Peculiarities of DNA-proflavine binding under different concentration ratios

Binding of proflavine to calf thymus DNA is investigated by differential scanning calorimetry and spectrophotometry. It is shown that proflavine interacts with DNA by three binding modes. At high DNA—ligand concentration ratios (P/D), proflavine prefers to intercalate into GC-sites but can also insert into other sites. At low P/D ratios, proflavine interacts with DNA by the external binding mode. The parameters of proflavine-DNA complexation have been calculated from spectrophotometric concentration dependences. Thermodynamic parameters of DNA melting have been calculated from differential scanning calorimetry data.     

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.     

Interaction of proflavine and 9-aminoacridine with DNA at temperatures below and above the melting temperature

The influence of temperature on the binding of 9-aminoacridine and of proflavine to E. coli DNA in 10^?3M NaCl solution has been determined by a spectrophotometric technique. The inadequacy of the expression normally used for the determination of the extent of binding is discussed with reference to measurements at temperatures above which dissociation of the double helix occurs. A method of determining the relative extents of binding to native and denatured DNA at elevated temperatures is described.     

The inhibition of ribonucleic acid polymerase by acridines

1. The aminoacridines, proflavine (3,6-diaminoacridine) and 9-aminoacridine, and a hydrogenated derivative, 9-amino-1,2,3,4-tetrahydroacridine, were shown to inhibit in vitro the DNA-primed RNA polymerase of Escherichia coli. The inhibition is strong with both proflavine and 9-aminoacridine, but weak with 9-amino-1,2,3,4-tetrahydroacridine. 2. The extent to which the three acridines bind to calf-thymus DNA in the enzyme medium was studied spectrophotometrically. The extent of binding decreases in the order: proflavine, 9-aminoacridine, 9-amino-1,2,3,4-tetrahydroacridine. Some evidence was also obtained for interaction between the nucleoside triphosphate substrates and proflavine or 9-aminoacridine; no such interaction was detectable with 9-amino-1,2,3,4-tetrahydroacridine. 3. Although the amount of acridine bound to DNA increases with increasing inhibition, a stage is reached where an increase in acridine concentration still causes an increase in inhibition, with practically no increase in the amount bound to DNA. 4. Plots of reciprocal rates against the reciprocal of DNA concentration were linear and had a common intercept when proflavine or 9-aminoacridine was present. Similar relations were obtained when the reciprocal concentration of nucleoside triphosphates was plotted. The observations are interpreted kinetically in terms of a competitive inhibition of the enzyme by proflavine or 9-aminoacridine and of a kinetic role for the DNA analogous to ;activation'. 5. This suggests that inhibitory acridine molecules can occupy the sites on the RNA polymerase that are specific for binding the nucleoside triphosphate substrate or the bases of the DNA, when these become accessible during the copying process.     

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.     

Dielectric behavior of DNA-proflavine complex

The dielectric relaxation of namtive DNA and DNA–proflavine complexes at different DNA phosphate (P) to dye (D) ratios, were investigated in the frequency range 100 c/sec to 100 Kc/sec. The proflavine molecules were found to have a profound effect on the static dielectric constant and the relaxation time of the polymers. The static dielectric constant was oberserved to decrese with increasing level of added proflavine. At P/D = 1, the variation of dielectric constant with frequency was small. Relaxation time (τ) was greater for the DNA–proflavine complexes compared to that for free DNA, Maximum value of the relaxation time was obtained at P/D = 10. The increase in the relaxation time and decrease in the static dielectric constant were attributed to the increase in length and meutralization of surface charges of the DNA molecules, respectively, as aresult of proflavine binding.     

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.     

Chromosome regions containing DNAs of known base composition,specifically evidenced by 2,7-di-t-butyl proflavine

After staining by a new proflavine derivative (2,7-di-t-butyl proflavine, DBP), which specifically binds to the A-T base pairs of DNA by an external process, the constrictions of the human chromosomes 1, 16 and to a lesser extent 9 and the centromeric regions of the chromosomes (except the Y) of Mus musculus are brightly fluorescent. These chromosome regions are known to contain repetitive DNAs rich in A-T. On the contrary, the centromeric regions of the autosomes of Bos taurus, which contain a G-C rich DNA, are faintly fluorescent. The arms of the chromosomes of the three species display a banding similar to, but fainter than, the Q-banding. These results are discussed in correlation with physico-chemical studies on the binding and fluorescence processes of the dye bound to DNA and to nucleohistone. The staining properties of DBP are compared to those of quinacrine, quinacrine mustard and proflavine, three intercalative dyes which are also supposed to reveal the A-T base pairs along the chromosomes, but are faintly fluorescent on the human and murine A-T rich regions. This comparison leads us to discuss the mechanisms responsible for the chromosomal banding in relation to DNA base composition and repetitiveness, protein distribution and packing of the chromatin fibers, along the chromosomes.     

