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Interaction studies of Epirubicin with DNA using spectroscopic techniques
Journal of Molecular Structure 1000 (2011) 150–154
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Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Interaction studies of Epirubicin with DNA using spectroscopic techniques Sonika Charak, Deepak K. Jangir, Gunjan Tyagi, Ranjana Mehrotra ⇑ Quantum Optics and Photon Physics, National Physical Laboratory, Council of Scientific and Industrial Research, Dr. K.S. Krishnan Marg, New Delhi 110 012, India
a r t i c l e
i n f o
Article history: Received 9 April 2011 Received in revised form 12 June 2011 Accepted 13 June 2011 Available online 5 July 2011 Keywords: Epirubicin FTIR spectroscopy UV–visible spectroscopy Circular dichroism spectroscopy DNA conformation
a b s t r a c t Epirubicin (EPR) is an anticancer chemotherapeutic drug which exerts its cytotoxic effect by inhibiting DNA synthesis and DNA replication. We report the structural and conformational effect of EPR binding on DNA duplex under physiological conditions. Fourier transform infrared spectroscopy (FTIR), ultraviolet–visible (UV–visible) spectroscopy and circular dichroism (CD) spectroscopy were used to determine the binding mode and binding constant of EPR with DNA. The effect of EPR–DNA complexation on stability and secondary structure of DNA was studied. FTIR measurements showed that EPR–DNA interaction occurs through guanine and cytosine bases. External binding of EPR with DNA was observed through phosphate backbone. UV–visible measurements revealed the intercalative mode of binding of EPR with DNA. The binding constant was estimated to be K = 3.4 104 which is indicative of moderate binding between EPR and DNA helix. FTIR and CD studies suggested partial transition from B-conformation of DNA to A-conformation of DNA after EPR binding to DNA duplex. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Epirubicin (EPR) is an anthracycline derivative used as chemotherapeutic drug. It is used for the treatment of variety of cancers, primarily breast cancer, ovarian cancer, gastric and lung cancer [1,2]. EPR is a stereoisomer of doxorubicin and differs from it in hydroxyl group orientation at carbon atom four (C4) of the hexapyranosyl sugar (Fig. 1). EPR is favored over doxorubicin due to more therapeutic index and lesser side effects [3,4]. It is also used in conjunction with other drugs for the treatment of non Hodgkin lymphoma and breast cancer [5–7]. The exact mechanism of action by which anthracycline drugs exert their cytotoxic effect is still unclear. It is proposed that rapidly replicating DNA in cancer cells is the major target of these drugs. The incorporation of drug into double stranded DNA disturbs its helical structure causing strand breakage. The primary mode of interaction of anthracycline drugs with DNA is intercalation. Intercalation of anthracycline drugs into the DNA bases lead to inhibition of DNA synthesis and replication [8]. Inhibition of topoisomerase-II enzyme activity, which assists in DNA unwinding during DNA replication, is another mechanism involved in the cytotoxic effects of anthracycline drugs [9]. In vitro and in vivo experiments have shown that EPR inhibit cell proliferation and DNA synthesis in carcinoma cell lines [10–12]. Along with anticancer properties, EPR also shows side effect like cardiotoxicity and myelosuppression [13]. Cytotoxicity caused by EPR effects the
⇑ Corresponding author. Tel.: +91 11 45608366; fax: +91 11 45609310. E-mail address: [email protected] (R. Mehrotra). 0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2011.06.013
structure and function of DNA, therefore detailed study on the interaction of EPR with DNA becomes important. FTIR spectroscopy has emerged as an efficient tool in life science due to its potential to provide fine structural information of the various biomolecules and biomolecule ligand complexes. This is widely used technique because it offers many advantages. This technique is fast, nondestructive and provide snapshot of all the conformations of the sample compound. It is very useful in characterizing the nature of drug and biomolecule interaction and effect of this interaction on the structure of the biomolecule [14–17]. It has been used in recent years to determine binding site of various drugs sequence specificity and changes in the structure of biomolecules after binding with the drugs [18–20]. Along with FTIR, circular dichroism spectroscopy is also a useful tool in determining the secondary structure of DNA and changes in the conformation states of DNA after DNA-drug complex formation [21,22]. In the present work, interaction of EPR with DNA has been investigated using FTIR, CD and UV–visible spectroscopy (Fig. 2). Different molar ratios of EPR with constant DNA concentration were prepared in aqueous solution at physiological conditions and subsequently analyzed. 2. Materials and methods EPR and highly polymerized type-1 calf thymus DNA (sodium content 6%) were purchased from Sigma Aldrich, USA. DNA purity estimation was done by recording the UV–visible absorbance at 260 nm and 280 nm. The ratio of absorbance at 260–280 nm was found to be 1.86, which indicates that the DNA was free from any contamination [23]. Other chemicals used were of analytical
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to noise ratio, a total of 128 scans were performed. For water subtraction, spectra of buffer at pH = 7.4 was recorded and then subtracted from the spectra of free DNA and EPR–DNA complexes. A satisfactory water subtraction is achieved when flat baseline around 2200 cm1 is produced, where water combination bands are positioned. This is a rough estimation method for water subtraction, but removes water spectral features in a satisfactory way [24]. 2.3. UV–visible spectroscopic measurements
Fig. 1. Chemical structure of: (a) doxorubicin (b) epirubicin.
