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β-Carboline alkaloids bind DNA
Journal of Photochemistry and Photobiology B: Biology 100 (2010) 84–91
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b-Carboline alkaloids bind DNA Shohreh Nafisi a,*, Mahyar Bonsaii a, Pegah Maali a, Mohammad Ali Khalilzadeh b, Firouzeh Manouchehri a a b
Department of Chemistry, Azad University, Central Tehran Branch (IAUCTB), Tehran 14676 86831, Iran Department of Chemistry, Azad University, Ghaemshahr Branch, Ghaemshahr, Iran
a r t i c l e
i n f o
Article history: Received 18 March 2010 Received in revised form 27 April 2010 Accepted 12 May 2010 Available online 19 May 2010 Keywords: DNA b-Carboline alkaloids FTIR UV–visible spectroscopy
a b s t r a c t b-Carboline alkaloids present in Peganum harmala (harmal) have recently drawn attention due to their antitumor activities. The mechanistic studies indicate that beta-carboline derivatives inhibit DNA topoisomerases and interfere with DNA synthesis. They interact with DNA via both groove binding and intercalative modes and cause major DNA structural changes. The aim of this study was to examine the interactions of five b-carboline alkaloids (harmine, harmane, harmaline, harmalol and tryptoline) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.25 mM) and various alkaloids/polynucleotide (phosphate) ratios of 1/240, 1/160, 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1/1. Fourier transform infrared (FTIR) and UV–visible spectroscopic methods were used to determine the ligand binding modes, the binding constants, and the stability of alkaloids–DNA complexes in aqueous solution. Spectroscopic evidence showed major binding of alkaloids to DNA with overall binding constants of Kharmine DNA = 3.44 107 M1, Kharmane DNA = 1.63 105 M1, Kharmaline DNA = 3.82 105 M1, Kharmalol DNA = 6.43 105 M1 and Ktryptoline DNA = 1.11 105 M1. The affinity of alkaloids–DNA binding is in the order of harmine > harmalol > harmaline > harmane > tryptoline. No biopolymer secondary structural changes were observed upon alkaloid interaction and DNA remains in the B-family structure in these complexes. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Peganum harmala L. commonly known as Harmal, is a wild flower plant belonging to the family Zygophyllaceae, perennial herbaceous, glabrous, that grows in semi-arid conditions, steppe areas and sandy soil. It is mainly found in eastern Mediterranean region and Central Asia, North Africa and Middle East. The pharmacologically active compounds of P. harmala include a number of carboline and quinazoline alkaloids like harmaline, harmalol, harmane, harmine [1,2]. Certain b-carbolines, such as tryptoline, pinoline, harmane and norharman have been reported as common constituents of human tissues and body fluids. Tryptoline is a natural organic derivative of beta-carboline. It is a competitive selective inhibitor of the enzyme monoamine oxidase type A (MAO-A) [3–7]. The common chemical structure of the alkaloids used in this study comprises fused one indole nucleus and a six-member pyrrole (Fig. 1). They can be divided into two groups: dihydro-b-carbolines (harmaline and harmalol) and aromatic b-carbolines (harmane, harmine). The b-carboline compounds are essentially * Corresponding author. Present address: Department of Biology, Northeastern University, Boston, MA 02115, United States. Tel./fax: +1 781 913 4080. E-mail addresses: s.nafi[email protected], drsnafi[email protected] (S. Nafisi). 1011-1344/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2010.05.005
planar, but dihydro-b-carbolines with a partial saturation ring are not planar. They are known to possess hypothermic, and hallucinogenic properties [3,8]. In folk medicine, the seeds of P. harmala have been used as an antiheamoroidal, helmicide for central nervous system (CNA) stimulating agent. In Iran, it is known as espand – mixed with other ingredients – are burnt so as to produce a scented smoke that is used as an air as well as mind purifier [3,8]. b-Carboline alkaloids exert a wide spectrum of action, including antimicrobial [4,9], anti-HIV and antiparasitic effects [10]. They show effective antioxidant properties. They can act as scavengers of reactive oxygen species and have a significant protective effect against H2O2 and paraquat oxidative agents in yeast cells, and that their ability to scavenge hydroxyl radicals contributes to their antimutagenic and antigenotoxic effects [11–14]. b-Carbolines exhibit cytotoxicity with regards to HL60 and K562 leukemia cell lines [15]. They have been reported to possess significant antitumor activities. Ground P. harmala seeds have been used occasionally to treat skin cancer and subcutaneous cancers traditionally in Morraco [16]. In Iran and china, the extracts containing beta-carbolines from the plant P. harmala have been widely used as a very potent antitumor folk medicine for cancers of digestive system [17,18].
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 100 (2010) 84–91
Fig. 1. Chemical structure of b-carboline alkaloids.
Biological responses caused by mutagenic, carcinogenic and antitumor agents are often associated with the binding of small molecules to DNA [19]. The determination of the binding constant of a drug for its target molecule is of considerable importance. It is a basic experimental parameter in a variety of studies, such as the prediction of drug efficiency, or in the pharmacokinetic drug interaction. The interaction of carboline alkaloids with DNA have been investigated by different methods. All of the results showed that these compounds intercalate into DNA with different degrees [20–26]. Even though much is reported about antitumor activities of bcarboline alkaloids, little is known about their interaction with individual DNA molecule by FTIR and UV spectroscopy. Thus, we studied the interaction of DNA adducts with five b-carboline derivatives: harmaline (3,4-dihydro-7-methoxy-1-methyl-9H-pyrido [3,4-b]indol-7-ol), harmalol (3,4-dihydro-1-methyl-9H-pyrido[3,4-b]indol-7-ol), harmane(1-methyl-9H-pyrido[3,4-b]indol7-ol), harmine (7-methoxy-1-methyl-9H-pyrido[3,4-b]indole) and tryptoline (1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole) at pH 7 in aqueous solution with alkaloids/DNA(P) molar ratios of 1/240–1/ 1 by FTIR and UV measurements. Structural analyses regarding the alkaloids-binding site, binding constant, DNA secondary structure are provided. Our spectroscopic results provide a major structural analysis of alkaloids–biopolymers interaction, which helps elucidate the nature of this biologically important complexation in vitro. 2. Materials and methods 2.1. Materials DNA sodium salt and alkaloids were purchased from Sigma Chemical (St. Louis, MO) and used without further purification. To check the protein content of DNA solutions, the absorbance bands at 260 and 280 nm were used. The A260/A280 ratio was 2.10 for DNA, showing that DNA samples were sufficiently free from protein [27]. Other chemicals were of reagent grade and used without further purification. 2.2. Preparation of stock solutions DNA was dissolved to 0.5% w/w (0.0125 M) polynucleotide (phosphate) in 0.1 M NaCl and 1 mM sodium cocodylate/L (pH 7.30) at 5 °C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the DNA solution was determined spectrophotometrically at 260 nm using molar extinction coefficient e260 = 9250 cm1 M1 (DNA) (expressed as molarity of phosphate groups) [28]. Due to low stability of harmalol, harmaline, harmane, tryptoline in water, appropriate amount of alkaloids in ethanol/water (20/80) and harmine in hot water (alkaloids, 0.05–12.5 mM) were prepared
