Mutagen–nucleic acid complexes at the polynucleotide duplex level in solution: Intercalation of proflavine into poly(dA-dT) and the melting transition of the complex

The nmr chemical shifts and line widths of the nucleic acid base and sugar proton resonances and the proflavine ring protons can be monitored through the melting transition of the proflavine + poly(dA-dT) complex, phosphate/dye (P/D) ratio = 24 and 8 in 1M salt solution. The nucleic acid and mutagen protons in the complex are in fast exchange between duplex and strand states with the midpoint of the melting transition monitored at the nucleic acid resonances increasing from 72.6°C for poly(dA-dT) to 78.1°C for the P/D = 24 complex and 83.4°C for the P/D = 8 complex in 1M salt solution. The melting transition monitored by the proflavine resonances were 80.0°C for the P/D = 24 complex and 84.3°C for the P/D = 8 complex in 1M salt solution. Since the nucleic acid is in excess at high P/D ratios, the nucleic acid transitions are an average for the opening of mutagen-free and mutagen-bound base-pair regions, while the proflavine transitions monitor the melting of mutagen-bound base-pair regions. The observed 0.75 to 0.95 ppm unfield shift at all four proflavine protons on formation of the complex with poly(dA-dT) provides direct evidence for intercalation of the mutagen between base pairs of the nucleic acid duplex. We have deduced the approximate overlap geometry between the proflavine ring and nearest-neighbor base pairs at the intercalation site from a comparison between experimental proflavine complexation shifts and those calculated for various stacking orientations. The experimental chemical shift of the poly(dA-dT) adenine H-2 resonance in the duplex state in the absence and presence of proflavine suggests that intercalation occurs preferentially at dT-dA sites. The selective chemical shift changes at the sugar H-2′,2″ and H-3′ resonances of the poly(dA-dT) duplex on complex formation demonstrates changes in the sugar pucker and/or torsion angles of the sugar phosphate backbone at the intercalation site.     

Interaction of phenosafranine with nucleic acids and model polyphosphates: II. Characterisation of phenosafranine binding to DNA

The binding of phenosafranine (PS) to DNA was studied by a combination of spectroscopic methods (absorption and fluorescence) together with hydrodynamic measurements (sedimentation and viscosity), Analysis of spectroscopic binding curves revealed that the strength of binding of PS to DNA is generally lower than that of proflavine. These measurements enabled recognition of several modes of interaction between PS and native DNA: strong monomer binding prevailing at high DNA phosphate/dye ratios (p) comprising binding outside the DNA helix as well as intercalation; two modes of dimer binding at lower values of p; and probably also weak surface-binding of monomers as p approaches unity. Longer surface-bound aggregates of PS were not detected because of the low tendency of the dye to form aggregates, though the presence of dimeric species distinct frorn pure surface-stacked PS dimer was indicated by various observations. It occurs over a broad range of p values Starting at p ≈110 for ionic strengths 10^?3–10^?1. Thermal denaturation data indicate that this species is bound more strongly than pure surface-bound stacked dimer. Its dimeric character may be explained in terms of interaction of an intercalated dye molecule with an adjacent outside-bound one as suggested for acridines by Armstrong et al. Various properties of this species are discussed. Both strong and weak modes or binding of PS to DNA are sensitive to the presence of organic solvents. The effectiveness of solvents to destabilise the complexes substantially coincides with their capacity to alter the water activity. Viscometric investigations reveal that in the region of strongest bindins (p ? 15) the elongation of the DNA helix by approximately 0.18 nm per bound PS molecule is accompanied by a strong negative change in persistence length, i.e. bending. Similar bending is also found at higher levels of binding (p ? 15) induced by less lightly bound PS molecules, in which region, however, the unusually high elongation of approximately 0.34 nm per bound PS molecule is observed.     

The circular dichroism of complexes of 2,7-di-tertiary-butyl proflavine with DNA

The binding isotherm of 2, 7-di-tert-butyl proflavine on calf thymus DNA has been measured by dialysis equilibrium. The CD spectra of complexes of the dye and DNA have been measured, and the variation of the induced circular dichroism of the dye with the amount of dye bound (r) has been found. The results show that di-tert-butyl proflavine binds to DNA in a completely different manner from proflavine itself, since both the visible and ultraviolet CD spectra of complexes of the two dyes with DNA differ markedly. The conformation of the nucleic acid is not affected by the binding of di-tert-butyl proflavine. It is possible that these results may allow determination, by using CD spectroscopy, of whether molecules intercalate into DNA.     

