International Journal of Biological Macromolecules 66 (2014) 166–171
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
DNA binding and nuclease activity of an oxovanadium valinato-Schiff base complex Urmila Saha, Kalyan K. Mukherjea ∗ Department of Chemistry, Jadavpur University, Kolkata 700032, India
a r t i c l e
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Article history: Received 11 November 2013 Received in revised form 11 February 2014 Accepted 12 February 2014 Available online 20 February 2014 Keywords: Oxovanadium complex Calf thymus DNA (CT-DNA) Nuclease activity Gel-electrophoresis pUC19 super coiled DNA
a b s t r a c t A new oxovanadium complex [VO(sal-l-val)(phen)] (sal-l-val = Schiff base derived from salicylaldehyde and l-valine; phen = 1,10-phenanthroline) has been designed and synthesized with the aim of developing potential DNA nuclease. The interaction of DNA with this structurally characterized oxovanadium complex has been studied by various physicochemical tools like UV–vis, fluorescence, viscosity and circular dichroism (CD). The intrinsic binding constant of the complex with DNA is determined by electronic absorption studies and calculated to be (4.74 ± 0.02) × 105 M−1 . The spectroscopic studies and the viscosity measurements indicate that the complex binds calf thymus DNA (CT-DNA) by intercalative mode. The ability of the complex to induce DNA cleavage was studied by gel electrophoresis techniques. The complex has been found to promote cleavage of pUC19 plasmid DNA from the super coiled (SC) form I to nicked coiled (NC) relaxed form II with good efficiency. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The synthesis and development of metal based synthetic nucleases laid special attention in the chemotherapeutic research [1] since, they can be potentially used for cancer therapy or as restriction nucleases. DNA nucleases catalyze the cleavage of phosphodiester bonds. These enzymes play crucial roles in various DNA repair processes, involving DNA replication, base excision, nucleotide excision, base-pairing mismatch and double strand breaks. Although, numerous naturally occurring nucleases are known [2], the development of synthetic nucleases would be of great utility and importance to voluntarily monitor or manipulate biological reactions in the molecular level. The application of metal complexes as synthetic nucleases has been an area of intense interest since, the potential anti-cancer drugs, developed this way, are found to be far less toxic and more prone to exhibit antiproliferative activity [3]. Moreover, the development of metal-based artificial nucleases and nuclease mimics has been paid considerable attention in the field of molecular biology and gene technology. Again, many oxovanadium complexes are known to possess potent insulin-mimetic effects [4,5], high affinity for several enzymes and anticancer activity [6], which deserve increasing attention for application to biomedical sciences. On the other hand, many other such complexes have been known to be the initiators in the photo-cleavage of DNA [7].
∗ Corresponding author. Tel.: +91 9831129321; fax: +91 33 24146223. E-mail address: k [email protected] (K.K. Mukherjea). http://dx.doi.org/10.1016/j.ijbiomac.2014.02.033 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Applications of polyoxometalates as potential drugs against Herpes Simplex Virus and AIDS have also increased the interest to study the association between vanadium containing species and proteins [8]. The transition metal complexes of 1,10-phenanthroline or their modified variants have been widely employed [9] in DNA studies due to their applicability in bioinorganic and biomedicinal chemistry [10] as well as in the design of stereospecific DNA binding drugs [11]. They can break the DNA chains in the presence of H2 O2 and are used broadly as foot printing agents for proteins and DNAs [12]. The nuclease activity of some vanadium complexes with Schiff and phenanthroline has also been reported [13]. Although, the synthesis of some oxovanadium complexes with various amino acid Schiff base has been reported earlier [14], the detail investigation on the DNA interaction and nuclease activity by such complexes is very scanty [15–17]. In the present work, we report the nuclease activity and the DNA-binding properties of the structurally characterized amino acid based oxovanadium complex to have a deep understanding on the binding mode of this kind of complex to DNAs in order to explore new anticancer agents, biomolecular modification and detection agents as well as molecular scissors.
2. Experimental 2.1. Materials and general methods All reagents and chemicals were of AR grade and procured from commercial sources (SD Fine Chemicals, India and Aldrich) and used without further purification. Solvents were purified by standard
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Scheme 1. Synthesis of [VO(sal-l-val)(phen)].
