Study of DNA-Deltamethrin Binding by Voltammetry,Competitive Fluorescence,Thermal Denaturation,Circular Dichroism,and Atomic Force Microscopy Techniques

Abstract

The interaction of deltamethrin (DM), a synthetic insecticide, with calf thymus DNA was studied. The cyclic voltammetric (CV) results revealed that DM has two irreversible cathodic peaks. The first peak (a) was devoted to reduction of −CN by 4 electrons and the second peak (b) was devoted to reduction of the −C=C− moiety by two electrons. By using non-linear regression analysis of CV data of peak (a), the binding constant, binding site size, and diffusion coefficient for free DM (D_f) and DNA-DM (D_b) were calculated as: 2.6×10⁴, 1.6, 3.2×10⁻⁴Cm² S⁻¹, and 8.5×10⁻⁶Cm² S⁻¹, respectively. The thermal denaturation, competitive fluorescence, and AFM results revealed that the mode of interaction may be non-intercalative. Also the circular dichroism spectra showed that the conformation of CT DNA was converted from right-handed B-DNA to A-DNA due to the destacking of the adjacent guanine bases in pH 7.3 solution.

Introduction

P yrethroids are synthetic insecticides widely used for pest control in many countries. These compounds are divided into two classes: type I and II. Type I does not contain an α-cyano group and affects the sodium channels in nerve membranes producing repetitive neuronal discharge and prolonged negative after-potential. Type II does contain an α-cyano group and produces an even longer delay in sodium channel inactivation leading to a persistent depolarization of the nerve membrane without repetitive discharge (Michelangeli et al., 1990). Deltamethrin (DM), (s)-α-cyano-3-phenoxybenzyl-[IR]-cis-3-(2-dibromovinyl)-2,2-imethylcyclopropanecarboxylate (DM; Fig. 1) belong to the type II class of pyrethroids and has very stable biological activity in the environment. Genotoxicity and carcinogenicity data revealed that DM causes many adverse effects on non-target species (Aksakal et al., 2010). Villarini and coworkers (1998) demonstrated that DM is able to induce DNA damage, single and double-strand breaks, and micronuclei induction in human peripheral blood leukocytes. A study on tumorigenic and co-carcinogenic (tumor initiating and tumor promoting) potential of DM demonstrated that DM has a tumor initiating potential in Swiss albino mice (Shukla et al., 2001). Naguib El-Khatib et al. (2006) reported that the pyrethroid pesticide cypermethrin induced a clear, significant, positive dose-dependent increase in DNA damage in rat liver cells. Unwanted small intercalator molecules may lead to DNA damage (Abdel-Aziz et al., 2004). These reports and the scanty in vitro studies on the effects of DM on DNA led us to study the interaction of DM with CT-DNA to assess the mode of interaction. In this work, the DM–DNA interaction was investigated in 5 mM Tris-HCl aqueous solutions at the physiological pH 7.3, using cyclic voltammetry, melting DNA, competitive fluorescence, and AFM methods.

FIG. 1.

The structure of deltamethrin.

Materials and Methods

Highly polymerized calf-thymus double strand deoxyribonucleic acid (dsDNA) and DM with high purity were purchased from Sigma and used as received. All other reagents were of the highest commercial grade and were used without further purification. Deionized double distilled water was used throughout the experiments. dsDNA was dissolved in Tris-HCl buffer and was dialyzed against the same buffer overnight (Kashanian et al., 2007). Solutions of dsDNA gave ratios of UV absorbance at 260 and 280 nm above 1.8, indicating that dsDNA is sufficiently free of protein. Ds-DNA concentration per nucleotide was determined by UV/Vis spectrophotometery using the molar absorption coefficient of 6600 M⁻¹ at 258 nm (Kashanian et al., 2007). An HP Agilent (8453) UV-Vis spectrophotometer equipped with a peltier (Agilent 89090A) was used for thermal denaturation of DNA. All competitive fluorescence measurements were carried out with a Beckman spectrofluorometer (LS 45). Maximum excitation wavelength used was 330 nm. The pH values of the solutions were adjusted employing a Metrohm model 827 using a combined glass electrode. The AFM imaging of DNA-PYR complexes were performed in phase contrast and dynamic force operating mode and noncontact Mounted cantilever (Nanosurf Mobile S) using high frequency (170 kHz) by silicon cantilevers with thickness 7 μm, length 225 μm and width 38. Images were treated using the software Installation Instructions for Nanosurf Mobile S version 1.8. The cyclic voltammetry measurements were performed by a Metrohm VA 797 computrace (Metrohm) with a three-electrode system: a Hanging Mercury drop electrode (HMDE) as working electrode, silver-silver chloride (Ag/AgCl) as reference electrode, and a platinum wire as counter-electrode. For all types of voltammetric measurements, the supporting electrolyte (5.0 mM Tris-HCl buffer solution, pH 7.3) was placed in a polarographic cell of volume 10 mL and deaerated via purging with pure N₂ gas for 2 min. The cyclic voltammetric (CV) measurements were carried out by keeping both concentration of the DM and the total volume of solution constant while the dsDNA concentration varied.