Stacking interactions of ethidium bromide bound to a polyphosphate and phage DNA in situ

Ethidium bromide forms spectroscopically detectable aggregates in aqueous solution and at a high dye concentration larger than 1 × 10^?3 moles/ρ. At moderate concentration in the order of 1 × 10^?4 moles/ρ the dye interacts with inorganic polyphosphate Graham salt and with phage sd DNA in situ by formation of stacking complexes. Maximal stacking was found at a phosphate to dye ratio, P/D, of approximately 1 for Graham salt and 1.5–2 for phages. In going to a higher P/D ratio Graham salt dye complex dissociates again and free dye reappears, while phage dye binding changes from stacking (type II complex) to intercalation (type I complex). Stacking is accompanied by a decrease and intercalation by an increase of relative fluorescence intensity with respect to free dye. However, both binding types lead to hypechromism and a red shift of the dye absorption band in the visible spectral region. Thus spectral behavior of ethidium aggregates deviate clearly from that known for other dyes, i.e., acridines.     

Interactions of molecules with nucleic acids. IV. Binding energies and conformations of acridine and phenanthridine compounds in the two principal and in several unconstrained dimer-duplex intercalation sites

The binding positions and relative minimum binding energies are calculated for complexes of 9-aminoacridine, proflavine, N-methylphenanthridinium, and ethidium in theoretically determined intercalation sites in B-DNA (sites I and II) and in unconstrained dimer-duplex sites. The selection of site I in B-DNA by these compounds agrees with the theoretical interpretation of studies of unwinding angles in closed circular DNA in all cases but ethidium, which is predicted to select site II. The most stable binding positions of the acridines and ethidium in unconstrained dimer-duplex units agree with experimental results of intercalation complexes of dinucleoside monophosphate units. Base-pair specificity for Watson-Crick pairing is examined. The energy of an intercalation complex is partitioned into ΔE₂₃, the energy required to open base pairs BP₂ and BP₃ in B-DNA to a site, and ΔE_In, the energy change when a free molecular intercalates. ΔE₂₃ depends strongly on the base-pair sequence, whereas ΔE_In for the four molecules studied does not. The three most stable sequences contain (pyrimidine)p(purine) units, and this provides a rationale for the exclusive formation of crystals of intercalation complexes with these units. In spite of this selectivity, the distribution of G?C and A?T base pairs is equal for these three units and persists as the more unstable sequences are included. Therefore, specificity arises from the interaction between the base pairs and the 2′-deoxyribose 5′-monophosphate backbone for the opening of B-DNA to an intercalation site and not from the interaction between the chromophore and the DNA.     

93 Spectroscopic investigation of methylene blue binding to DNA

The interaction of methylene blue (MB) with DNA has been investigated by UV absorption spectra, Fluorescence spectra and UV-melting method. Analysis of the results of the melting experiments shows that melting temperature (T _m) of the complexes increases with the [total ligand]: DNA ratio (r) at two concentrations of Na⁺ (2?mM Na⁺ and 20?mM Na⁺) providing support for conclusion that MB is a stabilizer of DNA helix structure. By contrast, the shapes of dependences of width of transition (ΔT) on r at low and high [Na⁺] are different which points to the existence of different types of binding modes of MB with DNA. UV-spectroscopy experiments and fluorescence spectra indicated that the binding modes of MB with DNA depended on r. At high r (r?>?0.25), remarkable hypochromic effect with no shift of λ _max in the absorption spectra of MB was observed. The fluorescence of MB was quenched which indicated that MB was bound to phosphate groups of DNA by electrostatic interaction. At low r ratios (r?<?0.2), the absorption spectra of MB upon increasing the concentration of DNA showed gradually decrease in the peak intensities with a red shift. This phenomenon is usually associated with molecular intercalation into the base stack of the ds-DNA. Using the Scatchard’s model, the complex formation constants for MB with DNA were determined: the binding constant K?≈?6.5?×?10⁵ and binding site size n?≈?4. Obtained data are not typical for intercalation model of ligands to DNA. Moreover, comparison between these data and our early experimental results of interaction of ethidium bromide with DNA made it possible to suggest that this binding type of MB is, more probably, semi-intercalation mode (Vardevanyan et al., 2003). This conclusion is in accordance with the analysis of the model structures of MB–DNA complexes which clearly shows the importance of solvent contributions in suggested structural form (Tong et al., 2010).     