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and added dropwise to DNA solution (alkaloids–DNA solutions), the alkaloids concentration was reduced to half to attain the desired alkaloids/DNA(P) molar ratios (r) of 1/240, 1/160, 1/80, 1/ 40, 1/20, 1/10, 1/5, 1/2 and 1/1 with a final DNA(P) concentration of 6.25 mM. At higher concentrations (for harmalol, harmaline and tryptoline more than r = 1/5, and for harmine more than r = 1/20), the experiments could not be continued due to DNA gel formation. The pH values of complex solutions were adjusted at 7.0 ± 0.2 using NaOH solution. The infrared spectra were recorded 2 h after mixing of the alkaloids with DNA solution. For UV measurements, the alkaloids concentrations of 8.16 106–5.1 104 at constant DNA concentration of 5.1 104 M were used. 2.3. FTIR spectroscopy measurements Infrared spectra were recorded on a Jasco FTIR spectrometer (Japan, Tokyo) equipped with a liquid-nitrogen-cooled HgCdTe (MCT) detector and a KBr beam splitter. The spectra of alkaloids/DNA solutions were taken using a cell assembled with AgBr windows. Spectra were collected and treated using the Spectra Manager software supplied by the manufacturer of the spectrophotometer. Solution spectra were recorded after 2 h incubation of the alkaloids with DNA solution, using AgBr windows. The bands were measured in triplicates (three individual samples of the same DNA, and alkaloids concentrations). For each spectrum, 100 scans were collected at a resolution of 4 cm1. The difference spectra [(polynucleotide solution + alkaloids solution) (polynucleotide solution)] were obtained using a sharp DNA band at 968 cm1 as internal reference [29,30]. These bands which are due to sugar C–C and C–O stretching vibrations, exhibit no spectral change (shifting or intensity variation) upon alkaloids–DNA complexation and cancelled out upon spectral subtraction [29,30]. The intensity ratios of the bands due to several DNA in-plane vibrations related to A–T, G–C base pairs and the PO2 stretching vibrations were measured with respect to the reference bands at 968 cm1 (DNA) as a function of alkaloids concentrations with an error of ±3%. Similar intensity variations have been used to determine the ligand binding to DNA bases and backbone phosphate groups [31]. The plots of the relative intensity (R) of several peaks of DNA in-plane vibrations related to A–T, G–C base pairs and the PO2 stretching vibrations such as 1714 (guanine), 1662 (thymine), 1608 (adenine), 1490 (cytosine), and 1227 cm1 (PO2 groups), vs. alkaloids concentrations were obtained after peak normalization using, Ri = Ii/I968, where Ii is the intensity of the absorption peak for pure DNA in the complex with i as ligand concentration, and I968 is the intensity of the 968 cm1 peak (DNA internal reference). 2.4. Absorption spectroscopy The absorption spectra were recorded on a LKB model 4054 UV–visible spectrometer, Quartz cuvettes of 1 cm were used and the absorption spectra recorded with drug concentrations of 8.16 106–5.1 104 M and constant polynucleotide concentration of 5.1 104 M. The binding constants of the alkaloids–DNA complexes were calculated as reported [32]. It is assumed that the interaction between the ligand L and the substrate S is 1:1; for this reason a single complex SL (1:1) is formed. It was also assumed that the sites (and all the binding sites) are independent and finally the Beer’s law is followed by all species. A wavelength is selected at which the molar absorptivities eS (molar absorptivity of the substrate) and e11 (molar absorptivity of the complex) are different. Then at
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total concentration St of the substrate, in the absence of ligand and the light path length is b = 1 cm, the solution absorbance is
Ao ¼ eS bSt
ð1Þ
In the presence of ligand at total concentration Lt, the absorbance of a solution containing the same total substrate concentration is
AL ¼ eS b½S þ eL b½L þ e11 b½SL
ð2Þ
(where [S] is the concentration of the uncomplexed substrate, [L] the concentration of the uncomplexed ligand and [SL] is the concentration of the complex) which, combined with the mass balance on S and L, gives
AL ¼ eS bSt þ eL bLt þ De11 b½SL
ð3Þ
where De11 = e11 eS eL (eL molar absorptivity of the ligand). By measuring the solution absorbance against a reference containing ligand at the same total concentration Lt, the measured absorbance becomes
A ¼ eS bSt þ De11 b½SL
ð4Þ
Combining Eq. (4) with the stability constant definition K11 = [SL]/[S][L], gives
DA ¼ K 11 De11 b½S½L
ð5Þ
where DA = A Ao. From the mass balance expression St = [S] + [SL], we get [S] = St/(1 + K11[L]), which is Eq. (5), giving Eq. (6) at the relationship between the observed absorbance change per centimeter and the system variables and parameters
DA St K 11 De11 ½L ¼ b 1 þ K 11 ½L
ð6Þ
Eq. (6) is the binding isotherm, which shows the hyperbolic dependence on free ligand concentration. The double-reciprocal form of plotting the rectangular hyperbola 1y ¼ df 1x þ de, is based on the linearization of Eq. (6) according to the following equation,
b 1 1 ¼ þ DA St K 11 De11 ½L St De11
ð7Þ
Thus the double reciprocal plot of 1/DA vs. 1/[L] is linear and the binding constant can be estimated from the following equation
K 11 ¼
intercept slope
ð8Þ
3. Results and discussion 3.1. Infrared spectra of alkaloids–DNA complexes Evidence of alkaloids–DNA complexation comes from the infrared spectroscopic results shown in Figs. 2 and 3. The spectral changes (intensity and shifting) of several prominent DNA in-plane vibrations at 1714 (G, T, mainly G), 1662 (T, G, A, and C, mainly T), 1608 (A, C, mainly A), 1490 (C, G, mainly C), 1227 (PO2 asymmetric stretch) and 1088 cm1 (PO2 symmetric stretch) [33,34] were monitored at different alkaloids–DNA molar ratios, and the results are shown in Figs. 2 and 3. In the harmine–DNA complexes (r = 1/240–/80), the intensity of the guanine, thymine, adenine and cytosine bands increased and reached a maximum at r = 1/160 for thymine and adenine and at r = 1/80 for guanine and cytosine (Figs. 2A and 3A). The observed intensity changes can be related to harmine interaction with G, C, A, T bases.