Result and Discussion

Electrochemical behavior of DM

The electrochemical behavior of 2.5×10⁻⁵ M of DM in binary mixture of acetonitrile-Tris-HCl buffer (20:80 v/v; pH=7.3) at the surface of HMDE was evaluated. A typical cyclic voltammogram of DM in the studied solution is shown in Figure 2. At the HMDE, the cyclic voltammogram of DM demonstrates two irreversible cathodic peaks in the −1.04 and −1.4 V. In 1999, Samatha and Sreedhar studied DM by polarography and also reported the electrochemical behavior of DM over the pH ranges of 2.0–12.0 at a drop mercury electrode. Their results revealed a single, well defined, irreversible peak at −0.7 V versus SCE in pH=4.0. They concluded that the cathodic peak arises from the reduction of the −C=C− moiety of DM by a two electron process. When the HMDE was used, two irreversible peaks were observed (see Fig. 2). To measure the symmetry of the energy barrier and all electrons in each peak, the equation (I) was used for both cathodic peaks. \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*}|E_{\rm p} - E_{{\rm p}/2}| = 1.857 \ {\rm RT} / \alpha {\rm nF} \ {\rm (mV)} \tag{{\rm I}}\end{align*}\end{document}

FIG. 2.

Cyclic voltammogram of 5.0×10⁻⁵ M of deltamethrin in the absence of DNA in 5 mM Tris-HCl buffer (pH 7.3), scan rate was 50 mV s⁻¹.

Where E _p/2 is the potential, where i=i _p/2 in cyclic voltammograms. For exact measurement of |E _p −E _p/2| all CV peaks were obtained by using the determination mode of the Metrohm VA 797 computrace instrument. As the |E _p −E _p/2| may be changed by scan rate, the average value of |E _p −E _p/2| in all scan rates (10 to 80 mV s⁻¹) for both cathodic peaks of a and b were calculated as 25 and 37.8 mV, respectively. The values of (αn)_a and (αn)_b, calculated from equation I, were 1.907 and 1.26, respectively. Generally, α in the totally irreversible electrode process is assumed as 0.5. Hence, 4 electrons are involved in the reduction process for peak a and 2 electrons are devoted to peak b. The values of α were 0.476 for peak a and 0.63 for peak b. The symmetry of the energy barrier (α) was calculated again, using the Anson equation (II) (Anson, 1983): \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*}E_p = E^0 - \frac { RT } { \alpha nF } \left[ ln \left( \frac { K_s } { { } _D \frac { 1 } { 2 } } \right) + 0.5 ln \left( \frac { \alpha nFv } { RT } \right) + 0.78\right] \tag { \rm II } \end{align*}\end{document}

The α value was calculated from the slope of the linear between E _p and lnν. The α value for peak a was 0.49 and for peak b was 0.66. Peak a may have resulted from the reduction of the −CN moiety and peak b may have resulted from the reduction of the −C=C− moiety. Therefore, on the basis of above results, the following mechanism may be proposed for each peak.

