Study of Interaction of Native DNA with Iron(III)-(2,4-Dihydroxysalophen)chloride
Study of Interaction of Native DNA with Iron(III)-(2,4-Dihydroxysalophen)chloride
Journal of the Korean Chemical Society. 2010. Oct, 54(5): 573-578
Copyright © 2010, The Korean Chemical Society
  • Received : May 10, 2010
  • Accepted : August 18, 2010
  • Published : October 20, 2010
Export by style
Cited by
About the Authors
Mohammad-Reza Azani
Azin Hassanpour
Abdol-Khalegh Bordbar
Laboratory of Biophysical Chemistry, Department of Chemistry, University of Isfahan, Isfahan, 81746-73441, I.R. Iran

In this study, iron(III)-2,4-dihydroxysalophen chloride (Fe(2,4-DHSalophen)Cl), has been synthesized by combination of 2,4-dihydroxysalophen (2,4-DHSalophen) with FeCl 2 in a solvent system. This complex combination was characterized using UV-vis and IR spectroscopies. Subsequently, the interaction between native calf thymus deoxyribonucleic acid (ct-DNA) and Fe(2,4-DHSalophen)Cl, was investigated in 10 mM Tris/HCl buffer solution, pH 7.2, using UV-visible absorption and fluorescence spectroscopies, thermal denaturation technique and viscosity measurements. From spectrophotometric titration experiments, the binding constant of Fe(2,4-DHSalophen)Cl with ct-DNA was found to be (1.6 ± 0.2) × 10 3 M -1 . The fluorescence study represents the quenching effect of Fe(2,4-DHSalophen)Cl on bound ethidium bromide to DNA. The quenching process obeys linear Stern-Volmer equation in extended range of Fe(2,4-DHSalophen)Cl concentration. Thermal denaturation experiments represent the increasing melting temperature of DNA (about 4.3 ℃) due to binding of Fe(2,4-DHSalophen)Cl. These results are consistent with a binding mode dominated by interactions with the groove of ct-DNA.
The interaction of transition metal complexes, containing multidentate aromatic ligands, with DNA has recently gained much attention following the important biological and medical roles played by potential metallointercalators. 1 Schiff base metal complexes have been recognized as powerful catalysts in a great number of chemical reactions such as, electrochemical reduction of alkyl halides in aprotic solvents, 2 oxygenation of indols, phenols, flavones, and others. 3
In addition, some of these complexes are able to bind in a reversible form to molecular oxygen. These complexes have been also investigated as model compounds in the study of the natural oxygen carriers such as, hemoglobin, myoglobin hemocyanin 4 and in the study of the catalytic properties of some cytochromes involved in the biological oxidative reactions. Finally, Schiff base tetradentate complexes of iron have been studied as mimetic models of peroxidases, catalases and superoxide dismutase. 5 In addition to these applications, the synthesis and further structural and chemical characterization of new Schiff base tetradentate ligands have been studied in many chemical and biological fields. Three major binding modes have been proposed for the binding of Schiff bases to DNA, intercalation, outside groove binding and outside binding with self-stacking in which the Schiff bases stacked along the DNA helix. 6 The central metal ion strongly influences both the binding characteristics of the Schiff base complex to DNA and the DNA cleavage properties. 7 The interaction of a number of iron(II) and iron(III) derivatives with nucleic acids has been widely investigated, but, no work has been published on the interaction of Fe(2,4-DHSalophen)Cl complexes with DNA. In this study, we have synthesized Fe(2,4-DHSalophen)Cl and investigated its interaction with DNA in aqueous solutions by UV-vis, and fluorescence spectroscopes, thermal denaturation and viscosity measurements.
- Chemicals and solutions
1,2-Diaminobenzene and 2,4-dihydroxybenzaldehyde, were obtained from Aldrich Chemical Co. and were used as received. Anhydrous dimethylformamide (DMF) was purchased from Sigma. All other chemicals used in this work, were reagent quality, obtained from Sigma- Aldrich Co. and used as received without further purification. 2,4-DHS has been synthesized by combination of 1,2-diaminobenzene and the 2,4-dihydroxybenzaldehyde. This ligand containing meta-quinone functional groups was characterized using UV-vis and IR spectroscopies, in non aqueous solvents, such as dimethylformamide (DMF). Fe(2,4-DHSalophen)Cl, has been synthesized by combination of 2,4-dihydroxysalophen (2,4-DHSalophen) with FeCl 2 .4H 2 O in a solvent system. This complex was characterized using UV-vis and IR spectroscopies. Stock solutions of Fe(2,4-DHSalophen) Cl derivatives (typically 10 mM) were prepared just prior to use by dissolving the solids in DMF. All the solutions were extensively degassed under vacuum before measurements.