procedures [18], wherever necessary. Calf thymus DNA (CT-DNA) was purchased from the Sigma Chemical Company, USA; supercoiled (SC) plasmid pUC19 DNA was obtained from Bangalore Genei (Bangalore, India). The solutions of CT-DNA in phosphate buffer saline (PBS) medium [0.15 M, pH 7.2] gave a ratio of A260 /A280 , of ca. 1.8–1.85, indicating that the DNA was sufficiently free from protein contamination. The DNA concentration per nucleotide was determined by absorption spectrophotometry using the molar absorption coefficient (ε = 6600 M−1 cm−1 ). Stock solutions were stored at 4 ◦ C and used within 4 days. 2.2. Preparation of the complex A solution of potassium hydroxide in 10 mL absolute alcohol and l-valine (1 mmol, 0.117 g) was stirred until dissolved in the methanol–KOH solution, thereafter a methanolic solution (2 mL) of salicylaldehyde (1 mmol) was added to it dropwise and stirred for 2 h. The resultant solution was added to a methanolic solution of vanadyl sulfate pentahydrate (1 mmol, 0.253 g) with continuous stirring, followed by the dropwise addition of a methanolic solution (5 mL) of 1,10 phenanthroline monohydrate (1 mmol, 0.198 g) and stirred for another 2 h, which on standing produced brown yellow solid (Scheme 1). Yield: 76%. IR [KBr, cm−1 ]: 1618.08 [as (COO)], 1538.19 [(C N)], and 960.91 [(V O)]. ESI-MS(+) in MeOH: m/z (relative intensity) 489 [M+ +Na, 100], 467 [m+ +1, 23], and 466 [M+ , 11] (Fig. 1). Elemental analysis: Calc: C 61.81%, H 4.54%, and N 9.01%; found: C 61.84%, H 4.50%, and N 8.99%. All physicochemical studies were done with this powdered solid. The crystal structure of the complex has been reported [19] by us earlier (Fig. 2).
Fig. 1. ESI-MS of the complex [VO(sal-l-val)(phen)].
Fig. 2. Ortep view of the structural representation of the complex.
2.3. UV–vis spectral study UV–vis spectra were recorded in a Schimadzu U-1200 spectrophotometer. The electronic spectra of the complex were monitored both in the presence and the absence of DNA. The binding constant for the interaction of the complex with DNA was obtained by monitoring the 383 nm absorption band of the complex. A fixed concentration of complex [10 M in 1% methanol–PBS buffer (v/v) solution] was titrated with increasing amounts of DNA over a range of 1–10 M. While measuring the absorption spectra, equal amounts of DNA were added to both the test and the reference solutions to eliminate any absorbance of DNA itself. The kinetic study of the decomposition of the complex for 2 h in 1% methanol–buffer (v/v) solution was undertaken to prove the stability of the complex (Fig. 3). The electronic absorption spectra of the complex solution were recorded after 5 s time intervals to see the change in absorbance with respect to time. The absorbance at 264 nm with respect to time was recorded, such values were plotted against respective time and fitted in first order rate equation, from which the dissociation constant (Kdiss ) was obtained. The dissociation constant (Kdiss ) was found to be (4.03 ± 0.02) × 10−6 s−1 and the t1/2 was calculated to be ∼2 days.
Fig. 3. Kinetic trace for the decomposition of [VO(sal-l-val)(phen)] in 1% MeOHbuffer (v/v) at 25 ◦ C. Inset is the corresponding time resolved spectra. Last spectra were taken at 7200 s.
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2.4. Fluorescence study The oxovanadium complex either in aqueous solution or in the presence of CT-DNA does not exhibit any fluorescence emission. So, the binding of the complex to CT-DNA has been studied by competitive ethidium bromide (EB) binding experiment. Perkin Elmer LS 55 spectrofluorimeter was used to measure fluorescence emission. In this binding experiment, total volume of the mixture was maintained constant 10 ml. The concentration of CT-DNA and ethidium bromide was 10.0 M and 15 M, respectively, using PBS buffer (0.15 M, pH 7.2). Aliquots of 100–1000 l from a 10% methanolic solution of 100 M stock solution of complex were added to the ethidium bromide bound CT-DNA solution so that the ratio of [complex]/EB–DNA were 0, 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0. The fluorescence was measured for each test solution after 2 h of incubation at 37 ◦ C. In either of the sets the amount of methanol was never exceeded 1% (methanol–buffer, v/v). The solutions were excited at 500 nm (with excitation and emission slit 10 nm) and spectra were recorded from 510 to 700 nm.
Fig. 4. Absorption spectra of the [VO(sal-l-val)(phen)]complex (10 M) in the presence of increasing amounts of CT-DNA, [DNA]/[complex] = (a − l): 0.0; 0.1; 0.2; 0.4; 0.6; 0.8; 0.9; 1.0.