As the reduction of the −CN moiety is carried out by 4e, the intensity of peak a in pH=7.3 is higher, and i _pa is remarkably more than the i _pb. Also, the influence of pH on the peak potentials of DM was studied in the pH range of 3.0 to 9.0 for a solution containing 5.0×10⁻⁵ M of DM by the cyclic voltammetry method. The results revealed that the peak potential (E _p) of the both cathodic peaks of DM as a function of pH were changed and were shifted toward negative, whereas, peak a was more shifted toward the pH range of 3–9. The plots of voltammetric peaks potential versus pH were straight lines with slope of 53.9 and 30.2 mV, respectively, which agrees with four and two reduction process with transfer coefficient α=0.5, respectively (Fig. 3). Therefore, the proposed reduction mechanism of DM was confirmed. The dependence of both cathodic peak currents of DM to the scan rate (ν) in the absence and presence of an excess of DNA was examined at HMDE., With increasing scan rate, both cathodic peak currents of DM in absence of DNA considerably increased, whereas in the presence of 5.0×10⁻⁵ M of DNA both the peak currents slowly increased. The negative shift of both peak potentials with increasing scan rate (ν) demonstrated an irreversible electrode process for DM. The plots of both peak currents versus ν and ν ^1/2 for DM are shown in Figure 4a and 4b. Both cathodic peak currents of DM linearly vary with square root of the scan rate, ν ^1/2, rather than with ν. The linear equation of i _pc versus ν ^1/2 for peak a is: i _pca (nA)=−4.761 ν ^1/2 (mV S⁻¹)^1/2–3.374 with R=0.995; and for peak b is i _pcb (nA)=−1.996 ν ^1/2 (mV S⁻¹)^1/2–0.948 with R=0.995. The results indicated that the mass transport of DM to the HMDE surface is a diffusion controlled process (Wang et al., 2003a). In the presence of DNA (R=[DNA]/[DM]=1), both cathodic peaks of DM decreased. The plots of i _pc versus ν and ν ^1/2 for each peak of DNA-DM complex are presented in Figure 5a and 5b. The linear equation of i _pc versus ν ^1/2 for peak a is: i _pc(a) (nA)=−4.8078 ν ^1/2 (mV S⁻¹)^1/2+ 1.1263 with R²=0.997; and for peak b is i _pc(b) (nA)=−2.4356 ν ^1/2 (mV S⁻¹)^1/2+ 4.261 with R²=0.998. The linearity of i _pc versus ν ^1/2 plots indicated that the main transport of complex to the electrode surface is controlled by diffusion (Wang et al., 2007). The lower diffusion coefficient of the DM-DNA species is responsible for the decay of current signals in CV behavior of DM. Also the larger slopes of plots of i _pc versus ν ^1/2 for DM in the presence of DNA could be attributed to the non-intercalation with DNA (Rehman et al., 2009). In our previous studies, we showed that decreases in the current of the complex is not from the viscosity of the solution or the blocking of the electrode surface by DNA adsorption (Ahmadi and Jafari, 2011; Ahmadi et al., 2011b; Ahmadi et al., 2011a). In order to show this decrease in current did not arise from the viscosity of the solution or the blockage of the electrode surface by DNA adsorption, we studied the CV of K₄Fe(CN)₆ in the absence and presence of DNA. The results revealed that DNA had no effects on the diffusion from the changed viscosity of the solution and/or the DNA adsorption. Therefore, a large decrease in current could be attributed to the diffusion of DM bound to the large, slowly diffusing DNA.

FIG. 3.

The dependences of cathodic peak potential Ep versus pH for peak a (▪) and peak b (▴). The conditions were as follows: 10 mL of electrolyte containing 20% (v/v) AN–Tris-HCl buffer, scan rate 50 mV s⁻¹.

FIG. 4.

The cathodic current variations of peak a (▪) and peak b (▴) of DM with (a) scan rate ν and (b) square root of the scan rate ν ^1/2.

FIG. 5.

The cathodic current variations of peak a (▪) and peak b (▴) of DM-DNA complex with (a) scan rate ν and (b) square root of the scan rate ν ^1/2.

CV titration of DM with DNA

Electrochemical titration is valuable in quantifying the interaction parameters of an electroactive molecule with DNA than other methods (Ahmadi and Bakhshandeh, 2009; Ahmadi and Jafari, 2011; Ahmadi et al., 2011b; Ahmadi et al., 2011a). By addition of different amounts of DNA to the voltammetric cell containing 1.0×10⁻⁵ M of DM, the cathodic peak currents of DM begin to decrease and the formal potential shifts to more positive values which suggest the interaction of the DM with DNA. When \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}$$R = { \frac { [DNA] } { [DM] } } = 5.0$$\end{document} , CV currents of DM decreased and were stable (Fig. 6). The decrease of both cathodic peak currents is due to the decrease of diffusion coefficients of the DM-DNA species and equilibrium concentration of DM in the solution. Under the experimental conditions and in the potential range of −0.9 to −1.7 V at the HMDE, the pure DNA is not electroactive (Ibrahim, 2001). This was mainly due to the merging of cathodic peaks at low DNA concentrations with the background discharge (Palecek and Postbieglova, 1986). According to these observations, it seems that the decrease of peak currents of DM after an addition of excess DNA are caused by the interaction of DM with the bulky, slowly diffusing DNA, which results in a considerable decrease in the apparent diffusion coefficient. The cathodic potential of peak a shifted to more positive values while peak b did not (Fig. 6). This positive shift in E _p of peak a suggested DM may interact more via the −CN moiety rather than other parts of the DM molecule. An intercalative mode involves a strong stacking interaction between aromatic rings of molecules and the base pairs of DNA. DM is a large molecule and has a tendency to interact with DNA via the −CN and −C=C− moieties rather than aromatic rings, the intercalative mode of interaction may not be predominant.