Water was purified with a Millipore Milli-Q system and all the experiments were carried out at room temperature. Double stranded calf thymus DNA (ct-DNA, activated and lyophilized) was purchased from Sigma. ct-DNA stock solutions (2 mg/mL) were prepared in 10 mM Tris/HCl , pH 7.2 buffer. The DNA solutions gave a UV absorbance ratio ( A260/A280 ) of about 1.9, indicating that the DNA was sufficiently free from protein. 8 The concentration in base pairs of DNA was determined using an extinction coefficient of 6600 cm -1 M-1 at 260 nm. 9
PPT Slide
Lager Image
The chemical structure of Fe(2 ,4-DHsalophen)Cl
- Synthesis of 2,4-DHSalophen ligand
According to the traditional procedure of synthesis of tetradentate Schiff base ligands, 10 the reaction of benzaldehydes with 0.5 mol equivalent of diamines, in refluxing MeOH for a few hours, gives rise to the final products (75 - 85% yield) which were analytically pure solids after recrystalization. To reflux the compounds, a 25 mL rounded bottom flask 1,2-diaminobenzene (0.1080 g; 1.00 mmol) was mixed with 2,4-dihydroxybenzaldehyde (0.2762 g; 2.00 mmol) and 25 mL ethanol 95%. Afterwards the mixture was refluxed for 3 hours to obtain a fine yellow solid mixture. The obtained precipitate was filtered and washed several times with ethanol 96% and ether. The product was recrystalized by MeOH to obtain yellow needles of pure 2,4-DHS (0.2819 g, 81%).
- Synthesis of Fe(2,4-DHSalophen)Cl complex
The complex of Fe(2,4-DHSalophen)Cl 1 , was synthesized by combination of 0.174 g (5.0 mmol) sample of 2,4-DHSalophen in 50 mL of ethanol and 0.099 g (5.0 mmol) of FeCl 2 .4H 2 O in 30ml of ethanol were added to the mixture drop by drop while stirring. Then the mixture was refluxed for 8 hours and cooled to room temperature. The synthesized complex was recrystallized from ethanol by diethyl ether and was filtered and dried under vacuum.
- Spectroscopy
The IR measurements were done on Philips spectrophotometer. The absorbance measurements were carried out using UV-vis, Carry-500 double beam spectrophotometer, operating from 200 to 800 nm in 1.0 cm quartz cells. The absorbance titrations were performed at a fixed concentration of the specific Fe(2,4-DHSalophen)Cl complex while varying the concentration of double stranded ct-DNA.
Fluorescence spectra were recorded with a RF-5000 Shimadzu spectrofluorimeter equipped with a Peltier system to control the temperature inside the cuvettes.
To achieve equilibration, samples of aqueous Fe(2,4-DHsalophen)Cl solutions were let for one week at room temperature. Stable and reproducible UV-vis absorption spectra were obtained at this equilibration. All the solutions were extensively degassed under vacuum before measurements.
- Melting experiments
Melting curves were established using an UV-vis Carry-500 double beam spectrophotometer in conjunction with a thermostated cell compartment. The measurements were carried out in 10 mM Tris/HCl, pH 7.2. The temperature inside the cuvette was measured with a platinum probe and was increased over the range 20 - 90 ℃ at a heating rate of 1 ℃/min. The melting temperature, T m , was obtained from the mid-point of the hyperchromic transition.
In all of the experiments, for the pH measurement, we used a potentiometer (Metrohm model, 744).