2.5. Viscometric study The viscosity of sonicated [20] DNA (average molecular weight of ∼200 base pairs obtained using a Labsonic 2000 sonicator) was measured by a fabricated micro-viscometer maintained at 28 (±0.5) ◦ C in a thermostatic water bath. Data were presented as (/o )1/3 vs. the ratio of the concentration of metal complex to that of the CT-DNA, where and o are the viscosities of the CT-DNA solutions in the presence and absence of the complex, respectively. The viscosity of DNA ( = t − to ) was calculated from the observed flow time of CT-DNA solutions (t) and buffer solution (to ). 2.6. Circular dichroism study The CD measurements of 60 M DNA were done in the presence of various concentration of oxovanadium compound and spectra were recorded from 200 to 350 nm. The unit of CD spectra is [] × 10−3 (deg cm2 /dmol) and the CD path lengths were 1 cm in each measurement. Fixed concentration of calf thymus DNA (CTDNA) was titrated with varying amounts of the compound, whereas the total volume of the test solution remained same in all the cases. 2.7. Gel electrophoresis study
of metal complexes with DNA is determined using the following equation [21]: [DNA] [DNA] 1 = + (εa − εb ) (εb − εf ) [Kb (εb − εf )] Here, [DNA] is the concentration of DNA in nucleotides, the apparent absorption coefficients εa , εf and εb correspond to Aobsd /[complex], the extinction coefficient for the free complex, and the extinction coefficient for the complex in the fully bound form, respectively. Plots of [DNA]/(εa − εb ) vs. [DNA] gave a slope of 1/(εb − εf ) with the intercept of 1/[Kb (εb − εf )]. The intrinsic binding constant Kb was obtained from the ratio of the slope to the intercept [21]. The electronic absorption of the complex at 383 nm was monitored in the presence and absence of DNA. A considerable increase in the absorptivity and a red shift [22] of the 383 nm band was observed followed by the addition of DNA (Fig. 4). The binding constant of the DNA-complex species was calculated to be (4.74 ± 0.02) × 105 M−1 from the plot of [DNA]/(εa − εb ) vs. [DNA] (Fig. 5). The binding constant of this complex is comparable to other classical intercalators and metal complex intercalators.
The DNA cleavage activity of the complex was monitored using agarose gel electrophoresis whereby the supercoiled pUC19 DNA (0.5 g) in PBS buffer was treated individually with H2 O2 (4 M) and the metal complex [40 M (C/D = 140) and 50 M (C/D = 170)] [9]. Thereafter, pUC19 DNA was treated with 40 and 50 M of metal complex along with H2 O2 (4 M). The solutions were then incubated for 45 min at 37 ◦ C. After incubation it was mixed with a sample loading dye. The samples were loaded on a 0.9% agarose gel containing 1.0 g ml−1 ethidium bromide solution. Electrophoresis was carried out at 80 mV for 3 h in 1× TAE buffer. The bands were visualized by UV light and photographed with UVP Bio Doc-It Imaging System. The extent of super coiled (SC) pUC19 DNA cleavage induced by the complex was determined by analyzing the intensity of the bands using UVP DOC-ItLS software. 3. Results and discussion 3.1. Spectrophotometric studies on DNA binding by the complex Absorption spectrophotometry is usually used to investigate the interaction of small molecules with DNA. The binding constant
Fig. 5. Plot of [DNA]/(εa − εb ) × 1011 vs. [DNA] × 10−5 .
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Fig. 7. Plot of I/I0 vs. [VO(sal-l-val)(phen)]/DNA. Fig. 6. Fluorescence emission spectra of the EB–DNA complex in the presence of [EB] = 15 M + [DNA] = 10.0 M; (a–j) the ratio of [VO(sal-l-val)(phen)]/EB–DNA = 0, 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0; excitation wave length = 500 nm.
3.2. Fluorescence emission titrations for DNA interaction Fluorimetric measurements using ethidium bromide (EB) as a probe are usually carried out to establish the binding mode of small molecule to the target molecule, the double-helical DNA. Ethidium bromide (EB) emits intense fluorescence in the presence of DNA due to its strong intercalation between the adjacent DNA base pairs. This enhanced fluorescence can be quenched by the addition of a third molecule which can bind DNA by intercalative mode by displacing EB [23]. The emission spectra of EB bound to DNA in the absence and presence of the complex are presented in Fig. 6. The addition of the complex to DNA pretreated with EB causes a gradual quenching in emission intensity, indicating that the complex competes with EB in binding DNA, which leads to a quenching in the fluorescence intensity of EB–DNA complex system. In fact, as expected for a displacement effect, the increase in the concentration of complex gradually quenches the fluorescence intensity of EB–DNA complex system. This significant decrease in fluorescence intensity lends strong support in favor of intercalation of the complex into the DNA double helix by displacing EB. Fluorescence quenching study in the presence of the complex was analyzed further by Stern–Volmer equation: I0 /I = 1 + KSV [Q] [24]; where I0 and I are the fluorescence intensities in the absence and presence of quencher (complex) respectively; [Q] is the concentration of the quencher, KSV is the Stern–Volmer quenching constant, which is obtained from the slope of plot of I0 /I vs. [Q]. A plot of I0 /I vs. [complex]/[DNA] appears linear (Fig. 7) and the Stern–Volmer quenching constant (KSV ) was found to be 1.09 (±0.02) × 105 M−1 at 37 ◦ C. This data are also in agreement with the value obtained by electronic spectral studies [25].
