Structural Analysis of DNA Interactions with Biogenic Polyamines and Cobalt(III)hexamine Studied by Fourier Transform Infrared and Capillary Electrophoresis

Biogenic polyamines, such as putrescine, spermidine, and spermine are small organic polycations involved in numerous diverse biological processes. These compounds play an important role in nucleic acid function due to their binding to DNA and RNA. It has been shown that biogenic polyamines cause DNA condensation and aggregation similar to that of inorganic cobalt(III)hexamine cation, which has the ability to induce DNA conformational changes. However, the nature of the polyamine·DNA binding at the molecular level is not clearly established and is the subject of much controversy. In the present study the effects of spermine, spermidine, putrescine, and cobalt(III)hexamine on the solution structure of calf-thymus DNA were investigated using affinity capillary electrophoresis, Fourier transform infrared, and circular dichroism spectroscopic methods. At low polycation concentrations, putrescine binds preferentially through the minor and major grooves of double strand DNA, whereas spermine, spermidine, and cobalt(III)hexamine bind to the major groove. At high polycation concentrations, putrescine interaction with the bases is weak, whereas strong base binding occurred for spermidine in the major and minor grooves of DNA duplex. However, major groove binding is preferred by spermine and cobalt(III)hexamine cations. Electrostatic attractions between polycation and the backbone phosphate group were also observed. No major alterations of B-DNA were observed for biogenic polyamines, whereas cobalt(III)hexamine induced a partial B → A transition. DNA condensation was also observed for cobalt(III)hexamine cation, whereas organic polyamines induced duplex stabilization. The binding constants calculated for biogenic polyamines are KSpm = 2.3 × 105m-1, KSpd = 1.4 × 105m-1, and KPut = 1.02 × 105m-1. Two binding constants have been found for cobalt(III)hexamine with K1 = 1.8 × 105m-1 and K2 = 9.2 × 104m-1. The Hill coefficients indicate a positive cooperativity binding for biogenic polyamines and a negative cooperativity for cobalt(III)hexamine. Biogenic polyamines, such as putrescine, spermidine, and spermine are small organic polycations involved in numerous diverse biological processes. These compounds play an important role in nucleic acid function due to their binding to DNA and RNA. It has been shown that biogenic polyamines cause DNA condensation and aggregation similar to that of inorganic cobalt(III)hexamine cation, which has the ability to induce DNA conformational changes. However, the nature of the polyamine·DNA binding at the molecular level is not clearly established and is the subject of much controversy. In the present study the effects of spermine, spermidine, putrescine, and cobalt(III)hexamine on the solution structure of calf-thymus DNA were investigated using affinity capillary electrophoresis, Fourier transform infrared, and circular dichroism spectroscopic methods. At low polycation concentrations, putrescine binds preferentially through the minor and major grooves of double strand DNA, whereas spermine, spermidine, and cobalt(III)hexamine bind to the major groove. At high polycation concentrations, putrescine interaction with the bases is weak, whereas strong base binding occurred for spermidine in the major and minor grooves of DNA duplex. However, major groove binding is preferred by spermine and cobalt(III)hexamine cations. Electrostatic attractions between polycation and the backbone phosphate group were also observed. No major alterations of B-DNA were observed for biogenic polyamines, whereas cobalt(III)hexamine induced a partial B → A transition. DNA condensation was also observed for cobalt(III)hexamine cation, whereas organic polyamines induced duplex stabilization. The binding constants calculated for biogenic polyamines are KSpm = 2.3 × 105m-1, KSpd = 1.4 × 105m-1, and KPut = 1.02 × 105m-1. Two binding constants have been found for cobalt(III)hexamine with K1 = 1.8 × 105m-1 and K2 = 9.2 × 104m-1. The Hill coefficients indicate a positive cooperativity binding for biogenic polyamines and a negative cooperativity for cobalt(III)hexamine. The biogenic polyamines putrescine NH2(CH2)4NH2, spermidine NH2(CH2)4NH(CH2)3NH2, and spermine NH2(CH2)3NH(CH2)4NH(CH2)3NH2 (Structure 1) are the most prevalent polyamines in mammalian cells. They are small aliphatic polycations involved in numerous diverse biological processes, such as the ability to modulate gene expression and enzyme activities, activation of DNA synthesis, cell proliferation and differentiation, and others (1Tabor H. Tabor C.W. Annu. Rev. 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Investigating on the mechanisms of this effect provides support for both electrostatic interaction between the polyamine amino-protonated groups and the negatively charged backbonePO2− group (38Thomas T.J. Bloomfield V.A. Biopolymers. 