Optical and electro-optical properties of the complexes of dibutylproflavine with DNA and nucleohistone

A number of optical and electro-optical measurements showed that the binding of dibutylproflavine to calf thymus DNA and nucleohistone in 1 mM NaCl could be resolved on the basis of two external and orientated bound species, whose binding parameters have been determined by a spectrophotometric method.The absorption and the electric dichroism properties were found to show up additivity of contributions from the two bound species. The orientation of the long axis of the strongly bound molecules of species I appeared close to the inclination of the DNA grooves, while that of species II differed by about ten degrees.A spatial proximity of the two binding sites was suggested by the dependence of the circular dichroism signals, the fluorescence quantum yield and the emission anisotropy on the degree of binding. The mobility of the first bound dibutylproflavine molecules was similar to that of the intercalated proflavine molecules, but lower in nucleohistone as compared to DNA.     

Extrinsic Cotton effects of proflavine bound to polynucleotides

The magnitude of the Cotton effect of proflavine which is bound to RNA or to denatured DNA depends on the ratio of bound proflavine to nucleic acid base. A statistical treatment which explains this behavior has been fitted to the experimental curves and indicates that optical activity arises through interaction between two or more bound proflavine molecules. The corresponding requirement with double helical DNA is for interaction between 3–4 proflavine molecules. Although proflavine binds to denatured DNA at pH 2.8, as shown by the shift of the proflavine spectrum, the strong binding process is absent, and to this is attributed the absence of the Cotton effect at low pH. Studies on the Cotton effects of proflavine bound to poly A and poly U at neutral pH, to poly A at acid pH and to poly (A + U) allow the generalization that a relatively rigid configuration of the binding macromolecule is required for the induction of these extrinsic Cotton effects.     

A temperature-jump kinetic study of the binding of proflavine to poly I-poly C

The kinetics of interaction between proflavine and poly I.poly C at 25°C, neutral pH, and moderate ionic strength have been studied by relaxation methods. The qualitative features of this system resemble those previously reported by Crothers and co-workers for proflavine–DNA and proflavine–poly A·poly U interactions–two relaxations are observed coresponding to a fast bimolecular step followed by a slower isomerization. These results can best be accommodated by a two-step mechanism leading from the free dye through an “outside-bound” complex to the intercalated complex. Quantitative comparison of the various rate constants for proflavine binding to different double-helical polynucleotides shows that the rates are slower for both ribohomopolymer pairs than for DNA. The rates for poly I·poly C are approximately three times faster than these for poly A·poly U.     

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.     

Effect of organic solvents on the properties of the complexes of DNA with proflavine and similar compounds

In order to obtain information on the binding forces involved in the formation of the complex proflavine–DNA by the stronger process I, the stability of the complexes was investigated in the presence of various organic solvents, methanol, ethanol, n-propanol, isopropanol, formamide, dimethyl sulfoxide, p-dioxane, glycerol, and ethylene glycol. Quantitative data on binding in terms of K/n and r were obtained by means of absorption and fluorescence spectra, as well as by a thermal denaturation technique. All organic solvents used decrease the binding ability of the dye. The effectiveness of the solvents increases with their hydrocarbon content, but can hardly be related to their dielectric constant. The complex formation is effectively suppressed by organic solvent concentrations, in which DNA still preserves its double-helical conformation. These results demonstrate the importance of hydrophobic forces in the formation of the complex proflavine–DNA in aqueous solution. The similarity in spectroscopic properties of proflavine bound to DNA by process I and the same dye dissolved in an organic solvent make it possible to interpret the observed red shift of the long-wavelength absorption peak as being due to the interaction of the dye molecules with the less polar environment. The same behavior was found for other dyes capable of intercalation like purified trypaflavine, phenosafranine and ethidium bromide. However, intercalation is not a necessary condition, as it was shown in the case of pinacyanol, which binds only at the surface of DNA.     