At r = 1/40, the intensity of the bases bands decreased that can be related to DNA stabilization upon harmine interaction. At r = 1/20, major intensity increase of the bases bands is related to helix destabilization upon harmine interaction (Figs. 2A and 3A). No major spectral changes (intensity and shifting) were observed for the phosphate vibrations at 1227 cm1 that indicates no major interaction of harmine with the PO2 backbone group (Figs. 2A and 3A). In the difference spectra of harmine–DNA complexes (Fig. 2A Diff. r = 1/160), positive features at 1681, 1650, 1633 cm1 are due to the intensity increase of the bases bands upon harmine–DNA complexation. The absorption spectral study of alkaloids in presence of DNA was performed to ascertain the nature of the binding of the fully bound drug. The results are presented in Fig. 4. The absorption maximum of harmine was at 245 and 319 nm (Fig. 4A), that presents a bathochromic (red) shift together with the decrease in the absorbance at 319 nm which is indicative of intercalation of harmine into DNA duplex [35–37]. In the harmane–DNA complexes (r = 1/240–1/40), the intensity of guanine band at 1714, thymine band at 1662 and cytosine band at 1490 cm1 increased and reached a maximum (for thymine at r = 1/160 and for guanine and cytosine at r = 1/80) that can be related to the interaction of harmane with these bases. No major spectral changes were observed at higher concentrations (r = 1/ 40–1/1), however at r = 1/10, decrease in the intensity of thymine, guanine and cytosine bands can be related to DNA stabilization upon harmane interaction. It should be noted that the adenine band at 1608 cm1 did not show any major spectral changes upon harmane interaction that can be related to the lack of participation of this base in harmane–DNA complexes at different concentrations (Figs. 2B and 3B). For the phosphate band, no major interaction was observed at lower harmane concentrations (r = 1/240–1/40), however, at higher concentrations (r = 1/20–1/2), the intensity of the phosphate band increased. Major intensity increase was accompanied by shifting of the phosphate band at 1227 in free DNA to 1230 (r = 1/20), 1234 (r = 1/10), 1237 (r = 1/5), 1236 (r = 1/2) (r = 1/20, 1/5, 1/2 are not shown here) (Figs. 2B and 3B). The observed spectral changes can be related to major interaction of harmane with phosphate backbone group (external binding) at higher concentrations. In the difference spectra of harmane–DNA complex (Fig. 2B, Diff. r = 1/80), the presence of positive features at 1716, 1683, 1653 cm1 are due to intensity increase of the bases upon harmane–DNA complexation. The absorption maximum of harmane was at 238, 287 and 347 nm (Fig. 4B) that presents a bathochromic (red) shift together with the decrease in the absorbance at 347 nm which is indicative of intercalation of harmane into DNA duplex that causes reduction in the intensity of the UV–visible absorption band characteristics of harmane [35–37]. In the harmaline–DNA complexes (r = 1/240, 1/160), increase in the intensity of guanine band at 1714 cm1 and adenine band at 1608 cm1 can be related to the interaction of harmaline with guanine and adenine bases at these concentrations (Figs. 2C and 3C). At r = 1/80, 1/40, the intensity of guanine and adenine decreased that can be related to DNA stabilization upon harmaline interaction. No major spectral changes (intensity and shifting) were observed for the thymine at 1662 cm1 at r = 1/ 240–1/40. By increasing the concentration (r = 1/20), the intensity of T, G and A bands, mainly T and G and to a lesser extent A increased that can be related to major harmaline interaction with thymine O2, guanine and adenine N7 at this concentration (Figs. 2C and 3C).
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Fig. 2. FTIR spectra in the region of 1800–600 cm1 for calf thymus DNA, and b-carboline alkaloids adducts (A) harmine–DNA, (B) harmane–DNA, (C) harmaline–DNA, (D) harmalol–DNA, (E) tryptoline–DNA in aqueous solution at pH=7. DNA and three complexes spectra obtained at various alkaloids–DNA (phosphate) molar ratios (top four spectra), drug and three difference spectra (bottom four spectra).
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Fig. 3. Intensity ratio variations for several DNA in-plane vibrations as a function of b-carboline alkaloids concentration. Intensity ratios for the DNA bands at 1714 (G), 1662 (T), 1608 (A), 1490 (C), and 1227 (PO2 asymmetric stretch) referenced to the DNA band at 968 cm1.
It should be noted that no major intensity changes were observed for the cytosine band at 1490 cm1 that is indicative of no major participation of cytosine in harmaline–DNA binding. For the antisymmetric phosphate band at 1227 cm1, no major spectral changes (intensity and shifting) were observed at lower concentrations (1/240–1/40), while at higher concentrations (r = 1/20–1/5), the intensity of the phosphate group increased and shifted to 1237–1238 cm1 that can be related to major phosphate–harmaline interaction (external binding) at higher concentrations (Figs. 2C and 3C). In the difference spectra of harmaline–DNA complexes (Fig. 2C Diff. r = 1/20, 1/5), positive features at 1687–1685, 1657–1654, 1256, 1247, 1103 cm1 are due to the intensity increase of the bases and phosphate bands upon harmaline–DNA complexation. The absorption maximum of harmaline was at 256 and 372 nm (Fig. 4C), the peak at 372 nm presents a minor bathochromic (red) shift together with the decrease in the absorbance at 347 nm which is indicative of intercalation of harmaline into DNA duplex [35–37]. At r = 1/240, 1/160, the intensity of the bases, mainly thymine, guanine and cytosine increased and reached a maximum at r = 1/ 160. The observed spectral changes can be related to interaction of harmalol with these bases (Figs. 2D and 3D). By increasing the concentration (r = 1/80), decrease in the intensity of the bases can be due to DNA stabilization upon harmalol interaction. At higher concentrations (r = 1/40–1/5), no major intensity changes were observed for the bases bands that is indicative of no major interaction of harmalol with DNA bases.