The absorption spectra of free DNA and EPR–DNA complex were collected using Cary 100 Bio UV–visible spectrophotometer. Quartz cuvette of 1 cm path length was used. The value of binding constant of EPR–DNA complex was determined by the method described by Kanakis et al. [25]. It was assumed that only one type of interaction (1:1 complex formation) occurs between EPR and DNA in aqueous solution [26,27]. On the basis of this assumption, Eqs. (1) and (2) can be established.
DNA þ EPR DNA : EPR
ð1Þ
The equilibrium constant is given by
K ¼ ðDNA : EPRÞ=ðDNAÞðEPRÞ
ð2Þ
We can write the Eq. (2) as
K ¼ ðC DE Þ=½C D ½C E
Fig. 2. Schematic diagram of EPR–DNA complex.
grade and were used as supplied. Ultrapure water was used to prepare EPR, DNA and buffer solutions. 2.1. Preparation of stock solutions For preparation of stock solution, highly polymerized calf thymus DNA (10 mg/ml) was dissolved in 10 mM Tris–HCl buffer (pH = 7.4). DNA stock solution was kept at 8 °C for 24 h to make homogenous solution. Final concentration of DNA stock solution was determined spectrophotometrically at 260 nm using a molar extinction coefficient of 6600 cm1 M1. EPR stock solution and different concentration of EPR were prepared in ultrapure water. Different molar ratios of EPR–DNA complex were prepared by drop by drop addition of EPR to DNA solution. FTIR studies were carried out with EPR/DNA molar ratios (r) = 1/50, 1/100, 1/150, and 1/200 with final DNA concentration of 14 mM. For circular dichroism studies, EPR/DNA molar ratios of 1/50, 1/75, 1/100 and 1/125 were used with constant DNA concentration of 5 mM. EPR concentration in the range of 10–60 lM was used with 100 lM constant DNA concentration for UV–visible studies.
ð3Þ
CDE, CD, CE are the analytical concentration of EPR–DNA complex, DNA and EPR in solution respectively. Beer Lambert law for the absorption of light is assumed to be followed by the ligand substrate binding.
C D ¼ C Do C DE
ð4Þ
C DE ¼ A A0 =eDE L
ð5Þ
and
C Do ¼ A0 =eD l
ð6Þ
where CDo is the concentration of pure DNA, A0 and A are the absorbance of pure DNA and in the presence of EPR respectively at 260 nm. eD and eDE are the molar extinction coefficient of DNA and EPR–DNA complex respectively. l is the path length of the cuvette (1 cm). By putting the values of CD and CDE from above equations in to the Eq. (3), following equation can be deduced:
A0 =A A0 ¼ eD =eDE þ eD =ðeDE KÞ 1=C E The double reciprocal plot of 1/(A A0) versus 1/CE is linear and the binding constant (K) can be estimated by calculating the ratio of the intercept to the slope [28].