Studies on the interactions of 3,6‐diaminoacridine derivatives with human serum albumin by fluorescence spectroscopy

This study reports the preparation and investigation of the modes of binding of the two symmetric 3,6‐diaminoacridine derivatives obtained from proflavine, which are 3,6‐diphenoxycarbonyl aminoacridine and 3,6‐diethoxycarbonyl aminoacridine to human serum albumin (HSA). The interaction of HSA with the derivatives was investigated using fluorescence quenching and ultraviolet‐visible absorption spectra at pH 7.2 and different temperatures. The results suggest that the derivatives used can interact strongly with HSA and are the formation of HSA‐derivative complexes and hydrophobic interactions as the predominant intermolecular forces in stabilizing for each complex. The Stern‐Volmer quenching constants, binding constants, binding sites and corresponding thermodynamic parameters ΔH, ΔS and ΔG were calculated at different temperatures. The binding distance (r) ~ 3 nm between the donor (HSA) and acceptors (3,6‐diethoxycarbonyl aminoacridine, 3,6‐diphenoxycarbonyl aminoacridine and proflavine) was obtained according to Förster's non‐radiative energy transfer theory. Moreover, the limit of detection and limit of quantification of derivatives were calculated in the presence of albumin. Copyright © 2014 John Wiley & Sons, Ltd.     

Viscosity and sedimentation study of sonicated DNA–proflavine complexes

The intrinsic viscosity of sonicated calf thymus DNA (molecular weight 4–5 × 10⁵) increases and the sedimentation constant decreases, with increasing binding of proflavine at 0. 2 ionic strength and at 25°C. The measurements correspond to a linear increase in length of the almost rodlike DNA molecules with the amount of proflavine bound; independent calculations from viscosity and sedimentation measurements yield almost identical results. Over the range of r (moles of proflavine bound per moles of nucleotides) equal to zero to r = 0.13, the length increases by about 20%. This extension is compatible with the intercalation hypothesis proposed by Lerman. Density increments at various values of r, at constant chemical potential of diffusible solutes, were determined. It was also found that, in addition to the known isosbestic point of DNA-proflavine complexes at 455.5 mμ, an additional isosbestic point exists at 225.5 mμ; this proved extremely useful for the evaluation of binding studies.     

Calorimetry of DNA-dye interactions in aqueous solution: I. Proflavine and ethidium bromide

The results of an investigation on the interaction of proflavine and of ethidium bromide with DNA (calf thymus) in dilute aqueous solution are reported. The binding of the two dyes by DNA has been studied by means of microcalorimetric and of equilibrium dialysis measurements. Data on the thermodynamics of dimerization of both proflavine and ethidium bromide in aqueous solution obtained on the basis of spectroscopic and/or calorimetric experiments are also reported.The enthalpy data show that dye-dimerization and dye “strong” interaction with DNA are energetically favourable and quite similar while only in the latter case the phenomenon is also entropy driven. This is taken as further evidence in support of the concept that “strong” interaction-of both proflavine and ethidium bromide with DNA means dye molecules intercalation into the native, double helical structure of the biopolymer.     

Induced circular dichroism of DNA-dye complexes

The binding of methylene blue, proflavine, and ethidium bromide with DNA has been studied by spectrophotometric titration. Methylene blue and proflavine or methylene blue and ethidium bromide were simultaneously titrated by DNA. The results indicate that all of these dyes compete for the same bindine sites. The binding properties are discussed in terms of symmetry. The optical properties of the dye–DNA complexes have been studied as a function of DNA/dye ratio. The induced circular dichriosm due to dye–dye interaction was measured at low dye/DNA ratios for cases involving both the same dye and different dyes. A positive Cotton effect for DNA–proflavine complex may be induced at 465 mμ by eithr proflavine or ethidium bromide, whereas a netgative Cotton effect at 465 mμ may be induced by methylene blue. The limiting circular dichroism, with no dye–dye interaction, and the induced circular dichroism spectra are discussed in terms of symmetry rules.     

Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed.     

Kinetic and hydrodynamic studies of the complex of proflavine with poly A·poly U

Relaxation kinetic experiments reveal general similarity between the mechanism of binding of proflavine to poly A·poly U and DNA. There are differences in detail, however. For example, the rate constants are roughly an order of magnitude smaller for the former, and the thermodynamic parameters of the individual steps are also different. The total heat and free energy for intercalation of free dye are quite similar in the two cases. As was the case with DNA, considerable dye (up to 25% of the bound form) is attached externally to the double helix, even in the strong binding region of the isotherm. Sedimentation measurements on small, rodlike fragments of poly A·poly U reveal a length increase on binding proflavine of a magnitude similar to that found with DNA. This length increase seems to become smaller under conditions (high temperature) where the relaxation measurements indicate a higher fraction of externally bound dye.     

Acetyl pyridine-based palladium(II) compounds as an artificial metallonucleases

Palladium(II) complexes of type [Pd(Lⁿ)Cl₂] and [Pd(bdt)(Lⁿ)] have been synthesized using 2-acetyl pyridine derivatives(Lⁿ) and benzene-12-dithiol(bdt). The synthesized complexes have been characterized by various analytical techniques like thermo gravimetric analysis, elemental analysis, conductance measurement, and spectroscopic techniques like elemental analysis, mass spectra, absorption spectra, IR, ¹H NMR, energy-dispersive X-ray spectroscopy. The interaction of the complexes with calf-thymus DNA (CT-DNA) has been explored by absorption titration, viscosity measurement methods. Based on the observations, an intercalative binding mode of DNA has been proposed. In order to provide additional evidence for the intercalation mode of binding between the complex and CT-DNA, fluorescence titration experiment was performed. In addition, molecular modeling study has been carried out with the aim of establishing the complex’s binding mode. Antibacterial activity study of the complexes have been screened against pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Serratia marcescens, and Pseudomonas aeruginosa. Gel electrophoresis assay demonstrates that all the complexes can cleave the pUC19 plasmid DNA.     

Über die Vitalfluorochromierung des Kernchromatins und der Chromosomen von HeLa-Zellen mit AMHA und die Bindung des Acridinfarbstoffs an DNA

Summary The fluorochrome AMHA (3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine) stains the nuclear chromatin and the chromosomes of living HeLa cells. At relatively low dye concentrations C _F10^–4 M and short incubation periods t _I2 h cell growth is not affected by the drug. But at higher C _F and longer t _I the population doubling time of the cell cultures rapidly increases, and finally the cells die.In vital staining experiments the dye AMHA preferentially binds to the DNA of the nuclei and to the chromosomes of the cells, respectively. The dye binding to DNA has been proved by the absorption and emission microspectra of the stained cells, and by the comparison with authentic spectra of AMHA bound to DNA in aqueous solutions. Within the limits of experimental errors both types of spectra are identical. The spectra of DNA-bound AMHA show a characteristic gap of ca. 3500 cm^–1 between the 0-0-transitions of the long wave length ¹ L _a absorption and the fluorescence. AMHA molecules dissolved in the polar solvent water have a gap of even 4100 cm^–1. This energy gap shows that the electron distribution of AMHA is strongly changed by light absorption and emission.Finally, using absorption spectroscopy, we investigated the binding of AMHA to DNA in aqueous solutions over a wide range of concentrations of the dye, of nuceleic acid (calf thymus), and of the competitor NaCl respectively. The Scatchard binding isotherms were determined. With the method of competitive salt effect three different bonds of AMHA to DNA can be distinguished even at low dye concentrations: The intercalation 1 of the fluorochrome F, binding constant K _F1=1,1·10⁵ M ^–1, binding parameter n ₁=0,15; the pre-intercalative or external binding 2, K _F2=6,9·10⁵ M ^–1, n ₂=0,21; the external binding 3, K _F3=2,8·10⁵ M ^–1, n ₃=0,55. Externally bound dye molecules 2 and 3 occupy two phosphodiester residues of the DNA. A detailed discussion of the data and the competitive salt effect shows that in living cells only intercalated and small amounts of pre-intercalatively bound molecules 1 and 2 exist. The binding constant K _F1=1,1·10⁵ M ^–1 of AMHA is unusual high in comparison with the constants of intercalation of other dyes, K _F1=(1–4)·10⁴ M ^–1. Therefore, the amount of intercalated AMHA is also relatively high, and it is possible to visualize the DNA-bound fluorochrome in the nuclei and chromosomes of the living cells under the fluorescence microscope.     