The adenine band at 1608 did not show spectral changes (intensity and shifting) upon harmalol interaction. It can be related to no major participation of adenine base in the harmalol–DNA complexation (Figs. 2D and 3D). No major harmalol phosphate backbone group was observed at all concentrations, except for the concentration r = 1/160, in which the intensity of the phosphate band at 1227 cm1 increased and shifted to 1234 cm1 that can be assigned to major interaction of harmalol with phosphate group at this concentration (Figs. 2D and 3D). In the difference spectra of harmalol–DNA complexes (Fig. 2D Diff. r = 1/160), positive features at 1698, 1684, 1653, 1242, 1065 cm1 are due to intensity increase of the bases and phosphate vibrations upon harmalol–DNA complexation. The UV bands at 259, 371 nm characteristics of harmalol absorptions (Fig. 4D) did not show any major shifting upon DNA interaction, but decrease in the absorbance band at 371 nm is indicative of intercalation of harmalol into DNA duplex [35–37]. In tryptoline–DNA complexes, at low drug concentration (r = 1/ 240), intensity increase of the guanine band at 1714, thymine band at 1662 and adenine band at 1608 cm1 can be related to interaction of tryptoline with these bases (Figs. 2E and 3E). At r = 1/160, the intensity of T, G and A bands decreased that can be related to DNA stabilization upon tryptoline interaction. No major intensity changes were observed at r = 1/80 for the bases, however at r = 1/40, 1/20, the intensity of thymine, guanine
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Fig. 4. UV–Visible results of (A) harmin (0.0816 mM), calf thymus DNA (0.51 mM) and its complex; (B) harmane (0.153 mM), calf thymus DNA (0.51 mM) and its complex; (C) harmaline (0.102 mM), calf thymus DNA (0.51 mM) and its complex; (D) harmalol (0.153 mM), calf thymus DNA (0.51 mM) and its complex; (E) tryptoline (0.102 mM), calf thymus DNA (0.51 mM) and its complex. Plot of 1/(A A0) vs (1/drug concentration) for b-carboline alkaloids and calf-thymus DNA complexes; (A’) Plot of 1/(A A0) vs (1/drug concentration) for harmine complexes; (B’) Plot of 1/(A A0) vs (1/drug concentration) for harmane complexes; (C’) Plot of 1/(A A0) vs (1/drug concentration) for harmaline complexes; (D’) Plot of 1/(A A0) vs (1/drug concentration) for harmalol complexes; (E’) Plot of 1/(A A0) vs (1/drug concentration) for tryptoline complexes; where A0 is the initial absorbance of DNA (258 nm) and A is the recorded absorbance at different b-carboline alkaloids concentrations (L = 1/120 to 1/1 mM) with constant DNA concentrations of 0.51mM at pH 7.
and cytosine increased and reached a maximum at r = 1/40 for thymine and at r = 1/20 for guanine and cytosine that can be related to extended interaction of harmaline with these bases. At higher concentrations (r = 1/10), decrease in intensity of the bases bands can be related to DNA condensation and aggregation upon tryptoline complexation. At higher concentrations (r = 1/80–1/5), no major intensity changes were observed for adenine band at 1608 cm1 that can be assigned to no major participation of adenine in tryptoline–DNA complexes at these concentrations (Figs. 2E and 3E). For the antisymmetric phosphate band at 1227 cm1, no major spectral changes (shifting and intensity) were observed at r = 1/ 240–1/80, however at r = 1/40, the intensity of antisymmetric phosphate vibration at 1227 cm1 increased upon tryptoline interaction that can be assigned to tryptoline interaction with phosphate group (external binding) at this concentration (Figs. 2E and 3E). At r = 1/240, the positive features at 1712, 1659, 1643 cm1 for bases (Fig. 2E, Diff. r = 1/240) can be due to increase in intensity of the bases vibrational frequencies upon tryptoline interaction. It is worth mentioning that the absorption bands with medium intensity at 1653 cm1 in the IR spectrum of free DNA and 1651– 1654 cm1 in spectra of the alkaloids–DNA adducts and in differ-
ence spectra are due to water deformation mode, and they are not coming from DNA vibrations [39]. The UV bands at 219 and 277 nm characteristics of tryptoline absorptions are overlapped by the DNA peak and it is not clear if the shift observed belongs to the peaks of tryptoline or to the DNA peak(Fig. 4E). It should be noted that compounds that bind nucleic acids via intercalative mode cause minor spectral changes (shifting and intensity) in the IR spectra of ligand–DNA complexes. Such spectral changes was observed in the infrared spectra of complexes of ethidium bromide (EB), acridine orange (AO) and methylene blue (MB) with DNA in which minor spectral changes was attributed to the intercalation of these compounds into DNA duplex [38]. Minor intensity changes (intensity and shifting) of the alkaloids–DNA complexes can show weak or partial intercalation of beta-carboline alkaloids into DNA duplex. 3.2. DNA conformation In the alkaloids–DNA complexes, DNA remains in B conformation. When a complete B to A transition occurs, the B-DNA marker bands such as 836 cm1 appears at about 820–810 cm1, while the
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PO2 stretching vibration at 1227 cm1 shifts toward a higher frequency at 1230–1240 cm1 and the guanine band at 1710 appears at 1700 cm1 [40,41]. No major shifting was observed in the B-DNA conformation marker bands upon alkaloids interaction. In the harmane and harmaline–DNA complexes, shifting of B-DNA marker bands at 1227 to 1230–1238 cm1 is indicative of drug interaction with phosphate and not arising from B to A-DNA conformational change [42–44] (Fig. 2). 3.3. Stability of alkaloids–DNA complexes The alkaloids binding constants were determined as described in Materials and Methods (UV–visible spectroscopy). The calculations of the overall binding constants were carried out using UV spectroscopy as reported [32]. Concentrations of the complexed ligand were determined by subtracting absorbance of the free DNA at 258 nm from those of the complexed. Concentration of the free ligand was determined by subtraction of complexed ligand from total ligand used in the experiment. Our data of 1/[ligand complexed] almost proportionally increased as a function of 1/[free ligand] (Fig. 4). The double reciprocal plot of 1/ (A A0) vs. 1/(drug concentration) is linear, and the binding constant (K) can be estimated from the ratio of the intercept to the slope (Fig. 4), where A0 is the initial absorbance of the free DNA at 258 nm, and A is the recorded absorbance of DNA in the presence of different drug concentrations. The overall binding constants are estimated to be Kharmine DNA = 3.44 107 M1 for harmine–DNA, Kharmane DNA = 1.63 105 M1 for harmane–DNA, Kharmaline DNA = 3.82 105 M1 for harmaline–DNA, Kharmalol DNA = 6.43 105 M1 for harmalol–DNA, Ktryptoline DNA = 1.11x105 M1 for tryptoline–DNA. Similar binding constants were estimated for other isoquinoline alkaloids–DNA complexes [45]. The binding constants of alkaloids–DNA binding is in the order of harmine > harmalol > harmaline > harmane > tryptoline. The binding constant of harmine is 100 times greater than harmane that can be attributed to the presence of polar group OCH3 at the benzene ring on the skeleton ring. The presence of polar substituent at the benzene ring increases the binding affinity. The binding constant of harmine is 100 times greater than harmaline because the former is planar, while, the latter is not planar. It can be concluded that the binding of planar compounds with DNA is stronger than that of non-planar compounds. By decreasing planarity of the b-carboline skeleton, the binding affinity to DNA decreases. The binding constant of harmalol is greater than harmaline that can be related to high electron donating property of OH rather than OCH3 which enhances the binding affinity of the indole ring. The above results are in compatible with Duportail and Taira experiments who calculated the same order of affinity for the b-carboline alkaloids–DNA complexes [20,22]. 4. Summary On the basis of our spectroscopic results, b-carboline alkaloids interact with bases via cytosine O2, thymine O2, guanine and adenine N7 atoms as well as backbone phosphate group (external binding) with partial and weak intercalation into G–C and A–T base pairs of DNA. The binding constants of alkaloids–DNA binding is in the order of harmine > harmalol > harmaline > harmane > tryptoline. No DNA conformational changes were observed upon alkaloids complexation. Disclosure statement No competing financial interests exist.