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.     

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.     

Mechanism of enhancement of polynucleotide binding to cells by mutagens.

The binding of polyuridylate to cells is substantially increased by proflavine. This enhanced binding is saturable with respect to time and to the concentration of both proflavine and polyuridylate. Enhancement is observed only when cells are exposed to both proflavine and polyuridylate together and depends cooperatively on the proflavine concentration. The resulting complex formed between the cell, proflavine, and polyuridylate can be dissociated with salt but not with sucrose solutions. An increase in the binding of polyuridylate to cells similar to that observed with proflavine was also obtained with cationic dyes such as acridine orange, 9-aminoacridine, and Hoechst 33258, while the introduction of a bulky polysaccharide residue, dextran, into the dyes cancels these effects. Similarly, cationic aromatic compounds such as primaquine and quinacrine which carry bulky nonplanar substituents or aliphatic cationic compounds like ethylenediamine do not enhance binding. Proflavine is unable to augment the binding of a basic macromolecule, diethylaminoethylaminoethyldextran, to cells. The model proposed for the enhanced binding of polyuridylate is based on the cooperative formation of stacked complexes of cationic dye located between the cell surface and the bound polyuridylate.     

Interaction of proflavine with 2-hydroxy-5-nitrobenzylated papain

Interaction of proflavine with papain, modified by hydroxynitrobenzylation of Trp-177 (HNB-papain), is characterized by a dissociation constant about twice that of proflavine's binding to native papain. Kinetic analyses revealed that proflavine's activation of papain-catalyzed hydrolysis of benzoyl-L-arginine ethyl ester persists with the HNB-enzyme; however, hydroxynitrobenzylation of papain precludes proflavine's inhibition of benzoyl-L-arginine p-nitroanilide (BzArgNan) hydrolysis. Yet, proflavine noncompetitively inhibits hydrolysis of BzPheValArgNan by both native and HNB-papain to about the same extent. Thus, proflavine appears to reduce nonproductive binding of smaller substrates, but may also interfere with conformational repositioning necessary for anilide hydrolysis.     

Effect of DNA base composition on the intercalation of proflavine. A kinetic study.

The effect of DNA base composition on the kinetics of the association between DNA and proflavine has been investigated using the temperature jump relaxation method. It is found that, regardless of the G + C base composition the results fit a two step mechanism, the second of which exhibits characteristics of intercalation of proflavine into DNA. However, they two equilibrium constants corresponding to these steps, KI and KII, depend on the nature of the DNAs. The constant KI is found to be an order of magnitude greater for M. lysodeikticus DNA (72% G + C) than for calf thymus DNA (48% G + C). Increasing G-C content thus appears to favor the intermediate non-intercalated complex of proflavine with DNA. Methylation of M. lysodeikticus DNA with dimethyl sulfate, preferentially yielding N7 methyl guanine as the modified base, again leads to an apparent two step mechanism, with the value of KI unchanged with respect to untreated DNA, while the affinity of proflavine for the intercalated complex measured by the value of KII increases for methylated DNA.     

Extrinsic cotton effects of aminoacridines bound to DNA

The optical rotatory dispersion curves of the proflavine cation were measured in the spectral range 400–500 mμ. No optical activity was observed for the free cation but a large positive Cotton effect appeared in the presence of DNA. The effect of ionic strength, denaturation of the DNA, and the DNA/proflavine ratio were studied. The dependence of the magnitude of the Cotton effect on the DNA/proflavine ratio suggests that a nearest-neighbor interaction between bound proflavine molecules is necessary for optical activity. A simple statistical treatment was made which indicated that only a small number of proflavine molecules are required in close proximity for optical activity to occur. Denaturation of the DNA did not destroy the optical activity, which shows that long runs of DNA double helix are not necessary for optical activity of the ligand molecules. The optical rotatory dispersion curves of acridine orange which was bound to DNA were also measured. Two Cotton effects of opposite sense could be distinguished, the relative magnitudes of which depended on the DNA/acridine orange ratio and the state of denaturation of the DNA. The apparent differences from the proflavine-DNA system can to a large extent be explained in terms of the tendency of acridine orange to form aggregates.