol Chem, 254:12269–12272.

42.

Singh

N.K.

, Singh

S.B.

2002. Antitumor and Immunomodulatory effects of Cu(II) complexes of thiobenzhydrazide. Met Based Drugs, 9:109–118.

43.

Tan

L.F.

, Chao

2007. DNA-binding and photocleavage studies of mixed polypyridyl ruthenium(II) complexes with calf thymus DNA. Inorg Chim Acta, 360:2016–2022.

44.

Tan

L.F.

, Chao

, Liu

Y.J.

, Li

, Sun

, Ji

L.N.

2005. DNA-binding and photocleavage studies of [Ru(phen)₂(NMIP)]²⁺ Inorg Chim Acta, 358:2191–2198.

45.

Wang

, Lin

, Hong

, Lu

, Li

, Okabe

, Odoko

2009. Synthesis, structures and DNA binding of ternary transition metal complexes M(II)L with the 2,2′-bipyridylamine (L = p-aminobenzenecarboxylic acid, M = Ni, Cu and Zn) Inorg Chim Acta, 362:377–384.

46.

Wang

, Okabe

2005. Synthesis, structures and DNA binding of ternary transition metal complexes M(II)L with the 2,2^′-bipyridylamine (L = p-HB, M = Co, Ni, Cu and Zn) Inorg Chim Acta, 358:3407–3416.

47.

Wolfe

, Shimer

G.H.

, Meehan

1987. Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA. Biochemistry, 26:6392–6396.

48.

Z.H.

, Xi

P.X.

, Chen

F.J.

, Liu

X.H.

, Zeng

Z.z.

2008. Synthesis, characterization, and DNA-binding properties of copper(II), cobalt(II), and nickel(II) complexes with salicylaldehyde 2-phenylquinoline-4-carboylhydrazone. Transit Met Chem, 33:267–273.

49.

Zhang

, Zhu

, Tu

, Wei

, Yang

, Lin

, Ding

, Zhang

, Guo

2004. A novel cytotoxic ternary copper(II) complex of 1,10-phenanthroline and l-threonine with DNA nuclease activity. Inorg Biochem, 98:2099–2106.