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
b-Carboline alkaloids bind DNA Shohreh Nafisi a,*, Mahyar Bonsaii a, Pegah Maali a, Mohammad Ali Khalilzadeh b, Firouzeh Manouchehri a a b
Department of Chemistry, Azad University, Central Tehran Branch (IAUCTB), Tehran 14676 86831, Iran Department of Chemistry, Azad University, Ghaemshahr Branch, Ghaemshahr, Iran
a r t i c l e
i n f o
Article history: Received 18 March 2010 Received in revised form 27 April 2010 Accepted 12 May 2010 Available online 19 May 2010 Keywords: DNA b-Carboline alkaloids FTIR UV–visible spectroscopy
a b s t r a c t b-Carboline alkaloids present in Peganum harmala (harmal) have recently drawn attention due to their antitumor activities. The mechanistic studies indicate that beta-carboline derivatives inhibit DNA topoisomerases and interfere with DNA synthesis. They interact with DNA via both groove binding and intercalative modes and cause major DNA structural changes. The aim of this study was to examine the interactions of five b-carboline alkaloids (harmine, harmane, harmaline, harmalol and tryptoline) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.25 mM) and various alkaloids/polynucleotide (phosphate) ratios of 1/240, 1/160, 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1/1. Fourier transform infrared (FTIR) and UV–visible spectroscopic methods were used to determine the ligand binding modes, the binding constants, and the stability of alkaloids–DNA complexes in aqueous solution. Spectroscopic evidence showed major binding of alkaloids to DNA with overall binding constants of Kharmine DNA = 3.44 107 M1, Kharmane DNA = 1.63 105 M1, Kharmaline DNA = 3.82 105 M1, Kharmalol DNA = 6.43 105 M1 and Ktryptoline DNA = 1.11 105 M1. The affinity of alkaloids–DNA binding is in the order of harmine > harmalol > harmaline > harmane > tryptoline. No biopolymer secondary structural changes were observed upon alkaloid interaction and DNA remains in the B-family structure in these complexes. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Peganum harmala L. commonly known as Harmal, is a wild flower plant belonging to the family Zygophyllaceae, perennial herbaceous, glabrous, that grows in semi-arid conditions, steppe areas and sandy soil. It is mainly found in eastern Mediterranean region and Central Asia, North Africa and Middle East. The pharmacologically active compounds of P. harmala include a number of carboline and quinazoline alkaloids like harmaline, harmalol, harmane, harmine [1,2]. Certain b-carbolines, such as tryptoline, pinoline, harmane and norharman have been reported as common constituents of human tissues and body fluids. Tryptoline is a natural organic derivative of beta-carboline. It is a competitive selective inhibitor of the enzyme monoamine oxidase type A (MAO-A) [3–7]. The common chemical structure of the alkaloids used in this study comprises fused one indole nucleus and a six-member pyrrole (Fig. 1). They can be divided into two groups: dihydro-b-carbolines (harmaline and harmalol) and aromatic b-carbolines (harmane, harmine). The b-carboline compounds are essentially * Corresponding author. Present address: Department of Biology, Northeastern University, Boston, MA 02115, United States. Tel./fax: +1 781 913 4080. E-mail addresses: s.nafi[email protected], drsnafi[email protected] (S. Nafisi). 1011-1344/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2010.05.005
planar, but dihydro-b-carbolines with a partial saturation ring are not planar. They are known to possess hypothermic, and hallucinogenic properties [3,8]. In folk medicine, the seeds of P. harmala have been used as an antiheamoroidal, helmicide for central nervous system (CNA) stimulating agent. In Iran, it is known as espand – mixed with other ingredients – are burnt so as to produce a scented smoke that is used as an air as well as mind purifier [3,8]. b-Carboline alkaloids exert a wide spectrum of action, including antimicrobial [4,9], anti-HIV and antiparasitic effects [10]. They show effective antioxidant properties. They can act as scavengers of reactive oxygen species and have a significant protective effect against H2O2 and paraquat oxidative agents in yeast cells, and that their ability to scavenge hydroxyl radicals contributes to their antimutagenic and antigenotoxic effects [11–14]. b-Carbolines exhibit cytotoxicity with regards to HL60 and K562 leukemia cell lines [15]. They have been reported to possess significant antitumor activities. Ground P. harmala seeds have been used occasionally to treat skin cancer and subcutaneous cancers traditionally in Morraco [16]. In Iran and china, the extracts containing beta-carbolines from the plant P. harmala have been widely used as a very potent antitumor folk medicine for cancers of digestive system [17,18].
S. Nafisi et al. / Journal of Photochemistry and Photobiology B: Biology 100 (2010) 84–91
Fig. 1. Chemical structure of b-carboline alkaloids.
Biological responses caused by mutagenic, carcinogenic and antitumor agents are often associated with the binding of small molecules to DNA [19]. The determination of the binding constant of a drug for its target molecule is of considerable importance. It is a basic experimental parameter in a variety of studies, such as the prediction of drug efficiency, or in the pharmacokinetic drug interaction. The interaction of carboline alkaloids with DNA have been investigated by different methods. All of the results showed that these compounds intercalate into DNA with different degrees [20–26]. Even though much is reported about antitumor activities of bcarboline alkaloids, little is known about their interaction with individual DNA molecule by FTIR and UV spectroscopy. Thus, we studied the interaction of DNA adducts with five b-carboline derivatives: harmaline (3,4-dihydro-7-methoxy-1-methyl-9H-pyrido [3,4-b]indol-7-ol), harmalol (3,4-dihydro-1-methyl-9H-pyrido[3,4-b]indol-7-ol), harmane(1-methyl-9H-pyrido[3,4-b]indol7-ol), harmine (7-methoxy-1-methyl-9H-pyrido[3,4-b]indole) and tryptoline (1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole) at pH 7 in aqueous solution with alkaloids/DNA(P) molar ratios of 1/240–1/ 1 by FTIR and UV measurements. Structural analyses regarding the alkaloids-binding site, binding constant, DNA secondary structure are provided. Our spectroscopic results provide a major structural analysis of alkaloids–biopolymers interaction, which helps elucidate the nature of this biologically important complexation in vitro. 2. Materials and methods 2.1. Materials DNA sodium salt and alkaloids were purchased from Sigma Chemical (St. Louis, MO) and used without further purification. To check the protein content of DNA solutions, the absorbance bands at 260 and 280 nm were used. The A260/A280 ratio was 2.10 for DNA, showing that DNA samples were sufficiently free from protein [27]. Other chemicals were of reagent grade and used without further purification. 2.2. Preparation of stock solutions DNA was dissolved to 0.5% w/w (0.0125 M) polynucleotide (phosphate) in 0.1 M NaCl and 1 mM sodium cocodylate/L (pH 7.30) at 5 °C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the DNA solution was determined spectrophotometrically at 260 nm using molar extinction coefficient e260 = 9250 cm1 M1 (DNA) (expressed as molarity of phosphate groups) [28]. Due to low stability of harmalol, harmaline, harmane, tryptoline in water, appropriate amount of alkaloids in ethanol/water (20/80) and harmine in hot water (alkaloids, 0.05–12.5 mM) were prepared