2.2. FTIR spectroscopic measurements 2.4. Circular dichroism spectroscopic measurements Infrared spectra of DNA and EPR–DNA complexes were recorded on FTIR spectrophotometer (Varian-660 model) equipped with deuterated triglycine sulfate (DTGS) detector and KBr beamsplitter. Spectra were collected after incubation of EPR–DNA complex for 2 h at room temperature. Spectra of EPR–DNA solutions were recorded using horizontal attenuated total internal reflection (HATR) crystal. Interferograms were accumulated in the spectral range of 1800–600 cm1 with 4 cm1 resolution. All the measurements were done in triplicate. To achieve better signal
CD spectroscopic measurements were recorded with 1 mm path length quartz cuvette using Jasco J812 CD spectrophotometer. Measurements were done in the far UV region (200–320 nm) in the nitrogen atmosphere. Sample spectra were collected at room temperature. An accumulation of average of three scans was done for each measurement with scan speed of 50 nm/min. Spectrum of Tris-buffer at pH 7.4 was recorded and was subtracted from the spectra of DNA and EPR–DNA complex.
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Fig. 3. Stacked view of FTIR spectra of free DNA and EPR–DNA complex at different molar ratios of 1/50, 1/100 and 1/150 in the spectral region of 1800–700 cm1.
3. Results and discussion 3.1. FTIR analysis FTIR spectra of free DNA and EPR–DNA complexes were monitored at different EPR/DNA ratios as shown in Fig. 3. FTIR spectrum of free DNA in B-conformation corresponds to four major nitrogenous bases, phosphate groups and sugar bands in the region of 1800–700 cm1. 3.1.1. Base analysis Fig. 3 shows the infrared spectrum of free DNA and EPR–DNA complexes. The peak at position 1710 cm1 corresponds to in plane vibration of the guanine (G) [20]. 1667 cm1 peak is attributed to thymine (T) stretching [20]. It is observed that after binding of EPR with DNA, peak at position 1710 cm1 shifts upwards to 1714–1716 cm1 and peak at position 1667 cm1 shifts downwards to 1663 cm1. Adenine (A) peak at position 1603 cm1 gets shifted towards higher wave number (1604). Peak at position 1489 cm1 which corresponds to cytosine (C) base vibrations shows upward shift of 4–5 cm1. The change in the peak position of the bases is observed in all EPR–DNA complexes. This shifting in peak position is also accompanied by the change in intensity in all EPR–DNA complexes. Presence of positive features at position 1723 cm1, 1655 cm1 and 1614 cm1 in the difference spectra correspond to increased intensity of guanine, thymine and adenine respectively (Fig. 4). Changes in major peak position and intensity variation indicate towards the intercalative binding mode of EPR. Spectral changes observed after EPR binding with DNA at guanine, cytosine bases indicate that EPR might get intercalated between G– C bases. 3.1.2. Phosphate analysis The peak at position 1225 cm1 due to asymmetric phosphate stretching in the spectrum of free DNA shows downward shift of 4–5 cm1 after binding with EPR in all the molar ratios studied.
The phosphate peak due to symmetrical stretching vibration at position 1087 cm1 shows downward shift of 1 cm1. Presence of positive features at 1211 cm1 and 1125 cm1 in the difference spectra indicates towards the increased intensity of asymmetric and symmetric phosphate peak after binding with DNA (Fig. 4). Peak shifting and intensity increase in intensity of asymmetric and symmetric phosphate peaks may be attributed to external binding of EPR with DNA helix backbone. 3.1.3. Conformational analysis Infrared peaks in this region primarily occur due to vibration in sugar bases. In B–A conformational change, guanine peak at 1710 cm1, phosphodiester peak at 837 cm1 and symmetric phosphate peak at 1087 cm1 exhibits change in their position. Similarly during B to Z transition peak at 837 cm1 shift to 800 cm1, guanine peak at 1710–1717 cm1 appears at 1690 cm1 and 1225 cm1 peak shifts towards 1215 cm1 [16]. It is observed that sugar phosphate band at position 1055 cm1 does not exhibits any significant change after binding with EPR. B-DNA marker peak at position 968 cm1 shows downward shift of 1–2 cm1 in EPR– DNA complex at different molar ratios examined in the study. Similarly the B-DNA marker peak at 837 cm1 due to sugar-phosphate chain vibrations shifts at 840 cm1. A new infrared peak emerges at 868 cm1 in all EPR–DNA complexes. Shifting in the peak positions of B-DNA conformation characteristic peaks and presence of new peak 868 cm1 in all EPR–DNA complexes studied indicates towards partial B–A transition of DNA after binding with EPR. 3.2. UV–visible studies UV–visible spectra of free DNA and EPR at different molar ratio are shown in Fig. 5. Hyperchromic effect was observed after EPR binding to free DNA. Proportional increase in absorption maxima (A260 nm) of free DNA is observed with increasing concentration of EPR. DNA helix is stabilized by hydrogen bonds between the two strands. EPR might intercalates between the two strands of
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Fig. 4. IR difference spectra [(DNA + EPR) – DNA solution] of EPR/DNA molar ratios of 1/50, 1/100 and 1/150 in the spectral region of 1800–700 cm1.