Interaction between tryptophan‐Sm(III) complex and DNA with the use of a acridine orange dye fluorophor probe

The interaction of the Trp–Sm(III) complex with herring sperm DNA (hs‐DNA) was investigated with the use of acridine orange (AO) dye as a spectral probe for UV‐vis spectrophotometry and fluorescence spectroscopy. The results showed that the both the Trp–Sm(III) complex and the AO molecule could intercalate into the double helix of the DNA. The Sm(III)–(Trp)₃ complex was stabilized by intercalation into the DNA with binding constants: K^?_25°C = 7.14 × 10⁵ L·mol^?1 and K^?_37°C = 5.28 × 10⁴ L·mol^?1, and it could displace the AO dye from the AO–DNA complex in a competitive reaction. Computation of the thermodynamic functions demonstrates that Δ_rH_m^? is the primary driving power of the interaction between the Sm(III)(Trp)₃ complex and the DNA. The results from Scatchard and viscometry methods suggested that the interaction mode between the Sm(III)(Trp)₃ complex and the hs‐DNA is groove binding and weak intercalation binding. Copyright © 2015 John Wiley & Sons, Ltd.     

86 Characterization and differentiation of binding modes of water-soluble porphyrins at complexation with DNA

The influence of water-soluble cationic meso-tetra-(4?N-oxyethylpyridyl)porphyrin (H₂TOEPyP4) and it’s metallocomplexes with Ni, Cu, Co, and Zn on hydrodynamic and spectral behavior of DNA solutions has been studied by UV/Vis absorption and viscosity measurement. It was shown that the presence of planar porphyrins such as H₂TOEPyP4, NiTOEPyP4, and СuTOEPyP4 leads to an increase in viscosity at relatively small concentrations, and then decrease to stable values. Such behavior is explained by intercalation of these porphyrins in DNA structure because the intercalation mode involves the insertion of a planar molecule between DNA base pairs which results in a decrease in the DNA helical twist and lengthening of the DNA. Further decrease of viscosity is explained by the saturation intercalation sites and occurs outside the binding mode. But, in the case of porphyrins with axial ligands such as CoTOEPyP4 and ZnTOEPyP4, the hydrodynamic parameters decrease, which is explained by self-stacking of these porphyrins in DNA surface. This data are proved by spectral measurements. The results obtained from titration experiments were used for calculation of binding parameters: the binding constant K _b and the number of binding sites per base pair n. Obtained data reveal that K _b varies between 3.4 and 5.4?×?10⁶?M^?1 for a planar porphyrins, a range typical for intercalation mode interactions, and 5.6?×?10⁵?M^?1 and 1.8?×?10⁶?M^?1 for axial porphyrins. In addition, the exclusion parameter n also testifies that at intercalation, (n～2) the adjacent base pairs are removed to place the planar molecules, and for outside binders to pack on the surface needs too few places (n～0.5–1). It is apparent that the binding is somewhat stronger at intercalation. The viscometric and spectrophotometric measurements are in good agreement.     

Studies of the optical properties of the proflavine–DNA complex

We report studies of the optical properties of the proflavine–DNA complex, using absorbance and circular dichroism spectroscopy. From comparison of the absorption spectra of proflavine complexed with calf thymus and T2 DNA, we conclude that stacking of the dyes external to the double helix is comparatively much weaker with T2 DXA, probably because of its glucosylation. Several sources are found for the circular dichroism induced in proflavine when it is complexed with DNA. There is a relatively weak circular dichroism induced when the dye is infinitely dilute on the DNA lattice; this presumably arises from the environmental asymmetry of the binding site. Stronger circular dichroism effects are induced by interaction of intercalated and stacked dyes; studies with T2 DNA, for which stacking seems to be blocked, permit a tentative resolution of effects due to the two modes of binding. One recurring theme of these studies is the observation that the optical properties are quite dependent on environment. The most dramatic example is a strong variation with salt concentration of the amplitude of the circular dichroism induced in the isolated (intercalated) monomer by the surrounding DNA. This suggests that the structure of the intercalated complex is quite sensitive to external conditions.     