85
and added dropwise to DNA solution (alkaloids–DNA solutions), the alkaloids concentration was reduced to half to attain the desired alkaloids/DNA(P) molar ratios (r) of 1/240, 1/160, 1/80, 1/ 40, 1/20, 1/10, 1/5, 1/2 and 1/1 with a final DNA(P) concentration of 6.25 mM. At higher concentrations (for harmalol, harmaline and tryptoline more than r = 1/5, and for harmine more than r = 1/20), the experiments could not be continued due to DNA gel formation. The pH values of complex solutions were adjusted at 7.0 ± 0.2 using NaOH solution. The infrared spectra were recorded 2 h after mixing of the alkaloids with DNA solution. For UV measurements, the alkaloids concentrations of 8.16 106–5.1 104 at constant DNA concentration of 5.1 104 M were used. 2.3. FTIR spectroscopy measurements Infrared spectra were recorded on a Jasco FTIR spectrometer (Japan, Tokyo) equipped with a liquid-nitrogen-cooled HgCdTe (MCT) detector and a KBr beam splitter. The spectra of alkaloids/DNA solutions were taken using a cell assembled with AgBr windows. Spectra were collected and treated using the Spectra Manager software supplied by the manufacturer of the spectrophotometer. Solution spectra were recorded after 2 h incubation of the alkaloids with DNA solution, using AgBr windows. The bands were measured in triplicates (three individual samples of the same DNA, and alkaloids concentrations). For each spectrum, 100 scans were collected at a resolution of 4 cm1. The difference spectra [(polynucleotide solution + alkaloids solution) (polynucleotide solution)] were obtained using a sharp DNA band at 968 cm1 as internal reference [29,30]. These bands which are due to sugar C–C and C–O stretching vibrations, exhibit no spectral change (shifting or intensity variation) upon alkaloids–DNA complexation and cancelled out upon spectral subtraction [29,30]. The intensity ratios of the bands due to several DNA in-plane vibrations related to A–T, G–C base pairs and the PO2 stretching vibrations were measured with respect to the reference bands at 968 cm1 (DNA) as a function of alkaloids concentrations with an error of ±3%. Similar intensity variations have been used to determine the ligand binding to DNA bases and backbone phosphate groups [31]. The plots of the relative intensity (R) of several peaks of DNA in-plane vibrations related to A–T, G–C base pairs and the PO2 stretching vibrations such as 1714 (guanine), 1662 (thymine), 1608 (adenine), 1490 (cytosine), and 1227 cm1 (PO2 groups), vs. alkaloids concentrations were obtained after peak normalization using, Ri = Ii/I968, where Ii is the intensity of the absorption peak for pure DNA in the complex with i as ligand concentration, and I968 is the intensity of the 968 cm1 peak (DNA internal reference). 2.4. Absorption spectroscopy The absorption spectra were recorded on a LKB model 4054 UV–visible spectrometer, Quartz cuvettes of 1 cm were used and the absorption spectra recorded with drug concentrations of 8.16 106–5.1 104 M and constant polynucleotide concentration of 5.1 104 M. The binding constants of the alkaloids–DNA complexes were calculated as reported [32]. It is assumed that the interaction between the ligand L and the substrate S is 1:1; for this reason a single complex SL (1:1) is formed. It was also assumed that the sites (and all the binding sites) are independent and finally the Beer’s law is followed by all species. A wavelength is selected at which the molar absorptivities eS (molar absorptivity of the substrate) and e11 (molar absorptivity of the complex) are different. Then at
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total concentration St of the substrate, in the absence of ligand and the light path length is b = 1 cm, the solution absorbance is
Ao ¼ eS bSt
ð1Þ
In the presence of ligand at total concentration Lt, the absorbance of a solution containing the same total substrate concentration is
AL ¼ eS b½S þ eL b½L þ e11 b½SL
ð2Þ
(where [S] is the concentration of the uncomplexed substrate, [L] the concentration of the uncomplexed ligand and [SL] is the concentration of the complex) which, combined with the mass balance on S and L, gives
AL ¼ eS bSt þ eL bLt þ De11 b½SL
ð3Þ
where De11 = e11 eS eL (eL molar absorptivity of the ligand). By measuring the solution absorbance against a reference containing ligand at the same total concentration Lt, the measured absorbance becomes
A ¼ eS bSt þ De11 b½SL
ð4Þ
Combining Eq. (4) with the stability constant definition K11 = [SL]/[S][L], gives
DA ¼ K 11 De11 b½S½L
ð5Þ
where DA = A Ao. From the mass balance expression St = [S] + [SL], we get [S] = St/(1 + K11[L]), which is Eq. (5), giving Eq. (6) at the relationship between the observed absorbance change per centimeter and the system variables and parameters
DA St K 11 De11 ½L ¼ b 1 þ K 11 ½L
ð6Þ
Eq. (6) is the binding isotherm, which shows the hyperbolic dependence on free ligand concentration. The double-reciprocal form of plotting the rectangular hyperbola 1y ¼ df 1x þ de, is based on the linearization of Eq. (6) according to the following equation,
b 1 1 ¼ þ DA St K 11 De11 ½L St De11
ð7Þ
Thus the double reciprocal plot of 1/DA vs. 1/[L] is linear and the binding constant can be estimated from the following equation
K 11 ¼
intercept slope
ð8Þ
3. Results and discussion 3.1. Infrared spectra of alkaloids–DNA complexes Evidence of alkaloids–DNA complexation comes from the infrared spectroscopic results shown in Figs. 2 and 3. The spectral changes (intensity and shifting) of several prominent DNA in-plane vibrations at 1714 (G, T, mainly G), 1662 (T, G, A, and C, mainly T), 1608 (A, C, mainly A), 1490 (C, G, mainly C), 1227 (PO2 asymmetric stretch) and 1088 cm1 (PO2 symmetric stretch) [33,34] were monitored at different alkaloids–DNA molar ratios, and the results are shown in Figs. 2 and 3. In the harmine–DNA complexes (r = 1/240–/80), the intensity of the guanine, thymine, adenine and cytosine bands increased and reached a maximum at r = 1/160 for thymine and adenine and at r = 1/80 for guanine and cytosine (Figs. 2A and 3A). The observed intensity changes can be related to harmine interaction with G, C, A, T bases.