Fig. 5. UV–visible spectra of free DNA and its EPR–DNA complex at different molar ratios of 1/3, 1/5, 1/10, 1/20.
DNA causing increase in the absorption maxima of DNA due to the unwinding of DNA double helical structure. Due to unwinding of DNA, the aromatic bases get more exposed to UV radiation, hence results in increased intensity of absorption. The absorption maxima peak at 260 nm of free DNA also exhibits downward shifting (2 nm) indicating towards the intercalative binding mode [29]. Binding constant of EPR–DNA complex was calculated to determine its stability by using the method described in the experimental section. The binding constant for EPR in the concentration range of 10–60 lM after binding with DNA is observed to be, K = 3.4 104 (Fig. 6). The order of the binding constant is intimation of moderate binding between EPR and DNA. 3.3. CD analysis Circular dichroism spectra of different molar ratios of EPR with DNA and free DNA are shown in Fig. 7. Four major peaks for the Bconformation of DNA, 211 nm (negative), 221 nm (positive),
Fig. 6. Double reciprocal plot between 1/A A0 and 1/CE. A0 is the initial absorption of free DNA and A is the absorption at different concentrations at 260 nm. CE is the analytical concentration of EPR in solution.
245 nm (negative) and 275 nm (positive) are characteristic peaks for B-conformation of DNA. The peak at position 221 nm (negative), 245 nm (negative) and 275 nm (positive) are ascribed to formation of hydrogen bond, stacking of the bases and right handed helicity of B-DNA respectively [30]. Conformational change from B-form to A-form is accompanied by change in intensity as well as wavelength of the four characteristic peaks of B-DNA. Gain in negativity with minor shifting at 210 nm and 245 nm, decrease in intensity at 222 nm and increase in intensity at 275 nm with minor shifting (red shift) indicates towards complete B to A-DNA transition [31–33]. Changes in all negative and positive peak positions were observed in CD spectrum of DNA after binding with EPR (Fig. 7). Proportional increase in positive and decrease in negative peak position without any significant red shift was observed with increasing concentration of EPR. Spectral changes in intensity at 210 nm, 222 nm, 245 nm and 275 nm without shifting in peak position after interaction of EPR with DNA indicates towards partial B–A transition. These results are in agreement with our FTIR results
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Research for providing financial support. The infrastructural facility provided by Central Instrumentation Facility, Biotech Centre, University of Delhi South Campus is also acknowledged. References [1] [2] [3] [4] [5] [6]
[7] [8] [9] [10] [11] [12] [13] Fig. 7. Circular dichroism spectra of the free calf-thymus DNA and EPR/DNA molar ratios of 1/50, 1/75 and 1/100.
for partial B–A transition, due to emergence of a new infrared peak 868 cm1 in the infrared spectra of all DNA–EPR complexes. 4. Conclusion In the present study, we examined the structural and conformational effect of EPR on double helical structure of DNA using FTIR, UV–visible and CD spectroscopic techniques. FTIR results reflect toward binding of the EPR with DNA through guanine (G) and cytosine (C) bases and phosphate backbone by intercalation and external mode of binding. UV–visible spectral results further support the intercalative property of the EPR interaction with DNA. Binding constant of EPR–DNA complex was estimated to be K = 3.4 104. FTIR and CD results also suggest partial transition of B-conformation of DNA towards A-conformation of DNA.