1984; 23: 1295-1306Crossref PubMed Scopus (104) Google Scholar, 39Tsuboi M. Bull. Chem. Soc. Jpn. 1964; 37: 1514-1522Crossref Google Scholar, 40Liquori A.M. Constantino L. Crescenzi V. Elia V. Giglio E. Puliti R. De Santis-Savino M. Vitagliano V. J. Mol. Biol. 1967; 24: 113-122Crossref Scopus (211) Google Scholar), as well as a direct interaction with DNA bases (21Jain S. Zon G. Sundaralingam M. Biochemistry. 1989; 28: 2360-2364Crossref PubMed Scopus (165) Google Scholar, 41Williams L.D. Frederick C.A. Ughetto G. Rich A. Nucleic Acids Res. 1990; 18: 5533-5541Crossref PubMed Scopus (60) Google Scholar). Both experimental and theoretical methods have been used to determine the binding positions of polyamines on B-form DNA. These studies have concentrated mainly on spermine cation, whereas a few structural details about the binding of the other polyamines to DNA are known. Rather contradictory views concerning the binding sites of spermine and other polyamines have emerged. X-ray diffraction from crystals of B-form dodecamer revealed a spermine bound into the major groove of DNA duplex (42Drew H.R. Dickerson R.E. J. Mol. Biol. 1981; 151: 535-556Crossref PubMed Scopus (799) Google Scholar). In photoaffinity cleavage experiments (43Schmid N. Behr J.-P. Biochemistry. 1991; 30: 4357-4361Crossref PubMed Scopus (106) Google Scholar), the binding position of spermine in solution appeared to be in the minor groove of B-DNA. From the NMR study of polyamine with DNA dodecamer, it has been proposed that the spermine cation appeared to be mobile (44Wemmer D.E. Srivenugopol K.S. Reid B.R. Morris D.R. J. Mol. Biol. 1985; 185: 457-459Crossref PubMed Scopus (95) Google Scholar). 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Thus, the binding positions of spermine and other polyamines on B-DNA have not been firmly resolved, and it is probable that both the base sequence and the environment have an important influence on polycation binding. In the present work, we have studied the interactions between calf-thymus DNA and biogenic polyamines as well as cobalt(III)hexamine cation using affinity capillary electrophoresis, Fourier transform infrared, and circular dichroism spectroscopic methods. Evidence for DNA condensation and helix stabilization is provided. Furthermore, the influence of polyamine charge and concentration on DNA structural changes has been discussed, and the presence of specific base-polyamine binding is reported. Materials—Polyamines spermine, spermidine, putrescine, and cobalt(III)hexamine were purchased from Sigma. Highly polymerized type I calf-thymus DNA sodium salt (7% sodium content) was purchased from Sigma, and deproteinated by the addition of CHCl3 and isoamyl alcohol in NaCl solution. To check the protein content of DNA solution, the absorbance at 260 and 280 nm was recorded. The A260/A280 ratio was 1.85, showing that the DNA was sufficiently free of protein (51Marmur J. J. Mol. Biol. 1961; 3: 208-218Crossref Scopus (8945) Google Scholar). Other chemicals were of reagent grade and used without further purification. Preparation of Stock Solutions—Sodium-DNA (8.3 mg/ml) was dissolved in 50 mm NaCl (pH 7.20) at 5 °C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the stock calf-thymus DNA solution was determined spectrophotometrically at 260 nm using a molar extinction coefficient ϵ260 = 6600 cm-1m-1 (expressed as the molarity of phosphate groups) (52Reichmann M.E. Rice S.A. Thomas C.A. Doty P. J. Am. Chem. Soc. 1954; 76: 3047-3053Crossref Scopus (2049) Google Scholar, 53Vijayalakshmi R. Kanthimathi M. Subramanian V. Biochem. Biophys. Res. Commun. 2000; 271: 731-734Crossref PubMed Scopus (163) Google Scholar). The UV absorbance at 260 nm of a diluted solution (1/250) of calf-thymus DNA used in our experiments was 0.661 (path length was 1 cm), and the final concentration of the stock DNA solution was 25 mm in DNA phosphate. The average length of the DNA molecules, estimated by gel electrophoresis, was 9000 bp (molecular mass ∼ 6 × 106 Da). The appropriate amount of polyamines (0.3-25 mm) was prepared in distilled water and added dropwise to the DNA solution, to attain the desired polyamine/DNA(P) molar ratios (r) of 1/80, 1/40, 1/20, 1/10, 1/4, 1/2, and 1 at a final DNA concentration of 12.5 mm (4.15 mg/ml) for infrared measurements. For capillary electrophoresis, the polyamine/DNA(P) ratios were 1/800, 1/400, 1/200, 1/100, 1/50, 1/25, 1/12.5, and 1/6.25 with a final DNA concentration of 1.25 mm. The pH of the solutions was adjusted at 7.0 ± 0.2 with a pH meter ORION model 210A, using NaOH solution. FTIR Spectra—Infrared spectra were recorded with a FTIR spectrometer (Impact 420 model) equipped with deuterated triglycine sulfate detector and KBr beam splitter, using AgBr windows. Spectra were recorded after 2-h incubation of polyamine with the polynucleotide solution and measured in triplicate (three individual samples of the same polynucleotide and polyamine concentrations). Interferograms were accumulated over the spectral range 400-4000 cm-1 with a