At r = 1/40, the intensity of the bases bands decreased that can be related to DNA stabilization upon harmine interaction. At r = 1/20, major intensity increase of the bases bands is related to helix destabilization upon harmine interaction (Figs. 2A and 3A). No major spectral changes (intensity and shifting) were observed for the phosphate vibrations at 1227 cm1 that indicates no major interaction of harmine with the PO2 backbone group (Figs. 2A and 3A). In the difference spectra of harmine–DNA complexes (Fig. 2A Diff. r = 1/160), positive features at 1681, 1650, 1633 cm1 are due to the intensity increase of the bases bands upon harmine–DNA complexation. The absorption spectral study of alkaloids in presence of DNA was performed to ascertain the nature of the binding of the fully bound drug. The results are presented in Fig. 4. The absorption maximum of harmine was at 245 and 319 nm (Fig. 4A), that presents a bathochromic (red) shift together with the decrease in the absorbance at 319 nm which is indicative of intercalation of harmine into DNA duplex [35–37]. In the harmane–DNA complexes (r = 1/240–1/40), the intensity of guanine band at 1714, thymine band at 1662 and cytosine band at 1490 cm1 increased and reached a maximum (for thymine at r = 1/160 and for guanine and cytosine at r = 1/80) that can be related to the interaction of harmane with these bases. No major spectral changes were observed at higher concentrations (r = 1/ 40–1/1), however at r = 1/10, decrease in the intensity of thymine, guanine and cytosine bands can be related to DNA stabilization upon harmane interaction. It should be noted that the adenine band at 1608 cm1 did not show any major spectral changes upon harmane interaction that can be related to the lack of participation of this base in harmane–DNA complexes at different concentrations (Figs. 2B and 3B). For the phosphate band, no major interaction was observed at lower harmane concentrations (r = 1/240–1/40), however, at higher concentrations (r = 1/20–1/2), the intensity of the phosphate band increased. Major intensity increase was accompanied by shifting of the phosphate band at 1227 in free DNA to 1230 (r = 1/20), 1234 (r = 1/10), 1237 (r = 1/5), 1236 (r = 1/2) (r = 1/20, 1/5, 1/2 are not shown here) (Figs. 2B and 3B). The observed spectral changes can be related to major interaction of harmane with phosphate backbone group (external binding) at higher concentrations. In the difference spectra of harmane–DNA complex (Fig. 2B, Diff. r = 1/80), the presence of positive features at 1716, 1683, 1653 cm1 are due to intensity increase of the bases upon harmane–DNA complexation. The absorption maximum of harmane was at 238, 287 and 347 nm (Fig. 4B) that presents a bathochromic (red) shift together with the decrease in the absorbance at 347 nm which is indicative of intercalation of harmane into DNA duplex that causes reduction in the intensity of the UV–visible absorption band characteristics of harmane [35–37]. In the harmaline–DNA complexes (r = 1/240, 1/160), increase in the intensity of guanine band at 1714 cm1 and adenine band at 1608 cm1 can be related to the interaction of harmaline with guanine and adenine bases at these concentrations (Figs. 2C and 3C). At r = 1/80, 1/40, the intensity of guanine and adenine decreased that can be related to DNA stabilization upon harmaline interaction. No major spectral changes (intensity and shifting) were observed for the thymine at 1662 cm1 at r = 1/ 240–1/40. By increasing the concentration (r = 1/20), the intensity of T, G and A bands, mainly T and G and to a lesser extent A increased that can be related to major harmaline interaction with thymine O2, guanine and adenine N7 at this concentration (Figs. 2C and 3C).
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Fig. 2. FTIR spectra in the region of 1800–600 cm1 for calf thymus DNA, and b-carboline alkaloids adducts (A) harmine–DNA, (B) harmane–DNA, (C) harmaline–DNA, (D) harmalol–DNA, (E) tryptoline–DNA in aqueous solution at pH=7. DNA and three complexes spectra obtained at various alkaloids–DNA (phosphate) molar ratios (top four spectra), drug and three difference spectra (bottom four spectra).
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Fig. 3. Intensity ratio variations for several DNA in-plane vibrations as a function of b-carboline alkaloids concentration. Intensity ratios for the DNA bands at 1714 (G), 1662 (T), 1608 (A), 1490 (C), and 1227 (PO2 asymmetric stretch) referenced to the DNA band at 968 cm1.
It should be noted that no major intensity changes were observed for the cytosine band at 1490 cm1 that is indicative of no major participation of cytosine in harmaline–DNA binding. For the antisymmetric phosphate band at 1227 cm1, no major spectral changes (intensity and shifting) were observed at lower concentrations (1/240–1/40), while at higher concentrations (r = 1/20–1/5), the intensity of the phosphate group increased and shifted to 1237–1238 cm1 that can be related to major phosphate–harmaline interaction (external binding) at higher concentrations (Figs. 2C and 3C). In the difference spectra of harmaline–DNA complexes (Fig. 2C Diff. r = 1/20, 1/5), positive features at 1687–1685, 1657–1654, 1256, 1247, 1103 cm1 are due to the intensity increase of the bases and phosphate bands upon harmaline–DNA complexation. The absorption maximum of harmaline was at 256 and 372 nm (Fig. 4C), the peak at 372 nm presents a minor bathochromic (red) shift together with the decrease in the absorbance at 347 nm which is indicative of intercalation of harmaline into DNA duplex [35–37]. At r = 1/240, 1/160, the intensity of the bases, mainly thymine, guanine and cytosine increased and reached a maximum at r = 1/ 160. The observed spectral changes can be related to interaction of harmalol with these bases (Figs. 2D and 3D). By increasing the concentration (r = 1/80), decrease in the intensity of the bases can be due to DNA stabilization upon harmalol interaction. At higher concentrations (r = 1/40–1/5), no major intensity changes were observed for the bases bands that is indicative of no major interaction of harmalol with DNA bases.