- Viscosity measurements
The viscosity of ct-DNA solutions was measured at 25 ± 0.1 ℃ using an Ubbelohde viscometer. Typically, 10 mM Tris/HCl buffer solution, pH 7.2 was transferred to the viscometer to obtain the reading of flow time. For determination of solution viscosity, 10.0 mL of buffered solution of 160 μM ct-DNA was taken to the viscometer and a flow time reading was obtained. An appropriate amount of Schiff base was then added to the viscometer to give a certain r ( r = [Schiff base]/[DNA base pair]) value while keeping the ct-DNA concentration constant and the flow time was read. The flow times of samples were measured after the achievement of thermal equilibrium (30 min). Each point measured was the average of at least five readings. The obtained data were presented as relative viscosity, η/η o , versus r , where η is the reduced specific viscosity of DNA in the presence of Schiff base and η o is the reduced specific viscosity of ct-DNA alone. 11 , 12
- Structurals characterization of 2,4-DHS and Fe(2,4-DHSalophen)Cl
IR spectra of tetradentate 2,4-DHSalophen ligand, synthesized base on the procedures described in Section 2. The IR information of this compound (KBr): ν C=N 1614 cm -1 ; ν Ar-O-H 1300 cm -1 ; ν C=C 1486 cm -1 ; ν Ar-H 730 cm -1 . The significant frequencies were selected by comparing the IR spectra of the purified ligands with those were obtained for the commercially available salophen. This Schiff base ligand present a characteristic band in the range of 1605 - 1630 cm -1 attributable to the stretching vibration of the azomethine (C=N) group. The presence of this band is concomitant with the absence of absorption bands in the range of 1680 - 1690 cm -1 corresponding to the starting carboxaldehyde groups. The presence of phenolic hydroxylic groups can be ascertained by the presence of strong absorption bands in the range of 1270 - 1290 cm -1 . The absorbance spectrum of 2,4-DHSalophen showed the maximum wave length at 331 nm. 13
IR spectra of tetra dentate Fe(2,4-DHSalophen)Cl complex, synthesized according with the procedures described in Section 3, contained this IR information (KBr): ν C=N 1605 cm -1 ; ν Ar-O-H 1270 cm -1 ; ν C=C 1440 cm -1 and ν Ar-H 720 cm -1 . The decreasing of ν C=N in Fe(2,4-DHSalophen)Cl (1605 cm -1 ) compared to 2,4-DHS (1614 cm -1 ) is a valuable sign for formation of Fe complex. The absorbance spectrum of Fe(2,4-DHSalophen)Cl also shows the maximum wave length at 316 nm.
- UV-vis spectral studies
The binding of certain complex to DNA can be produced hypochromism, a broadening of the envelope, and a red shift of the complex absorption band. These effects are particularly pronounced for intercalators; with groove binders, a small changes observed on Soret band. A spectral change of Fe(2,4-DHSalophen)Cl due to addition of DNA is shown in . 1 . For obtaining these spectra, the fixed amount of Schiff base in Tric-HCl buffer pH 7.2 was titrated with a stock solution of ct-DNA. The changes in absorbance of the Soret band upon addition of ct-DNA were monitored at the maximum of the Soret band. It exhibited the low hypochromism and negligible red shift due to the incremental addition of ct-DNA indicating groove binding mode.
PPT Slide
Lager Image
The spectrum of Fe(2,4-DHsalophen)Cl in the presence of various concentration of ct-DNA. Measurements were done in 10 mM Tris/HCl buffer, pH 7.2 and at 25 ℃.
The apparent binding constant, Kapp , for the interaction between the Fe (2,4-DHsalophen)Cl complex and ct-DNA can be determined by analysis of absorption spectrophotometric titration data at room temperature using Eq. (1): 14
PPT Slide
Lager Image
Where [DNA] T app f and ε b correspond to total concentration of ct-DNA, A obsd /[Schiff base], the extinction coefficient for the free Schiff base and the extinction coefficient for the Schiff base in the fully bound form, respectively. In the plot of [DNA] T /(|ε app f |) versus [DNA] T that is shown in . 2 , K app is given by the ratio of the slope to the intercept. The apparent binding constant of Fe(2,4-DHSalophen)Cl was determined by (1.6 ± 0.2) × 10 3 M -1 .
PPT Slide
Lager Image
The plot of [DNA]T/(|εappf|) versus [DNA]T.
- Fluorescence spectroscopic studies
Ethidium bromide (EB) emits intense fluorescence light in the presence of DNA, due to its strong intercalation between the adjacent DNA base pairs. As it has been reported, the enhanced fluorescence can be quenched by the addition of a second molecule. 15 , 16 In fluorescence titration experiment, the specified volumes of concentrated Fe(2,4-DHSalophen) Cl solution were added consecutively to the cell which contained 2000 μL DNA (12 μM) and EB (5 μM) solution. Representative emission spectra of EB-DNA solution in the presence of various amounts of Fe(2 ,4-DHSalophen)Cl is shown in . 3 .