3.4. Circular dichroism study The CD spectra of the free calf-thymus DNA and DNA-complex systems at different concentrations are shown in Fig. 9. The CD spectrum of the free DNA (curve a) is composed of major peaks at 221(positive), 247 (negative), and 280 nm (positive). This is consistent with CD spectrum of double-helical DNA in B conformation [28]. Upon addition of the compound (2, 4, 6, 8 and 10 M), a shifting of CD bands along with an increase in the molar ellipticity was observed. In the intercalative mode of binding of small molecules to CT-DNA, the small molecules stack between the base pairs of DNA leading to an enhancement in the molar ellipticity of the bands [29]. The present CD spectral results suggest that the oxovanadium complex binds the CT-DNA by intercalative mode with the retention of B conformation of DNA. 3.5. Gel electrophoresis study for nuclease activity The double-stranded plasmid pUC19 DNA exists in a compact supercoiled (SC) form. Upon introduction of strand breaks, the supercoiled form of DNA is disrupted into the nicked circular (NC) form or the linear form. If one strand is cleaved, the super coiled form will relax to produce a nicked circular form. If both the strands are cleaved, a linear form will be produced. Relatively fast migration is observed for super coiled form when the plasmid DNA is
3.3. Viscometric studies to confirm the DNA binding pattern Viscometric studies play a very important role in ascertaining DNA binding pattern in solution. More precisely, it can lend strong support in favor of intercalative binding. The intercalative binding by the ligand lengthens the DNA helix, which in turn, causes an increase in the viscosity of DNA [26]. The values of relative specific viscosities of DNA in the absence and presence of the complex are plotted against [complex]/[DNA] and are presented in Fig. 8. It is observed that the addition of the complex to the CT-DNA solution leads to an increase in the viscosity of the CT-DNA, thereby clearly demonstrating the intercalative binding of the complex to CT-DNA [26,27].
Fig. 8. Effect of increasing amount of [VO(sal-l-val)(phen)] complex on the specific viscosity of CT-DNA. Samples were prepared so as to give total complex/base pair ratios of 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6.
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U. Saha, K.K. Mukherjea / International Journal of Biological Macromolecules 66 (2014) 166–171 Table 1 Results of the cleavage of pUC19 DNA determined by gel electrophoresis study. Sl No.
Reaction condition
Form I (% SC)
Form II (% NC)
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6
Control DNA DNA + H2 O2 (4 M) DNA + complex (40 M) DNA + complex (40 M) + H2 O2 DNA + complex (50 M) DNA + complex (50 M) + H2 O2
88 70 55 49 51 14
12 30 45 51 49 86
(Lanes 3 and 5). Whereas on the addition of different concentrations of compound and H2 O2 together (4 M H2 O2 + 40 M) and (4 M H2 O2 + 50 M), the percentage of relaxed form of DNA increases to 51% (Lane 4) and 86% (Lane 6), respectively. This clearly indicates that the compound alone shows moderate nuclease activity but exhibits profound nuclease activity at higher concentration when used in combination with H2 O2 . To the best of our knowledge, this complex shows the highest nuclease activity among the complexes of similar classes [9,23,30]. 4. Conclusions Fig. 9. CD spectra of CT-DNA (60 M) in the absence (line a) and presence of the [VO(sal-l-val)(phen)] complex over the range of 2–10 M.
subjected to electrophoresis, while the nicked circular form migrates slowly and the linear form migrates in between SC and NC [30]. Hence, DNA strand breaks could be quantified by measuring the transformation of the super coiled form into nicked circular and linear forms. The ability of the vanadium complex to induce DNA cleavage was studied by gel electrophoresis using super coiled pUC19 DNA in PBS (0.15 M, pH 7.2) buffer. The results of the Gel electrophoresis (Fig. 10) experiment and quantitative cleavage data are presented in Table 1. In the gel electrophoresis six bands are observed. Lane 1 contains only DNA and has 88% supercoiled form, i.e., Form I and 12% relaxed form, i.e., Form II. On the addition of H2 O2 (4 M), percentage of relaxed form increases to 30% (Lane 2) while on the addition of the compound (40 and 50 M), the percentage of relaxed form increases to 45 and 49, respectively
The DNA binding and nuclease activity of a new oxovanadium complex [VO (sal-l-val)(phen)] has been established. Absorption titration studies indicate that the complex binds CT-DNA by intercalative mode. Fluorimetric studies also support the proposition of intercalative binding. The intercalative binding of the complex with CT-DNA has been further substantiated by CD and viscometric studies. The nuclease activity of the complex in the presence of H2 O2 has been studied by gel electrophoresis of supercoiled (SC) pUC19 DNA, whereby the results show that the complex exhibits excellent nuclease activity in the presence of peroxide. Acknowledgments The authors are thankful to UGC, New Delhi for financial support in the form of a Major Research Project [Sanction no.F.NO. 39-706/2010(SR)] to K.K.M., where U.S. is a project fellow. Thanks are also due to the UGC-CAS, Department of Chemistry, Jadavpur University for circular dichroism instrument. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2014. 02.033. References
Fig. 10. (a) Agarose gel (0.9%) electrophoregram of supercoiled DNA (0.5 g) incubated for 45 min at 37 ◦ C, in PBS buffer (0.15 M, pH 7.2) at 37 ◦ C. Lane 1: DNA control; Lane 2: DNA + H2 O2 ; Lane 3: DNA + complex (40 M); Lane 4: DNA + complex (40 M) + H2 O2 ; Lane 5: DNA + complex (50 M); Lane 6: DNA + complex (50 M) + H2 O2. (b) Graphical representation of % of cleavage in different lanes.