[14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]
Acknowledgements S.C. is thankful to Department of Biotechnology (DBT-JRF/0809/150), D.K.J. and G. Tyagi is thankful to Indian Council of Medical
[31] [32] [33]
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Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Interaction studies of Epirubicin with DNA using spectroscopic techniques Sonika Charak, Deepak K. Jangir, Gunjan Tyagi, Ranjana Mehrotra ⇑ Quantum Optics and Photon Physics, National Physical Laboratory, Council of Scientific and Industrial Research, Dr. K.S. Krishnan Marg, New Delhi 110 012, India
a r t i c l e
i n f o
Article history: Received 9 April 2011 Received in revised form 12 June 2011 Accepted 13 June 2011 Available online 5 July 2011 Keywords: Epirubicin FTIR spectroscopy UV–visible spectroscopy Circular dichroism spectroscopy DNA conformation
a b s t r a c t Epirubicin (EPR) is an anticancer chemotherapeutic drug which exerts its cytotoxic effect by inhibiting DNA synthesis and DNA replication. We report the structural and conformational effect of EPR binding on DNA duplex under physiological conditions. Fourier transform infrared spectroscopy (FTIR), ultraviolet–visible (UV–visible) spectroscopy and circular dichroism (CD) spectroscopy were used to determine the binding mode and binding constant of EPR with DNA. The effect of EPR–DNA complexation on stability and secondary structure of DNA was studied. FTIR measurements showed that EPR–DNA interaction occurs through guanine and cytosine bases. External binding of EPR with DNA was observed through phosphate backbone. UV–visible measurements revealed the intercalative mode of binding of EPR with DNA. The binding constant was estimated to be K = 3.4 104 which is indicative of moderate binding between EPR and DNA helix. FTIR and CD studies suggested partial transition from B-conformation of DNA to A-conformation of DNA after EPR binding to DNA duplex. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Epirubicin (EPR) is an anthracycline derivative used as chemotherapeutic drug. It is used for the treatment of variety of cancers, primarily breast cancer, ovarian cancer, gastric and lung cancer [1,2]. EPR is a stereoisomer of doxorubicin and differs from it in hydroxyl group orientation at carbon atom four (C4) of the hexapyranosyl sugar (Fig. 1). EPR is favored over doxorubicin due to more therapeutic index and lesser side effects [3,4]. It is also used in conjunction with other drugs for the treatment of non Hodgkin lymphoma and breast cancer [5–7]. The exact mechanism of action by which anthracycline drugs exert their cytotoxic effect is still unclear. It is proposed that rapidly replicating DNA in cancer cells is the major target of these drugs. The incorporation of drug into double stranded DNA disturbs its helical structure causing strand breakage. The primary mode of interaction of anthracycline drugs with DNA is intercalation. Intercalation of anthracycline drugs into the DNA bases lead to inhibition of DNA synthesis and replication [8]. Inhibition of topoisomerase-II enzyme activity, which assists in DNA unwinding during DNA replication, is another mechanism involved in the cytotoxic effects of anthracycline drugs [9]. In vitro and in vivo experiments have shown that EPR inhibit cell proliferation and DNA synthesis in carcinoma cell lines [10–12]. Along with anticancer properties, EPR also shows side effect like cardiotoxicity and myelosuppression [13]. Cytotoxicity caused by EPR effects the
⇑ Corresponding author. Tel.: +91 11 45608366; fax: +91 11 45609310. E-mail address: [email protected] (R. Mehrotra). 0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2011.06.013
structure and function of DNA, therefore detailed study on the interaction of EPR with DNA becomes important. FTIR spectroscopy has emerged as an efficient tool in life science due to its potential to provide fine structural information of the various biomolecules and biomolecule ligand complexes. This is widely used technique because it offers many advantages. This technique is fast, nondestructive and provide snapshot of all the conformations of the sample compound. It is very useful in characterizing the nature of drug and biomolecule interaction and effect of this interaction on the structure of the biomolecule [14–17]. It has been used in recent years to determine binding site of various drugs sequence specificity and changes in the structure of biomolecules after binding with the drugs [18–20]. Along with FTIR, circular dichroism spectroscopy is also a useful tool in determining the secondary structure of DNA and changes in the conformation states of DNA after DNA-drug complex formation [21,22]. In the present work, interaction of EPR with DNA has been investigated using FTIR, CD and UV–visible spectroscopy (Fig. 2). Different molar ratios of EPR with constant DNA concentration were prepared in aqueous solution at physiological conditions and subsequently analyzed. 2. Materials and methods EPR and highly polymerized type-1 calf thymus DNA (sodium content 6%) were purchased from Sigma Aldrich, USA. DNA purity estimation was done by recording the UV–visible absorbance at 260 nm and 280 nm. The ratio of absorbance at 260–280 nm was found to be 1.86, which indicates that the DNA was free from any contamination [23]. Other chemicals used were of analytical
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to noise ratio, a total of 128 scans were performed. For water subtraction, spectra of buffer at pH = 7.4 was recorded and then subtracted from the spectra of free DNA and EPR–DNA complexes. A satisfactory water subtraction is achieved when flat baseline around 2200 cm1 is produced, where water combination bands are positioned. This is a rough estimation method for water subtraction, but removes water spectral features in a satisfactory way [24]. 2.3. UV–visible spectroscopic measurements
Fig. 1. Chemical structure of: (a) doxorubicin (b) epirubicin.