PPT Slide
Lager Image
The emission spectrum of EB (5 μM) bound to DNA (12 μM) in the presence of various amounts of Fe(2,4-DHsalophen)Cl. Measurements were done in 10 mM Tris/HCl buffer, pH 7.2 and at 25 ℃. The peak area is decreased with increasing of Fe(2,4-DHsalophen)Cl concentration in the direction of arrow. The excitation wavelength was 515 nm.
The pecks area was measured and analyzed using the classical Stern-Volmer equation Eq. (2): 17
PPT Slide
Lager Image
Where I 0 and I are the fluorescence area peck in the absence and the presence of complex, respectively, K is a linear Stern-Volmer quenching constant and r is the ratio of total concentration of complex to that of ct-DNA. The quenching extent of fluorescence of EB bound to ct-DNA was used to determine the extent of binding between the second molecule and ct-DNA. The Stern-Volmer plot for fluorescence quenching of EB bound to ct-DNA by the Schiff base is shown in . 4 .The quenching plots illustrate that the quenching of EB bound to ct-DNA by the Schiff base is in good agreement with the linear Stern-Volmer equation which confirms the Schiff base bind to DNA. In the plot of I 0 / I versus [Schiff base]/[DNA], K is calculated by the ratio of the slope to intercept. The K value for Fe(2,4-DHSalophen)Cl was 6.2 The relative value of K compared with other known intercalators represents the non-intercalation mode for Fe(2,4-DHsalophen)Cl. 13 , 18
PPT Slide
Lager Image
The Stern-Volmer plot for quenching of EB bound to DNA by Fe(2 ,4-DHsalophen)Cl complex.
- Thermal denaturation experiments
When a molecule intercalates to ct-DNA, the stability of the helix increases and the temperature at which the helix denatures ( T m ) goes up varies around 5 - 12 ℃ in r = 1. 19 , 20 Thus, this parameter is most useful in analyzing the mode of interaction. The melting plot of ct-DNA was monitored by plotting the variation of maximum absorption of ct-DNA solution (100 μM) at 258 nm vs . temperature. The denaturation curves were measured at various [Fe(2,4-DHSalophen) Cl]/[DNA] molar ratios and is shown in . 5 .
PPT Slide
Lager Image
Thermal denaturation curves of DNA (10.0 × 10-5 M) in the various [Fe(2,4-DHsalophen)Cl]/[DNA] molar ratios of r1 = 0.0(■), r2 = 0.2(▲) and r3 = 1.0(●). Measurements were done in 10 mM Tris/HCl buffer, pH 7.2 and at 25 ℃.
The melting temperature of ct-DNA ( T m ) was estimated from the midpoint of transition curves. With respecting to the results, the melting temperature of ct-DNA has been increased about 4.3 ℃ in the presence of Fe (2,4-DHSalophen)Cl. The observed small change in the T m of ct-DNA in the presence of Fe (2,4-DHSalophen)Cl suggests that the interaction of these compounds with ct-DNA does not involve intercalation between the base pairs and could be ascribed to interactions with the DNA grooves. 21
- Viscosity measurements
Optical or photo physical probes generally provide necessary, but not sufficient, clues to support kind of binding model. Hydrodynamic measurements that are sensitive to length increases (i.e. viscosity, sedimentation, rotational diffusion as measured by transient electric diffusion) are the least ambiguous and most critical tests of the binding models in solution. The DNA helix lengthens as the base pairs are separated to accommodate the bound complex for the groove binding of the molecule, leading to lower increase in DNA viscosity. Hydrodynamic methods are thus suitable to detect such changes and, in the absence of crystallographic structural data, provide essential evidence to support the intercalation model. In contrast, partial and/or nonclassical intercalation of complex could bend (or kink) the DNA helix, reduce its effective length and in turn, its viscosity. The effect of Fe(2,4-DHsalophen)Cl on the viscosity of DNA is shown in . 6 The relative viscosity of DNA shows small rises with increase in the concentration of the Fe (2,4-DHsalophen)Cl, which is not similar to that of the classical intercalation (i.e. ethidium bromide). 22 , 23 The viscosity results unambiguously show that Fe(2,4-DHsalophen)Cl bind with DNA by groove binding mode, these results are in agreement with optical absorption experiments.
PPT Slide
Lager Image
Relative viscosity of calf thymus DNA (7.8 × 10-5 M) in Tris-HCl 10 mM in the presence of increasing amounts of Fe(2,4-DHSalophen)Cl at stoichiometric ratios r = [Fe(Salen)Cl]/[DNAphosphate] = 0.0 - 2.0, plotted as (η/η o)1/3 vs. r.