FIG. 6.

Cyclic voltammetric titration of 5.0×10⁻⁵ M of DM with DNA (DNA concentration varied from 0.0 to 2.5×10⁻⁴M).

Equilibrium constant measurements

Cyclic voltammeric titration is valuable for quantifying binding of the DM to DNA. By addition of different amounts of DNA to the electrolyte solution, the peak currents begin to decrease and the formal potential shifts to more positive values, which suggests strong interaction of the DM with DNA. When R=5.0 (R is defined as the ratio of the total concentration of nucleic acid to that of DM), the currents were stable (Fig. 7). If an electroactive molecule (E) nonspecifically reacts with a DNA duplex at a binding site, which is composed of base pairs (S), a DNA-electroactive molecule complex (E −S) is produced as follows: \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*}E + S \leftarrow \ \rightarrow E - S \tag{\rm III}\end{align*}\end{document}

As DM non-specifically binds to the DNA duplex and covers S consecutive base pairs (i.e., one binding site), the binding constant, K, can be given by the equation (IV): \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*}K = \frac { C_b } { C_f \ C_s } \tag { \rm IV } \end{align*}\end{document}

Where C_b, C_f , and C_s represent the equilibrium concentrations of the DNA-DM complex, free DM and free binding site, respectively. The total concentration of DM, C_t , is: \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*}C_t = C_b + C_f \tag{\rm V}\end{align*}\end{document}

The average of number of binding sites (x) along a DNA duplex molecule with an average total number of base pairs L can be described by the following: \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}$$x = \frac { L } { s } $$\end{document} , where s is the binding site size of the electroactive molecule interacting with DNA. It corresponds to the number of DNA base pairs is occupied (or covered) by a binding molecule. Thus, the total concentration of binding sites (xC_DNA ) can be expressed as follows: \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*}xC_{DNA} = C_b + C_s \tag{\rm VI}\end{align*}\end{document}

where \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*}C_ { DNA } = \frac { C_ { NP } } { 2L } \tag { \rm VII } \end{align*}\end{document}

C_NP represents the concentration of nucleotide phosphate, which is determined by the UV absorption at 260 nm. The total concentration of binding sites can also be expressed as follows: \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*} \frac { C_ { NP } } { 2s } = C_b + C_s \tag { \rm VIII } \end{align*}\end{document}

The ratio of the NP concentration and the total concentration of electroactive molecules can be defined as R: \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*}R= \frac { C_ { NP } } { C_t } \tag { \rm IX } \end{align*}\end{document}

For an irreversible reaction in CV, the total cathodic current (I_pc ) under the fixed potential with any R can be calculated: \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*}i_ { pc } =B\left[(an)^ \frac { 1 } { 2 } _f D^ \frac { 1 } { 2 } _f C_f + (an)^ \frac { 1 } { 2 } _b D^ \frac { 1 } { 2 } _b C_b\right] \tag { \rm X } \end{align*}\end{document}

B represents the appropriate, concentration-independent terms in the voltammetric expression. A Nernstain reaction in CV at 25°C is shown as follows (Wang et al., 2003b): \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*}B = 2.99 \times 10^5nAv^ \frac { 1 } { 2 } \tag { \rm XI } \end{align*}\end{document}

Where n is the number of electron transferred, A the electrode area, v the scan rate, D_f and D_b are the diffusion coefficients for free and bound molecules, α_f and α_b are the electron transfer coefficients for free and bound molecules, and C_f and C_b are the bulk concentrations of the free and bound irreversibly electroactive species.