The absorption spectra of free DNA and EPR–DNA complex were collected using Cary 100 Bio UV–visible spectrophotometer. Quartz cuvette of 1 cm path length was used. The value of binding constant of EPR–DNA complex was determined by the method described by Kanakis et al. [25]. It was assumed that only one type of interaction (1:1 complex formation) occurs between EPR and DNA in aqueous solution [26,27]. On the basis of this assumption, Eqs. (1) and (2) can be established.
DNA þ EPR DNA : EPR
ð1Þ
The equilibrium constant is given by
K ¼ ðDNA : EPRÞ=ðDNAÞðEPRÞ
ð2Þ
We can write the Eq. (2) as
K ¼ ðC DE Þ=½C D ½C E
Fig. 2. Schematic diagram of EPR–DNA complex.
grade and were used as supplied. Ultrapure water was used to prepare EPR, DNA and buffer solutions. 2.1. Preparation of stock solutions For preparation of stock solution, highly polymerized calf thymus DNA (10 mg/ml) was dissolved in 10 mM Tris–HCl buffer (pH = 7.4). DNA stock solution was kept at 8 °C for 24 h to make homogenous solution. Final concentration of DNA stock solution was determined spectrophotometrically at 260 nm using a molar extinction coefficient of 6600 cm1 M1. EPR stock solution and different concentration of EPR were prepared in ultrapure water. Different molar ratios of EPR–DNA complex were prepared by drop by drop addition of EPR to DNA solution. FTIR studies were carried out with EPR/DNA molar ratios (r) = 1/50, 1/100, 1/150, and 1/200 with final DNA concentration of 14 mM. For circular dichroism studies, EPR/DNA molar ratios of 1/50, 1/75, 1/100 and 1/125 were used with constant DNA concentration of 5 mM. EPR concentration in the range of 10–60 lM was used with 100 lM constant DNA concentration for UV–visible studies.
ð3Þ
CDE, CD, CE are the analytical concentration of EPR–DNA complex, DNA and EPR in solution respectively. Beer Lambert law for the absorption of light is assumed to be followed by the ligand substrate binding.
C D ¼ C Do C DE
ð4Þ
C DE ¼ A A0 =eDE L
ð5Þ
and
C Do ¼ A0 =eD l
ð6Þ
where CDo is the concentration of pure DNA, A0 and A are the absorbance of pure DNA and in the presence of EPR respectively at 260 nm. eD and eDE are the molar extinction coefficient of DNA and EPR–DNA complex respectively. l is the path length of the cuvette (1 cm). By putting the values of CD and CDE from above equations in to the Eq. (3), following equation can be deduced:
A0 =A A0 ¼ eD =eDE þ eD =ðeDE KÞ 1=C E The double reciprocal plot of 1/(A A0) versus 1/CE is linear and the binding constant (K) can be estimated by calculating the ratio of the intercept to the slope [28].