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DNA binding and nuclease activity of an oxovanadium valinato-Schiff base complex Urmila Saha, Kalyan K. Mukherjea ∗ Department of Chemistry, Jadavpur University, Kolkata 700032, India
a r t i c l e
i n f o
Article history: Received 11 November 2013 Received in revised form 11 February 2014 Accepted 12 February 2014 Available online 20 February 2014 Keywords: Oxovanadium complex Calf thymus DNA (CT-DNA) Nuclease activity Gel-electrophoresis pUC19 super coiled DNA
a b s t r a c t A new oxovanadium complex [VO(sal-l-val)(phen)] (sal-l-val = Schiff base derived from salicylaldehyde and l-valine; phen = 1,10-phenanthroline) has been designed and synthesized with the aim of developing potential DNA nuclease. The interaction of DNA with this structurally characterized oxovanadium complex has been studied by various physicochemical tools like UV–vis, fluorescence, viscosity and circular dichroism (CD). The intrinsic binding constant of the complex with DNA is determined by electronic absorption studies and calculated to be (4.74 ± 0.02) × 105 M−1 . The spectroscopic studies and the viscosity measurements indicate that the complex binds calf thymus DNA (CT-DNA) by intercalative mode. The ability of the complex to induce DNA cleavage was studied by gel electrophoresis techniques. The complex has been found to promote cleavage of pUC19 plasmid DNA from the super coiled (SC) form I to nicked coiled (NC) relaxed form II with good efficiency. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The synthesis and development of metal based synthetic nucleases laid special attention in the chemotherapeutic research [1] since, they can be potentially used for cancer therapy or as restriction nucleases. DNA nucleases catalyze the cleavage of phosphodiester bonds. These enzymes play crucial roles in various DNA repair processes, involving DNA replication, base excision, nucleotide excision, base-pairing mismatch and double strand breaks. Although, numerous naturally occurring nucleases are known [2], the development of synthetic nucleases would be of great utility and importance to voluntarily monitor or manipulate biological reactions in the molecular level. The application of metal complexes as synthetic nucleases has been an area of intense interest since, the potential anti-cancer drugs, developed this way, are found to be far less toxic and more prone to exhibit antiproliferative activity [3]. Moreover, the development of metal-based artificial nucleases and nuclease mimics has been paid considerable attention in the field of molecular biology and gene technology. Again, many oxovanadium complexes are known to possess potent insulin-mimetic effects [4,5], high affinity for several enzymes and anticancer activity [6], which deserve increasing attention for application to biomedical sciences. On the other hand, many other such complexes have been known to be the initiators in the photo-cleavage of DNA [7].
∗ Corresponding author. Tel.: +91 9831129321; fax: +91 33 24146223. E-mail address: k [email protected] (K.K. Mukherjea). http://dx.doi.org/10.1016/j.ijbiomac.2014.02.033 0141-8130/© 2014 Elsevier B.V. All rights reserved.
Applications of polyoxometalates as potential drugs against Herpes Simplex Virus and AIDS have also increased the interest to study the association between vanadium containing species and proteins [8]. The transition metal complexes of 1,10-phenanthroline or their modified variants have been widely employed [9] in DNA studies due to their applicability in bioinorganic and biomedicinal chemistry [10] as well as in the design of stereospecific DNA binding drugs [11]. They can break the DNA chains in the presence of H2 O2 and are used broadly as foot printing agents for proteins and DNAs [12]. The nuclease activity of some vanadium complexes with Schiff and phenanthroline has also been reported [13]. Although, the synthesis of some oxovanadium complexes with various amino acid Schiff base has been reported earlier [14], the detail investigation on the DNA interaction and nuclease activity by such complexes is very scanty [15–17]. In the present work, we report the nuclease activity and the DNA-binding properties of the structurally characterized amino acid based oxovanadium complex to have a deep understanding on the binding mode of this kind of complex to DNAs in order to explore new anticancer agents, biomolecular modification and detection agents as well as molecular scissors.
2. Experimental 2.1. Materials and general methods All reagents and chemicals were of AR grade and procured from commercial sources (SD Fine Chemicals, India and Aldrich) and used without further purification. Solvents were purified by standard
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Scheme 1. Synthesis of [VO(sal-l-val)(phen)].