nominal resolution of 2 cm-1 and a minimum of 100 scans. The water subtraction was carried out using 0.1 m NaCl solution at pH 7.0 ± 0.2 as a reference (54Alex S. Dupuis P. Inorg. Chim. Acta. 1989; 157: 271-281Crossref Scopus (196) Google Scholar). A good water subtraction is considered to be achieved if there is a flat baseline around 2200 cm-1, where the water combination mode is located. This method yields a rough estimate of the subtraction scaling factor, but it removes the spectral features of water in a satisfactory way (54Alex S. Dupuis P. Inorg. Chim. Acta. 1989; 157: 271-281Crossref Scopus (196) Google Scholar). The infrared spectra of polyamine·DNA complexes with molar ratios higher than 1/4 for spermine, spermidine, and cobalt-hexamine and higher than 1 for putrescine could not be recorded as a homogenous solution, due to DNA precipitation and solid gel formation. The difference spectra (DNA solution + polyamine) - (DNA solution) were obtained using a sharp DNA band at 968 cm-1 as an internal reference. This band, which is due to deoxyribose C-C and C-O stretching vibrations, exhibits no spectral changes (shifting or intensity variation) upon polyamine·DNA complexation and cancelled out upon spectral subtraction (55Ahmed Ouameur A. Nafisi S. Mohajerani N. Tajmir-Riahi H.A. J. Biomol. Struct. Dyn. 2003; 20: 561-565Crossref PubMed Scopus (27) Google Scholar). The spectra presented here were smoothed with a Savitzky-Golay procedure (54Alex S. Dupuis P. Inorg. Chim. Acta. 1989; 157: 271-281Crossref Scopus (196) Google Scholar). The plots of the relative intensity (R) of several peaks of DNA in-plane vibrations related to A-T, G-C base pairs and thePO2− stretching vibrations such as 1717 (guanine), 1663 (thymine), 1609 (adenine), 1492 (cytosine), and 1222 cm-1 (PO2− groups), versus the polyamine concentrations were obtained after peak normalization using,Ri=IiI968eq.1 where Ii is the intensity of absorption peak for pure DNA and DNA in the complex with i concentration of polyamine, and I968 is the intensity of the 968 cm-1 peak (internal reference). Circular Dichroism Measurements—Spectra were recorded with a Jasco J-720 spectropolarimeter. For measurements in the near-UV region, a quartz cell with a path length of 0.1 cm was used. Five scans were accumulated at a scan speed of 50 nm/min, with data being collected at every nanometer from 200 to 320 nm. Sample temperature was maintained at 25 °C using a Neslab RTE-111 circulating water bath connected to the water-jacketed quartz cuvettes. The concentration of the calf-thymus DNA solutions was kept at 1.25 mm (0.4 mg/ml) in 25 mm phosphate buffer, pH 7.0. CD spectra of DNA·cobalt(III)hexamine complexes were recorded withCo(NH3)63+/DNA(P) molar ratios in the range of 0, 1/15, 1/10, 1/8, 1/6, and 1/4. Spectra were corrected for buffer signal, and conversion to the molar ellipticity θ was performed with Jasco Standard Analysis software. Capillary Electrophoresis—A P/ACE System MDQ (Beckman) with photodiode array detector was used to study polyamine·DNA capillary of length of and length of 50 cm the was used. The capillary was by with 1 sodium for followed by a with 0.1 sodium it was with buffer for at high followed by a baseline for at the used for the experiments the capillary was with NaOH 1 for 2 followed by with buffer for at high The capillary was with distilled water for at the for and with water were using a at for 5 was carried out at a of 25 for using were carried out at 25 and low in the capillary was the complex to during The capillary and were after every five The polyamine binding experiments were performed in a buffer pH 7.0 ± using concentration of calf-thymus DNA mm) and concentrations of The stock solutions of polyamines mm) and DNA mm) were prepared in the The solutions were to attain polyamine/DNA(P) molar ratios of to 1/6.25 in the presence of 1.25 mm DNA. was to for and with for the same stock solution. The were at 260 nm and were collected and with the P/ACE software. capillary was used to a in polyamine binds to DNA. 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The and of the infrared of the DNA are in spectra in the of cm-1 for pure DNA, free polyamine, and and in solution at pH 7.0 ± 0.2 and difference spectra for polyamine·DNA obtained at polyamine/DNA(P) molar ratios relative and for the infrared of calf-thymus DNA in solution at pH ± = = = m = and = have been from the and are stretching of and of deoxyribose deoxyribose = = = m = and = have been from the and are in a At low polyamine concentrations = these polyamines bind to DNA Evidence for this from the changes in the intensity and of the absorption in the cm-1, due to the in-plane DNA (54Alex S. Dupuis P. Inorg. Chim. Acta. 1989; 157: 271-281Crossref Scopus (196) Google Scholar, Ouameur A. Nafisi S. Mohajerani N. Tajmir-Riahi H.A. J. Biomol. Struct. Dyn. 2003; 20: 561-565Crossref PubMed Scopus (27) Google Scholar, E. J. PubMed Scopus Google Scholar, E. J. J.A. Raman 1985; 12: Scholar, H. R. M. Tajmir-Riahi H.A. J. Biol.