2.2. FTIR spectroscopic measurements 2.4. Circular dichroism spectroscopic measurements Infrared spectra of DNA and EPR–DNA complexes were recorded on FTIR spectrophotometer (Varian-660 model) equipped with deuterated triglycine sulfate (DTGS) detector and KBr beamsplitter. Spectra were collected after incubation of EPR–DNA complex for 2 h at room temperature. Spectra of EPR–DNA solutions were recorded using horizontal attenuated total internal reflection (HATR) crystal. Interferograms were accumulated in the spectral range of 1800–600 cm1 with 4 cm1 resolution. All the measurements were done in triplicate. To achieve better signal
CD spectroscopic measurements were recorded with 1 mm path length quartz cuvette using Jasco J812 CD spectrophotometer. Measurements were done in the far UV region (200–320 nm) in the nitrogen atmosphere. Sample spectra were collected at room temperature. An accumulation of average of three scans was done for each measurement with scan speed of 50 nm/min. Spectrum of Tris-buffer at pH 7.4 was recorded and was subtracted from the spectra of DNA and EPR–DNA complex.
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Fig. 3. Stacked view of FTIR spectra of free DNA and EPR–DNA complex at different molar ratios of 1/50, 1/100 and 1/150 in the spectral region of 1800–700 cm1.
3. Results and discussion 3.1. FTIR analysis FTIR spectra of free DNA and EPR–DNA complexes were monitored at different EPR/DNA ratios as shown in Fig. 3. FTIR spectrum of free DNA in B-conformation corresponds to four major nitrogenous bases, phosphate groups and sugar bands in the region of 1800–700 cm1. 3.1.1. Base analysis Fig. 3 shows the infrared spectrum of free DNA and EPR–DNA complexes. The peak at position 1710 cm1 corresponds to in plane vibration of the guanine (G) [20]. 1667 cm1 peak is attributed to thymine (T) stretching [20]. It is observed that after binding of EPR with DNA, peak at position 1710 cm1 shifts upwards to 1714–1716 cm1 and peak at position 1667 cm1 shifts downwards to 1663 cm1. Adenine (A) peak at position 1603 cm1 gets shifted towards higher wave number (1604). Peak at position 1489 cm1 which corresponds to cytosine (C) base vibrations shows upward shift of 4–5 cm1. The change in the peak position of the bases is observed in all EPR–DNA complexes. This shifting in peak position is also accompanied by the change in intensity in all EPR–DNA complexes. Presence of positive features at position 1723 cm1, 1655 cm1 and 1614 cm1 in the difference spectra correspond to increased intensity of guanine, thymine and adenine respectively (Fig. 4). Changes in major peak position and intensity variation indicate towards the intercalative binding mode of EPR. Spectral changes observed after EPR binding with DNA at guanine, cytosine bases indicate that EPR might get intercalated between G– C bases. 3.1.2. Phosphate analysis The peak at position 1225 cm1 due to asymmetric phosphate stretching in the spectrum of free DNA shows downward shift of 4–5 cm1 after binding with EPR in all the molar ratios studied.
The phosphate peak due to symmetrical stretching vibration at position 1087 cm1 shows downward shift of 1 cm1. Presence of positive features at 1211 cm1 and 1125 cm1 in the difference spectra indicates towards the increased intensity of asymmetric and symmetric phosphate peak after binding with DNA (Fig. 4). Peak shifting and intensity increase in intensity of asymmetric and symmetric phosphate peaks may be attributed to external binding of EPR with DNA helix backbone. 3.1.3. Conformational analysis Infrared peaks in this region primarily occur due to vibration in sugar bases. In B–A conformational change, guanine peak at 1710 cm1, phosphodiester peak at 837 cm1 and symmetric phosphate peak at 1087 cm1 exhibits change in their position. Similarly during B to Z transition peak at 837 cm1 shift to 800 cm1, guanine peak at 1710–1717 cm1 appears at 1690 cm1 and 1225 cm1 peak shifts towards 1215 cm1 [16]. It is observed that sugar phosphate band at position 1055 cm1 does not exhibits any significant change after binding with EPR. B-DNA marker peak at position 968 cm1 shows downward shift of 1–2 cm1 in EPR– DNA complex at different molar ratios examined in the study. Similarly the B-DNA marker peak at 837 cm1 due to sugar-phosphate chain vibrations shifts at 840 cm1. A new infrared peak emerges at 868 cm1 in all EPR–DNA complexes. Shifting in the peak positions of B-DNA conformation characteristic peaks and presence of new peak 868 cm1 in all EPR–DNA complexes studied indicates towards partial B–A transition of DNA after binding with EPR. 3.2. UV–visible studies UV–visible spectra of free DNA and EPR at different molar ratio are shown in Fig. 5. Hyperchromic effect was observed after EPR binding to free DNA. Proportional increase in absorption maxima (A260 nm) of free DNA is observed with increasing concentration of EPR. DNA helix is stabilized by hydrogen bonds between the two strands. EPR might intercalates between the two strands of
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Fig. 4. IR difference spectra [(DNA + EPR) – DNA solution] of EPR/DNA molar ratios of 1/50, 1/100 and 1/150 in the spectral region of 1800–700 cm1.