procedures [18], wherever necessary. Calf thymus DNA (CT-DNA) was purchased from the Sigma Chemical Company, USA; supercoiled (SC) plasmid pUC19 DNA was obtained from Bangalore Genei (Bangalore, India). The solutions of CT-DNA in phosphate buffer saline (PBS) medium [0.15 M, pH 7.2] gave a ratio of A260 /A280 , of ca. 1.8–1.85, indicating that the DNA was sufficiently free from protein contamination. The DNA concentration per nucleotide was determined by absorption spectrophotometry using the molar absorption coefficient (ε = 6600 M−1 cm−1 ). Stock solutions were stored at 4 ◦ C and used within 4 days. 2.2. Preparation of the complex A solution of potassium hydroxide in 10 mL absolute alcohol and l-valine (1 mmol, 0.117 g) was stirred until dissolved in the methanol–KOH solution, thereafter a methanolic solution (2 mL) of salicylaldehyde (1 mmol) was added to it dropwise and stirred for 2 h. The resultant solution was added to a methanolic solution of vanadyl sulfate pentahydrate (1 mmol, 0.253 g) with continuous stirring, followed by the dropwise addition of a methanolic solution (5 mL) of 1,10 phenanthroline monohydrate (1 mmol, 0.198 g) and stirred for another 2 h, which on standing produced brown yellow solid (Scheme 1). Yield: 76%. IR [KBr, cm−1 ]: 1618.08 [as (COO)], 1538.19 [(C N)], and 960.91 [(V O)]. ESI-MS(+) in MeOH: m/z (relative intensity) 489 [M+ +Na, 100], 467 [m+ +1, 23], and 466 [M+ , 11] (Fig. 1). Elemental analysis: Calc: C 61.81%, H 4.54%, and N 9.01%; found: C 61.84%, H 4.50%, and N 8.99%. All physicochemical studies were done with this powdered solid. The crystal structure of the complex has been reported [19] by us earlier (Fig. 2).
Fig. 1. ESI-MS of the complex [VO(sal-l-val)(phen)].
Fig. 2. Ortep view of the structural representation of the complex.
2.3. UV–vis spectral study UV–vis spectra were recorded in a Schimadzu U-1200 spectrophotometer. The electronic spectra of the complex were monitored both in the presence and the absence of DNA. The binding constant for the interaction of the complex with DNA was obtained by monitoring the 383 nm absorption band of the complex. A fixed concentration of complex [10 M in 1% methanol–PBS buffer (v/v) solution] was titrated with increasing amounts of DNA over a range of 1–10 M. While measuring the absorption spectra, equal amounts of DNA were added to both the test and the reference solutions to eliminate any absorbance of DNA itself. The kinetic study of the decomposition of the complex for 2 h in 1% methanol–buffer (v/v) solution was undertaken to prove the stability of the complex (Fig. 3). The electronic absorption spectra of the complex solution were recorded after 5 s time intervals to see the change in absorbance with respect to time. The absorbance at 264 nm with respect to time was recorded, such values were plotted against respective time and fitted in first order rate equation, from which the dissociation constant (Kdiss ) was obtained. The dissociation constant (Kdiss ) was found to be (4.03 ± 0.02) × 10−6 s−1 and the t1/2 was calculated to be ∼2 days.
Fig. 3. Kinetic trace for the decomposition of [VO(sal-l-val)(phen)] in 1% MeOHbuffer (v/v) at 25 ◦ C. Inset is the corresponding time resolved spectra. Last spectra were taken at 7200 s.
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2.4. Fluorescence study The oxovanadium complex either in aqueous solution or in the presence of CT-DNA does not exhibit any fluorescence emission. So, the binding of the complex to CT-DNA has been studied by competitive ethidium bromide (EB) binding experiment. Perkin Elmer LS 55 spectrofluorimeter was used to measure fluorescence emission. In this binding experiment, total volume of the mixture was maintained constant 10 ml. The concentration of CT-DNA and ethidium bromide was 10.0 M and 15 M, respectively, using PBS buffer (0.15 M, pH 7.2). Aliquots of 100–1000 l from a 10% methanolic solution of 100 M stock solution of complex were added to the ethidium bromide bound CT-DNA solution so that the ratio of [complex]/EB–DNA were 0, 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0. The fluorescence was measured for each test solution after 2 h of incubation at 37 ◦ C. In either of the sets the amount of methanol was never exceeded 1% (methanol–buffer, v/v). The solutions were excited at 500 nm (with excitation and emission slit 10 nm) and spectra were recorded from 510 to 700 nm.
Fig. 4. Absorption spectra of the [VO(sal-l-val)(phen)]complex (10 M) in the presence of increasing amounts of CT-DNA, [DNA]/[complex] = (a − l): 0.0; 0.1; 0.2; 0.4; 0.6; 0.8; 0.9; 1.0.