The adenine band at 1608 did not show spectral changes (intensity and shifting) upon harmalol interaction. It can be related to no major participation of adenine base in the harmalol–DNA complexation (Figs. 2D and 3D). No major harmalol phosphate backbone group was observed at all concentrations, except for the concentration r = 1/160, in which the intensity of the phosphate band at 1227 cm1 increased and shifted to 1234 cm1 that can be assigned to major interaction of harmalol with phosphate group at this concentration (Figs. 2D and 3D). In the difference spectra of harmalol–DNA complexes (Fig. 2D Diff. r = 1/160), positive features at 1698, 1684, 1653, 1242, 1065 cm1 are due to intensity increase of the bases and phosphate vibrations upon harmalol–DNA complexation. The UV bands at 259, 371 nm characteristics of harmalol absorptions (Fig. 4D) did not show any major shifting upon DNA interaction, but decrease in the absorbance band at 371 nm is indicative of intercalation of harmalol into DNA duplex [35–37]. In tryptoline–DNA complexes, at low drug concentration (r = 1/ 240), intensity increase of the guanine band at 1714, thymine band at 1662 and adenine band at 1608 cm1 can be related to interaction of tryptoline with these bases (Figs. 2E and 3E). At r = 1/160, the intensity of T, G and A bands decreased that can be related to DNA stabilization upon tryptoline interaction. No major intensity changes were observed at r = 1/80 for the bases, however at r = 1/40, 1/20, the intensity of thymine, guanine
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Fig. 4. UV–Visible results of (A) harmin (0.0816 mM), calf thymus DNA (0.51 mM) and its complex; (B) harmane (0.153 mM), calf thymus DNA (0.51 mM) and its complex; (C) harmaline (0.102 mM), calf thymus DNA (0.51 mM) and its complex; (D) harmalol (0.153 mM), calf thymus DNA (0.51 mM) and its complex; (E) tryptoline (0.102 mM), calf thymus DNA (0.51 mM) and its complex. Plot of 1/(A A0) vs (1/drug concentration) for b-carboline alkaloids and calf-thymus DNA complexes; (A’) Plot of 1/(A A0) vs (1/drug concentration) for harmine complexes; (B’) Plot of 1/(A A0) vs (1/drug concentration) for harmane complexes; (C’) Plot of 1/(A A0) vs (1/drug concentration) for harmaline complexes; (D’) Plot of 1/(A A0) vs (1/drug concentration) for harmalol complexes; (E’) Plot of 1/(A A0) vs (1/drug concentration) for tryptoline complexes; where A0 is the initial absorbance of DNA (258 nm) and A is the recorded absorbance at different b-carboline alkaloids concentrations (L = 1/120 to 1/1 mM) with constant DNA concentrations of 0.51mM at pH 7.
and cytosine increased and reached a maximum at r = 1/40 for thymine and at r = 1/20 for guanine and cytosine that can be related to extended interaction of harmaline with these bases. At higher concentrations (r = 1/10), decrease in intensity of the bases bands can be related to DNA condensation and aggregation upon tryptoline complexation. At higher concentrations (r = 1/80–1/5), no major intensity changes were observed for adenine band at 1608 cm1 that can be assigned to no major participation of adenine in tryptoline–DNA complexes at these concentrations (Figs. 2E and 3E). For the antisymmetric phosphate band at 1227 cm1, no major spectral changes (shifting and intensity) were observed at r = 1/ 240–1/80, however at r = 1/40, the intensity of antisymmetric phosphate vibration at 1227 cm1 increased upon tryptoline interaction that can be assigned to tryptoline interaction with phosphate group (external binding) at this concentration (Figs. 2E and 3E). At r = 1/240, the positive features at 1712, 1659, 1643 cm1 for bases (Fig. 2E, Diff. r = 1/240) can be due to increase in intensity of the bases vibrational frequencies upon tryptoline interaction. It is worth mentioning that the absorption bands with medium intensity at 1653 cm1 in the IR spectrum of free DNA and 1651– 1654 cm1 in spectra of the alkaloids–DNA adducts and in differ-
ence spectra are due to water deformation mode, and they are not coming from DNA vibrations [39]. The UV bands at 219 and 277 nm characteristics of tryptoline absorptions are overlapped by the DNA peak and it is not clear if the shift observed belongs to the peaks of tryptoline or to the DNA peak(Fig. 4E). It should be noted that compounds that bind nucleic acids via intercalative mode cause minor spectral changes (shifting and intensity) in the IR spectra of ligand–DNA complexes. Such spectral changes was observed in the infrared spectra of complexes of ethidium bromide (EB), acridine orange (AO) and methylene blue (MB) with DNA in which minor spectral changes was attributed to the intercalation of these compounds into DNA duplex [38]. Minor intensity changes (intensity and shifting) of the alkaloids–DNA complexes can show weak or partial intercalation of beta-carboline alkaloids into DNA duplex. 3.2. DNA conformation In the alkaloids–DNA complexes, DNA remains in B conformation. When a complete B to A transition occurs, the B-DNA marker bands such as 836 cm1 appears at about 820–810 cm1, while the
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PO2 stretching vibration at 1227 cm1 shifts toward a higher frequency at 1230–1240 cm1 and the guanine band at 1710 appears at 1700 cm1 [40,41]. No major shifting was observed in the B-DNA conformation marker bands upon alkaloids interaction. In the harmane and harmaline–DNA complexes, shifting of B-DNA marker bands at 1227 to 1230–1238 cm1 is indicative of drug interaction with phosphate and not arising from B to A-DNA conformational change [42–44] (Fig. 2). 3.3. Stability of alkaloids–DNA complexes The alkaloids binding constants were determined as described in Materials and Methods (UV–visible spectroscopy). The calculations of the overall binding constants were carried out using UV spectroscopy as reported [32]. Concentrations of the complexed ligand were determined by subtracting absorbance of the free DNA at 258 nm from those of the complexed. Concentration of the free ligand was determined by subtraction of complexed ligand from total ligand used in the experiment. Our data of 1/[ligand complexed] almost proportionally increased as a function of 1/[free ligand] (Fig. 4). The double reciprocal plot of 1/ (A A0) vs. 1/(drug concentration) is linear, and the binding constant (K) can be estimated from the ratio of the intercept to the slope (Fig. 4), where A0 is the initial absorbance of the free DNA at 258 nm, and A is the recorded absorbance of DNA in the presence of different drug concentrations. The overall binding constants are estimated to be Kharmine DNA = 3.44 107 M1 for harmine–DNA, Kharmane DNA = 1.63 105 M1 for harmane–DNA, Kharmaline DNA = 3.82 105 M1 for harmaline–DNA, Kharmalol DNA = 6.43 105 M1 for harmalol–DNA, Ktryptoline DNA = 1.11x105 M1 for tryptoline–DNA. Similar binding constants were estimated for other isoquinoline alkaloids–DNA complexes [45]. The binding constants of alkaloids–DNA binding is in the order of harmine > harmalol > harmaline > harmane > tryptoline. The binding constant of harmine is 100 times greater than harmane that can be attributed to the presence of polar group OCH3 at the benzene ring on the skeleton ring. The presence of polar substituent at the benzene ring increases the binding affinity. The binding constant of harmine is 100 times greater than harmaline because the former is planar, while, the latter is not planar. It can be concluded that the binding of planar compounds with DNA is stronger than that of non-planar compounds. By decreasing planarity of the b-carboline skeleton, the binding affinity to DNA decreases. The binding constant of harmalol is greater than harmaline that can be related to high electron donating property of OH rather than OCH3 which enhances the binding affinity of the indole ring. The above results are in compatible with Duportail and Taira experiments who calculated the same order of affinity for the b-carboline alkaloids–DNA complexes [20,22]. 4. Summary On the basis of our spectroscopic results, b-carboline alkaloids interact with bases via cytosine O2, thymine O2, guanine and adenine N7 atoms as well as backbone phosphate group (external binding) with partial and weak intercalation into G–C and A–T base pairs of DNA. The binding constants of alkaloids–DNA binding is in the order of harmine > harmalol > harmaline > harmane > tryptoline. No DNA conformational changes were observed upon alkaloids complexation. Disclosure statement No competing financial interests exist.
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