Heterogeneity of binding sites of proflavin on DNA

The desorption and melting with temperature of proflavine–DNA complexes has been studied by spectrophotometry and spectrofluorometry. Two methods are described to determine at each temperature the concentration of free and bound dye. The first one is based on the quenching of fluorescence of the free dye by the iodine ion, the second on fluorescence polarization measurements. It is shown that the sites where the bound dye fluoresces are thermally less stable than those where it is quenched, in such a way that a redistribution of the dye between the two types of sites occurs at intermediate temperatures, leading to a drop in the total fluorescence. This confirms the nature of the “emitting” sites which correspond to AT-rich region, while “quenched” sites correspond to GC-rich region. The first have a larger binding constant at room temperature, but only the latter are stabilized by dye intercalation. The desorption and melting have also been followed through the relative changes of absorption. The curves obtained at different wavelengths are not superimposed which is at variance with what is observed with complexes of proflavine with poly dAT and poly dG.dC. The beginning of the desorption process corresponds to minor variations at 445 nm, the maximum of absorption of the free dye, but large changes occur at 460 nm, the maximum of the difference spectrum of the complexes proflavine–poly dAT and proflavine-poly dG.dC. The spreading of the melting curves for different wave lengths must therefore reflect the dependence of the absorption spectra of the dye on the nature of the neighboring bases. However, the action spectrum of the fluorescence, which gives the absorption spectrum of the “emitting” sites only, is identical with the total absorption spectrum of the bound dye.     

Intercalation of cationic dyes in the DNA double helix: introductory theory

The effect of salt on the intercalation of acridine dyes and DNA is rather well explained by the Gouy-Chapman double-layer theory as applied to a cylinder model of the DNA–dye complex. The free energy of transfer of a dye ion from the bulk solution to the complex is divided into several parts, one of which, ΔF₀, accounts for the short-range, nonelectrostatic interactions. The assumption that ΔF₀ should not depend on the amount of dye in the complex leads to an internal dielectric constant of the cylinder of about D_i = 7. The scatter in ΔF₀ values, as calculated from individual experimental points, is of order 0.5 kT per dye ion. This scatter is large enough to mask possible effects of heterogeneity in DNA sequences. The calculations are made for a long cylinder with radius 10 Å, with the DNA phosphate charges smeared uniformly at the surface, a uniform spacing of dye charges at the cylinder axis, and a length of b = 3.37 Å per base pair. Each intercalated dye ion also adds a length b to the total length of the cylinder. The salt-dependent part of the electric free energy of intercalation, ΔF₁, is tabulated for complexes with r = 0–0.24 dye ions per DNA phosphate in 0.002–0.2M monovalent salt and dye solutions.     

DNA binding of a proflavine derivative bearing a platinum hanging residue

New platinum(II) complex of 3,6-diamine-9-[6,6-bis(2-aminohethyl)-1,6-diaminohexyl]acridine, AzaPt, has been synthesised and characterised. Behaviour of AzaPt in solution (protonation and possible self-aggregation phenomena) has been investigated by spectral methods (absorbance and fluorescence) at I = 0.1 M and 25 °C, and the equilibrium parameters of binding to calf thymus DNA have been established. Two different modes of DNA binding by the complex were detected, which depend on the polymer to dye molar ratio (P/D). At relatively low P/D values the mode was interpreted as binding by the polyamine residue external to the base pairs, while at high P/D values the binding corresponds to intercalation of the proflavine residue. Such interpretation is supported by the observed salt effect on binding and the temperature variation of the binding constants, which allowed estimating the ΔH and ΔS values contributions. Spectrophotometric analysis of the long time range binding revealed that AzaPt is involved in a slow reaction, interpreted as an attack by the platinum ion on the nucleobases. The time constant for such interaction was calculated and found to be the same order of magnitude as for processes responsible for the action of anti-tumour drugs that do covalently bind to polynucleotides.