2.5. Viscometric study The viscosity of sonicated [20] DNA (average molecular weight of ∼200 base pairs obtained using a Labsonic 2000 sonicator) was measured by a fabricated micro-viscometer maintained at 28 (±0.5) ◦ C in a thermostatic water bath. Data were presented as (/o )1/3 vs. the ratio of the concentration of metal complex to that of the CT-DNA, where and o are the viscosities of the CT-DNA solutions in the presence and absence of the complex, respectively. The viscosity of DNA ( = t − to ) was calculated from the observed flow time of CT-DNA solutions (t) and buffer solution (to ). 2.6. Circular dichroism study The CD measurements of 60 M DNA were done in the presence of various concentration of oxovanadium compound and spectra were recorded from 200 to 350 nm. The unit of CD spectra is [] × 10−3 (deg cm2 /dmol) and the CD path lengths were 1 cm in each measurement. Fixed concentration of calf thymus DNA (CTDNA) was titrated with varying amounts of the compound, whereas the total volume of the test solution remained same in all the cases. 2.7. Gel electrophoresis study
of metal complexes with DNA is determined using the following equation [21]: [DNA] [DNA] 1 = + (εa − εb ) (εb − εf ) [Kb (εb − εf )] Here, [DNA] is the concentration of DNA in nucleotides, the apparent absorption coefficients εa , εf and εb correspond to Aobsd /[complex], the extinction coefficient for the free complex, and the extinction coefficient for the complex in the fully bound form, respectively. Plots of [DNA]/(εa − εb ) vs. [DNA] gave a slope of 1/(εb − εf ) with the intercept of 1/[Kb (εb − εf )]. The intrinsic binding constant Kb was obtained from the ratio of the slope to the intercept [21]. The electronic absorption of the complex at 383 nm was monitored in the presence and absence of DNA. A considerable increase in the absorptivity and a red shift [22] of the 383 nm band was observed followed by the addition of DNA (Fig. 4). The binding constant of the DNA-complex species was calculated to be (4.74 ± 0.02) × 105 M−1 from the plot of [DNA]/(εa − εb ) vs. [DNA] (Fig. 5). The binding constant of this complex is comparable to other classical intercalators and metal complex intercalators.
The DNA cleavage activity of the complex was monitored using agarose gel electrophoresis whereby the supercoiled pUC19 DNA (0.5 g) in PBS buffer was treated individually with H2 O2 (4 M) and the metal complex [40 M (C/D = 140) and 50 M (C/D = 170)] [9]. Thereafter, pUC19 DNA was treated with 40 and 50 M of metal complex along with H2 O2 (4 M). The solutions were then incubated for 45 min at 37 ◦ C. After incubation it was mixed with a sample loading dye. The samples were loaded on a 0.9% agarose gel containing 1.0 g ml−1 ethidium bromide solution. Electrophoresis was carried out at 80 mV for 3 h in 1× TAE buffer. The bands were visualized by UV light and photographed with UVP Bio Doc-It Imaging System. The extent of super coiled (SC) pUC19 DNA cleavage induced by the complex was determined by analyzing the intensity of the bands using UVP DOC-ItLS software. 3. Results and discussion 3.1. Spectrophotometric studies on DNA binding by the complex Absorption spectrophotometry is usually used to investigate the interaction of small molecules with DNA. The binding constant
Fig. 5. Plot of [DNA]/(εa − εb ) × 1011 vs. [DNA] × 10−5 .
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Fig. 7. Plot of I/I0 vs. [VO(sal-l-val)(phen)]/DNA. Fig. 6. Fluorescence emission spectra of the EB–DNA complex in the presence of [EB] = 15 M + [DNA] = 10.0 M; (a–j) the ratio of [VO(sal-l-val)(phen)]/EB–DNA = 0, 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0; excitation wave length = 500 nm.
3.2. Fluorescence emission titrations for DNA interaction Fluorimetric measurements using ethidium bromide (EB) as a probe are usually carried out to establish the binding mode of small molecule to the target molecule, the double-helical DNA. Ethidium bromide (EB) emits intense fluorescence in the presence of DNA due to its strong intercalation between the adjacent DNA base pairs. This enhanced fluorescence can be quenched by the addition of a third molecule which can bind DNA by intercalative mode by displacing EB [23]. The emission spectra of EB bound to DNA in the absence and presence of the complex are presented in Fig. 6. The addition of the complex to DNA pretreated with EB causes a gradual quenching in emission intensity, indicating that the complex competes with EB in binding DNA, which leads to a quenching in the fluorescence intensity of EB–DNA complex system. In fact, as expected for a displacement effect, the increase in the concentration of complex gradually quenches the fluorescence intensity of EB–DNA complex system. This significant decrease in fluorescence intensity lends strong support in favor of intercalation of the complex into the DNA double helix by displacing EB. Fluorescence quenching study in the presence of the complex was analyzed further by Stern–Volmer equation: I0 /I = 1 + KSV [Q] [24]; where I0 and I are the fluorescence intensities in the absence and presence of quencher (complex) respectively; [Q] is the concentration of the quencher, KSV is the Stern–Volmer quenching constant, which is obtained from the slope of plot of I0 /I vs. [Q]. A plot of I0 /I vs. [complex]/[DNA] appears linear (Fig. 7) and the Stern–Volmer quenching constant (KSV ) was found to be 1.09 (±0.02) × 105 M−1 at 37 ◦ C. This data are also in agreement with the value obtained by electronic spectral studies [25].