The results obtained from fluorescence, UV-vis, thermal denaturation and viscosity measurements exclude DNA outside groove binding. From spectrophotometric titration experiments, the binding constants of Fe(2,4-DHSalophen)Cl with ct-DNA were found to be (1.6 ± 0.2) ×10 3 M -1 . These results are consistent with a binding mode dominated by interactions with the groove of ct-DNA, analogously to what reported for a number of porphyrazines and metal-porphyrazine complexes interacting with ct-DNA. 18
Metcalfe C. , Thomas J. A. 2003 Chem. Soc. Rev. 32 215 -    DOI : 10.1039/b201945k
Isse A. , Gennaro A. , Vianello E. 1998 J. Electroanal. Chem. 444 241 -    DOI : 10.1016/S0022-0728(97)00572-X
Ramnath R. , Al-Junaid S. , Motevalli M. , Parkin B. C. , Sullivan A. C. 2004 Inorg. Chem. 43 4072 -    DOI : 10.1021/ic035318h
Chen D. , Martell A. E. , Sun Y. 1989 Inorg. Chem. 28 2647 -    DOI : 10.1021/ic00312a029
Haikarainen A. , Sipila J. , Pitkanen P. , Pajunen A. , Mutikainen I. 2001 Med. Chem. 9 1633 -    DOI : 10.1016/S0968-0896(01)00053-0
García M. , Lorenzo T. , Pariente E. 2007 Biosensors and Bioelectronics 22 2675 -    DOI : 10.1016/j.bios.2006.11.002
Silvestri A. , Barone G. , Raisi G. , Giudice M. , Tumminello S. 2004 Inorg. Biochem. 98 589 -    DOI : 10.1016/j.jinorgbio.2004.01.010
Marmur J. 1961 J. Mol. Biol. 3 208 -    DOI : 10.1016/S0022-2836(61)80047-8
Duty P. , Rice S. A. 1955 Biochim. Biophys. Acta 16 446 -    DOI : 10.1016/0006-3002(55)90261-4
Bailes R. H. , Calvin M. 1947 J. Am. Chem. Soc. 69 1886 -    DOI : 10.1021/ja01200a013
Banville D. L. , Marzilli L. G. , Strickland J. A. , Wilson W. D. 1986 J. Biopolymers. 25 1837 -    DOI : 10.1002/bip.360251003
Gray T. A. , Yue K. T. , Marzilli L. G. 1991 J. Inorg. Biochem. 41 205 -    DOI : 10.1016/0162-0134(91)80013-8
Azani M. R. , Hassanpour A , Bordbar A K , Mirkhan V 2009 Bull. Korean Chem. Soc. 30 1973 -    DOI : 10.5012/bkcs.2009.30.9.1973
Meehan T. , Gamper H. , Becker J. F. 1982 J. Biol. Chem. 257 10479 -
Bagley B. C. , Lebret M. 1984 Biochemistry 23 937 -    DOI : 10.1021/bi00300a022
Lowicz J. R. , Webber G. 1973 Biochemistry 12 4161 -    DOI : 10.1021/bi00745a020
Cramb D. T. , Beck S. C. 2000 J. Photochem. Photobiol. A 134 87 -    DOI : 10.1016/S1010-6030(00)00249-5
Kang J. , Wu H. , Lu X. , Wang Y. , Zhou L. 2005 Spectrochimica Acta A 61 2041 -    DOI : 10.1016/j.saa.2004.08.009
Kuswandi B. , Tombelli S. , Marazza G. , Mancini M. 2005 Chimia. 59 236 -    DOI : 10.2533/000942905777676597
Wu H. L. , Li W. Y. , Miao K. , He X. W. , Liang H. 2002 Spectrosc. Lett. 35 781 -    DOI : 10.1081/SL-120016280
Nair R. B. , Teng E. S. , Kirkland S. L. , Murphy C. 1998 J. Inorg. Chem. 37 139 -    DOI : 10.1021/ic970432j
Cory M. , Mckee D. D. , Kagan J. , Henry D. W. , Miller J. 1985 J. Am.Chem. Soc. 107 2528 -    DOI : 10.1021/ja00294a054
Waring M. 1965 J. Mol. Biol. 13 269 -    DOI : 10.1016/S0022-2836(65)80096-1