Based on Carter and Bar (1987) the binding constant, K, can be expressed as the following form: \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*}K = \frac { c_b } { c_f \left( \frac { [NP] } { 23 } - c_b\right) } \tag { \rm XII } \end{align*}\end{document}

Where s is the size of binding site in terms of base pairs. Making appropriate substitutions and eliminating C_b and C_f from Eq. (X), a new equation was obtained: \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*}i_ { pc } = B \left \{ (an)_f^ \frac {1} {2} D_f^ \frac {1} {2} C_t + \left[(an)_b^ \frac {1} {2} D_b^ \frac{1} {2} - (an)_f^ \frac {1} {2} D_f^ \frac {1} {2} \right] \times \left[ \frac {b - \left(b^2 - \frac {2k^2C_E^2 R} {s} \right)^ \frac {1} {2}} {2K} \right] \right\} \tag {\rm XIII} \end{align*}\end{document}

Where \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}$$b = 1 + KC_t + \frac { KRC_t } { 2s } $$\end{document} .

Since i_pc , C_t and [NP] are experimentally measurable and (αn)_f, (αn)_b, have already been acquired as mentioned above, the binding constant (K) and binding site size (s) of the DM-DNA, D_f and D_b can be obtained from a nonlinear regression analysis of the experimental data (i_pc versus [R] plot; Fig. 7) according to Eq. (XIII). The nonlinear fit analysis yielded K=2.6×10⁴ and s=1.6, D_f=3.2×10⁻⁴Cm² S⁻¹ and D_b=8.5×10⁻⁶ Cm² S⁻¹.

FIG. 7.

Dependence of i_pca of 5.0×10⁻⁵ M DM on the concentration of added DNA by cyclic voltammetry, scan rate=50 mVs⁻¹.

In previous works the k_b of DNA with several pesticides at 25°C such as: fenitrothion (1.9×10⁴) (Ahmadi and Jafari, 2011), 2-imidazolidinethione (1.4×10³) (Ahmadi et al., 2010), 2,4-D (3.5×10³ and 5.0×10³) (Ahmadi and Bakhshandeh, 2009), clodinafop-propargyl (5.6×10³) (Askari et al., 2008), diazinon (1.6×10⁴) (Gholivand et al., 2008), and aminocarb (7.2×10³) (Zhang et al., 2010) have been reported. These carcinogenic compounds have a significant binding affinity to DNA in the range of 3.5×10³ to 1.6×10⁴. The K_b of DMis 2.6×10⁴ and is less than those observed for typical classical intercalators, ethidium–DNA, 7×10⁷ in 40 mM Tris–HCl buffer, pH 7.9 (Waring, 1965) and 1.4×10⁶ in 40 mM NaCl–25 mM Tris–HCl (Lepecq and Paoletti, 1967).

Competitive binding between ethidium bromide and DM for DNA

Ethidium bromide (EB) displays a dramatic enhancement of DNA fluorescence efficiency when intercalated into DNA. The mechanism for fluorescence enhancement of EB upon bonding with nucleic acids can be described with excited state quenching by solute-to-solvent proton transfer. According to this mechanism, the low fluorescence intensity of free EB in water is attributed to efficient quenching of excited state molecules by proton transfer to water molecules (quenching rate constant of 10⁷ s⁻¹ m⁻¹), and the enhancement of the fluorescence intensity on going to nonaqueous solvents, deuterated EB, or binding to DNA is attributed to a reduction in the proton transfer rate. Intercalated EB is substantially less accessible to water molecules than is outside-bound EB. Hence, the lifetime in the intercalated sites is substantially longer than in electrostatically bound sites. Therefore, the popular strategy for monitoring DNA interaction with target molecules by fluorescence spectroscopy is based on the quenching of EB fluorescence via competition for binding sites (Wu et al., 2005). DM does not show any fluorescence at room temperature in solution or in the presence of CT DNA, so that the binding to DNA cannot be directly predicted through the emission spectra. The emission spectra of EB bound to CT DNA in the absence and presence of DM have been recorded for [EB]=1.0×10⁻⁵ M, [DNA]=5.0×10⁻⁵ M for increasing amounts of DM (Fig. 8). There is no significant displacement of EB by DM, reflecting the absence of an intercalative mode of binding.

FIG. 8.

Emission spectra of (a) free ethidium bromide and (b) ethidium bromide bound to DNA in the presence of various amounts of DM in Tris-HCl buffer (pH 7.3).