3.4. Circular dichroism study The CD spectra of the free calf-thymus DNA and DNA-complex systems at different concentrations are shown in Fig. 9. The CD spectrum of the free DNA (curve a) is composed of major peaks at 221(positive), 247 (negative), and 280 nm (positive). This is consistent with CD spectrum of double-helical DNA in B conformation [28]. Upon addition of the compound (2, 4, 6, 8 and 10 M), a shifting of CD bands along with an increase in the molar ellipticity was observed. In the intercalative mode of binding of small molecules to CT-DNA, the small molecules stack between the base pairs of DNA leading to an enhancement in the molar ellipticity of the bands [29]. The present CD spectral results suggest that the oxovanadium complex binds the CT-DNA by intercalative mode with the retention of B conformation of DNA. 3.5. Gel electrophoresis study for nuclease activity The double-stranded plasmid pUC19 DNA exists in a compact supercoiled (SC) form. Upon introduction of strand breaks, the supercoiled form of DNA is disrupted into the nicked circular (NC) form or the linear form. If one strand is cleaved, the super coiled form will relax to produce a nicked circular form. If both the strands are cleaved, a linear form will be produced. Relatively fast migration is observed for super coiled form when the plasmid DNA is
3.3. Viscometric studies to confirm the DNA binding pattern Viscometric studies play a very important role in ascertaining DNA binding pattern in solution. More precisely, it can lend strong support in favor of intercalative binding. The intercalative binding by the ligand lengthens the DNA helix, which in turn, causes an increase in the viscosity of DNA [26]. The values of relative specific viscosities of DNA in the absence and presence of the complex are plotted against [complex]/[DNA] and are presented in Fig. 8. It is observed that the addition of the complex to the CT-DNA solution leads to an increase in the viscosity of the CT-DNA, thereby clearly demonstrating the intercalative binding of the complex to CT-DNA [26,27].
Fig. 8. Effect of increasing amount of [VO(sal-l-val)(phen)] complex on the specific viscosity of CT-DNA. Samples were prepared so as to give total complex/base pair ratios of 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6.
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U. Saha, K.K. Mukherjea / International Journal of Biological Macromolecules 66 (2014) 166–171 Table 1 Results of the cleavage of pUC19 DNA determined by gel electrophoresis study. Sl No.
Reaction condition
Form I (% SC)
Form II (% NC)
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6
Control DNA DNA + H2 O2 (4 M) DNA + complex (40 M) DNA + complex (40 M) + H2 O2 DNA + complex (50 M) DNA + complex (50 M) + H2 O2
88 70 55 49 51 14
12 30 45 51 49 86
(Lanes 3 and 5). Whereas on the addition of different concentrations of compound and H2 O2 together (4 M H2 O2 + 40 M) and (4 M H2 O2 + 50 M), the percentage of relaxed form of DNA increases to 51% (Lane 4) and 86% (Lane 6), respectively. This clearly indicates that the compound alone shows moderate nuclease activity but exhibits profound nuclease activity at higher concentration when used in combination with H2 O2 . To the best of our knowledge, this complex shows the highest nuclease activity among the complexes of similar classes [9,23,30]. 4. Conclusions Fig. 9. CD spectra of CT-DNA (60 M) in the absence (line a) and presence of the [VO(sal-l-val)(phen)] complex over the range of 2–10 M.
subjected to electrophoresis, while the nicked circular form migrates slowly and the linear form migrates in between SC and NC [30]. Hence, DNA strand breaks could be quantified by measuring the transformation of the super coiled form into nicked circular and linear forms. The ability of the vanadium complex to induce DNA cleavage was studied by gel electrophoresis using super coiled pUC19 DNA in PBS (0.15 M, pH 7.2) buffer. The results of the Gel electrophoresis (Fig. 10) experiment and quantitative cleavage data are presented in Table 1. In the gel electrophoresis six bands are observed. Lane 1 contains only DNA and has 88% supercoiled form, i.e., Form I and 12% relaxed form, i.e., Form II. On the addition of H2 O2 (4 M), percentage of relaxed form increases to 30% (Lane 2) while on the addition of the compound (40 and 50 M), the percentage of relaxed form increases to 45 and 49, respectively
The DNA binding and nuclease activity of a new oxovanadium complex [VO (sal-l-val)(phen)] has been established. Absorption titration studies indicate that the complex binds CT-DNA by intercalative mode. Fluorimetric studies also support the proposition of intercalative binding. The intercalative binding of the complex with CT-DNA has been further substantiated by CD and viscometric studies. The nuclease activity of the complex in the presence of H2 O2 has been studied by gel electrophoresis of supercoiled (SC) pUC19 DNA, whereby the results show that the complex exhibits excellent nuclease activity in the presence of peroxide. Acknowledgments The authors are thankful to UGC, New Delhi for financial support in the form of a Major Research Project [Sanction no.F.NO. 39-706/2010(SR)] to K.K.M., where U.S. is a project fellow. Thanks are also due to the UGC-CAS, Department of Chemistry, Jadavpur University for circular dichroism instrument. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2014. 02.033. References
Fig. 10. (a) Agarose gel (0.9%) electrophoregram of supercoiled DNA (0.5 g) incubated for 45 min at 37 ◦ C, in PBS buffer (0.15 M, pH 7.2) at 37 ◦ C. Lane 1: DNA control; Lane 2: DNA + H2 O2 ; Lane 3: DNA + complex (40 M); Lane 4: DNA + complex (40 M) + H2 O2 ; Lane 5: DNA + complex (50 M); Lane 6: DNA + complex (50 M) + H2 O2. (b) Graphical representation of % of cleavage in different lanes.
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