Fig. 5. UV–visible spectra of free DNA and its EPR–DNA complex at different molar ratios of 1/3, 1/5, 1/10, 1/20.
DNA causing increase in the absorption maxima of DNA due to the unwinding of DNA double helical structure. Due to unwinding of DNA, the aromatic bases get more exposed to UV radiation, hence results in increased intensity of absorption. The absorption maxima peak at 260 nm of free DNA also exhibits downward shifting (2 nm) indicating towards the intercalative binding mode [29]. Binding constant of EPR–DNA complex was calculated to determine its stability by using the method described in the experimental section. The binding constant for EPR in the concentration range of 10–60 lM after binding with DNA is observed to be, K = 3.4 104 (Fig. 6). The order of the binding constant is intimation of moderate binding between EPR and DNA. 3.3. CD analysis Circular dichroism spectra of different molar ratios of EPR with DNA and free DNA are shown in Fig. 7. Four major peaks for the Bconformation of DNA, 211 nm (negative), 221 nm (positive),
Fig. 6. Double reciprocal plot between 1/A A0 and 1/CE. A0 is the initial absorption of free DNA and A is the absorption at different concentrations at 260 nm. CE is the analytical concentration of EPR in solution.
245 nm (negative) and 275 nm (positive) are characteristic peaks for B-conformation of DNA. The peak at position 221 nm (negative), 245 nm (negative) and 275 nm (positive) are ascribed to formation of hydrogen bond, stacking of the bases and right handed helicity of B-DNA respectively [30]. Conformational change from B-form to A-form is accompanied by change in intensity as well as wavelength of the four characteristic peaks of B-DNA. Gain in negativity with minor shifting at 210 nm and 245 nm, decrease in intensity at 222 nm and increase in intensity at 275 nm with minor shifting (red shift) indicates towards complete B to A-DNA transition [31–33]. Changes in all negative and positive peak positions were observed in CD spectrum of DNA after binding with EPR (Fig. 7). Proportional increase in positive and decrease in negative peak position without any significant red shift was observed with increasing concentration of EPR. Spectral changes in intensity at 210 nm, 222 nm, 245 nm and 275 nm without shifting in peak position after interaction of EPR with DNA indicates towards partial B–A transition. These results are in agreement with our FTIR results
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Research for providing financial support. The infrastructural facility provided by Central Instrumentation Facility, Biotech Centre, University of Delhi South Campus is also acknowledged. References [1] [2] [3] [4] [5] [6]
[7] [8] [9] [10] [11] [12] [13] Fig. 7. Circular dichroism spectra of the free calf-thymus DNA and EPR/DNA molar ratios of 1/50, 1/75 and 1/100.
for partial B–A transition, due to emergence of a new infrared peak 868 cm1 in the infrared spectra of all DNA–EPR complexes. 4. Conclusion In the present study, we examined the structural and conformational effect of EPR on double helical structure of DNA using FTIR, UV–visible and CD spectroscopic techniques. FTIR results reflect toward binding of the EPR with DNA through guanine (G) and cytosine (C) bases and phosphate backbone by intercalation and external mode of binding. UV–visible spectral results further support the intercalative property of the EPR interaction with DNA. Binding constant of EPR–DNA complex was estimated to be K = 3.4 104. FTIR and CD results also suggest partial transition of B-conformation of DNA towards A-conformation of DNA.
[14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]
Acknowledgements S.C. is thankful to Department of Biotechnology (DBT-JRF/0809/150), D.K.J. and G. Tyagi is thankful to Indian Council of Medical
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