DNA thermal denaturation

To further characterize the DM-DNA complex, thermal denaturation of CT DNA in absence and presence of DM was investigated (Fig. 9). DM-DNA stoichiometries of r _i=0.0, 0.5, and 1.0 were investigated. When the solution temperature increases, double-stranded DNA gradually dissociates into single strands; T_m is defined as the temperature where half of the total base pairs is unpaired. The DNA melting temperature (T_m) is strictly related to the stability of the double helix, and interaction of DM with DNA may alter T_m by stabilizing or destabilizing the final complex. The melting temperature of DNA 5.0×10⁻⁵ M in Tris–HCl buffer (70±0.5°C) increases about 4.0 and 7.0°C, at r _i=0.5 and 1.0, respectively. These data are lower than that reported for intercalatores and indicated that DM stabilizes the native DNA conformation via nonintercalation mechanism.

FIG. 9.

Melting plots, by ultraviolet–visible absorbance spectrophotometry, of calf thymus DNA 5×10⁻⁵ M in Tris-HCl in the presence of DM, at r_i=[DM]/[DNA]=0.0 (•), 0.5(♦), and 1.0 (▴).

Atomic force microscopy

Recently, DNA molecules and their interactions with some small molecules have been studied by AFM (Liu et al., 2005; Ruiz et al., 2005; Sorel et al., 2006; Liu et al., 2010; Ahmadi et al., 2011c). AFM is capable of imaging nonconducting and conducting surfaces. However, the most practical limitation to the application of AFM to structural and conformational studies of DNA and its complexes with target macromolecules is the sample preparation. The macromolecules must be tethered to the substrate surface to avoid resolution-limiting motion caused by the sweeping tip during scanning. The free DNA and DM–DNA complexes at mole ratios of 1:1 and 2:1 at final DNA concentration of 0.1 mM were prepared in 2.0 mL Tris–HCl (pH 7.3). The samples were prepared on muscovite mica according to the methods of Marty et al. (2009). Figure 10a–e shows ultra structures of free DNA and DM–DNA complexes of 1:1 and 1:2 and their phase transition images, respectively. Figure 10a shows the free DNA structure which adhereto the surface of the mica. The worm-like structure in Figure 10a for free DNA is similar to that previously observed by Fang and Hoh (1998). In the 1:1 complex the DNA molecules coated with DM and formed a stable DNA adduct (Fig. 10b, arrow heads), whereas in 1:2 a grid shape was observed (Fig. 10c). As reported in previous studies, DNA can form many intermediates and finally change to the more compact structures such as the rods and toroids on the condensing process (Fang and Hoh, 1998; Hansma et al., 1998). This is because the dense DNA core is surrounded by a more deformable DM shell and condensed the DNA structure. The core of the 1:1 DNA-DM complex has an average size of 223 nm. The core of the 1:2 DNA-DM has an average size of 485 nm.

FIG. 10.

The atomic force microscopy images and transition phase of free DNA and DM-DNA complexes (a) Free DNA, (b) DM-DNA with molar ratio of 1:1, (c) DM-DNA with molar ratio of 1:2.

Evaluation of streoselectivity interaction by CD

The effect of DM on DNA structure was evaluated by CD spectroscopy and the results are shown in Figure 11. The CD spectrum of free CT-DNA (dotted line) consists of a negative Cotton effect at 248 nm and a positive Cotton effect at 276 nm. The negative spectrum corresponds to the helical structure of DNA and the positive spectrum corresponds to stacking of the base-pairs that is characteristic of DNA in the right-handed B-form. The DM molecule do not exhibit any CD signal in the region of 220–320. Figure 11 illustrates how the spectra of dsDNA changed with the addition of DM to the DNA solution. The addition of DM has little effect on the positive peak, but increased the negative peak. With increasing DM, the height of the negative peak also increased, indicating a transition of the secondary DNA structure from B- to A-form. This effect was attributed to intrastrand linking of adjacent guanines so that the DNA conformation is modified and destacking of the adjacent guanines bases occurs.

FIG. 11.

The circular dichroism spectrum of CT DNA (5.0×10⁻⁵ M) in absence (dot line) and presence of 1.5×10⁻⁵, 2.1×10⁻⁵, and 5.0×10⁻⁵ M of DM.

Conclusion

The electrochemical, thermal denaturation, and competitive fluorescence data revealed that DM interacted with DNA by the non-intercalation mode. The binding constant measured with cyclic voltammetry was 2.6×10⁴. The present AFM images provided further insights into the mechanism of interaction of DM with DNA.

Footnotes

Acknowledgments

We gratefully acknowledge Vice Chancellor for Research and Technology, Kermanshah University of Medical Sciences for financial support. This work was performed in partial fulfillment of the requirements for a Pharm. D by Nasibeh Jamali in Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermansha, Iran.

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