Competitive interaction of serotonin and the dye acridine orange with DNA

The interaction of serotonin and acridine orange dye with DNA isolated from bacterium Escherichia coli and the yeast Candida utilis has been analysed by spectrofluorimetric method. Using data on competitive binding to DNA of serotonin and acridine orange, known as DNA intercalator, a conclusion concerning the formation of intercalated complex between serotonin and DNA has been made. It is shown that for yeast DNA the constant of intercalated binding of serotonin is 3,5-fold smaller than for the bacterial one.     

Synthesis of a novel intercalator based on 2,2'-binaphthalene bearing dimethylammonium groups

A novel type of DNA intercalator, 8,8'-bis(dimethylaminomethyl)-2,2'-binaphthalene (1) based on 2,2'-binaphthalene skeleton, was prepared via homocoupling of 7-bromo-1-methylnaphthalene as a key step. The binding ability of 1 for calf thymus (CT) DNA was evaluated by UV-vis and fluorescence spectroscopic titrations and the melting temperature of CT DNA. The apparent association constant of 1 with CT DNA was larger than that of ethidium bromide (EB).     

DNA Bifunctional intercalators. 2. Fluorescence properties and DNA binding interaction of an ethidium homodimer and an acridine ethidium heterodimer

An ethidium homodimer and acridine ethidium heterodimer have been synthesized (Gaugain, B., Barbet, J., Oberlin, R., Roques, B. P., & Le Pecq, J. B. (1978) Biochemistry 17 (preceding paper in this issue)). The binding of these molecules to DNA has been studied. We show that these dimers intercalate only one of their chromophores in DNA. At high salt concentration (Na+ greater than 1 M) only a single type of DNA-binding site exists. Binding affinity constants can then be measured directly using the Mc Ghee & Von Hippel treatment (Mc Ghee, J. D., & Von Hippel, P. H. (1974) J. Mol. Biol. 86, 469). In these conditions the dimers cover four base pairs when bound to DNA. Binding affinities have been deduced from competition experiments in 0.2 M Na+ and are in agreement with the extrapolated values determined from direct DNA-binding measurements at high ionic strength. As expected, the intrinsic binding constant of these dimers is considerably larger than the affinity of the monomer (ethidium dimer K = 2 X 10(8) M-1; ethidium bromide K = 1.5 X 10(5) M-1 in 0.2 M Na+). The fluorescence properties of these molecules have also been studied. The efficiency of the energy transfer from the acridine to the phenanthridinium chromophore, in the acridine ethidium heterodimer when bound to DNA, depends on the square of the AT base pair content. The large increase of fluorescence on binding to DNA combined with a high affinity constant for nucleic acid fluorescent probes. In particular, such molecules can be used in competition experiments to determine the DNA binding constant of ligands of high binding affinity such as bifunctional intercalators.     

Thermodynamical model of mixed aggregation of ligands with caffeine in aqueous solution. Part II

A statistical-thermodynamical model of mixed association in which one component's self-association is unlimited while the second component does not self-aggregate is described. The model was tested with 4',6-diamidino-2-phenyl-indole-dihydrochloride (DAPI) and ethidium bromide (EB) using light absorption spectroscopy and calorimetry. The system is controlled by two parameters, which represent self-aggregation 'neighborhood' association constant KCC and mixed 'neighborhood' association constant KAC. Calculated, using this model, KAC = 58.2 +/- 1 M-1, KAC = 64.6 +/- 2 M-1 for DAPI and EB, respectively, are in good agreement with known values of stacking interactions. The titration microcalorimetric measurement of DAPI-CAF interaction delta H = -11.1 +/- 0.4 kcal/mol is also consistent with this type of reaction. The structures of the stacking complexes were also confirmed by semi-empirical molecular modeling in the presence of water. The data indicate that CAF forms stacking complexes with DAPI and EB, thus effectively lowering the concentration of the free ligands in the solution, and therefore, CAF can be used to modulate aromatic compound activity.     

Pressure-jump study of the kinetics of ethidium bromide binding to DNA

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

An evaluation of DNA fluorochromes, staining techniques, and analysis for flow cytometry. I. Unperturbed cell populations

Several preparative techniques (detergent treatment, ethanol fixation, and hypotonic cell lysis), DNA fluorochromes, and methods of numerical analysis (planimetric or curve-fitting) were compared for the estimation of cell-cycle kinetic parameters (G1, S, G2 + M) by flow cytometry. In addition, coefficients of variation (CV), relative fluorescence, and G1/chicken erythrocyte (CRBC) ratios were measured and the effects of the proportion of cycling cells and cellular RNA content were examined. DNA fluorochromes were ranked by relative fluorescence: 4,6-diamidino-2-phenylindole > ethidium bromide/mithramycin > Hoechst 33342 > mithramycin > ethidium bromide > acridine orange approximately equal to propidium iodide. The first four (DNA-specific stains) gave lower CVs than the remainder (DNA intercalators). Detergent treatment also increased relative fluorescence and slightly lowered CVs. Comparable results were obtained for the kinetic parameters independently of stain or staining procedure; intercalating dyes with cells of a high RNA content not treated with RNAse and acridine orange being the exceptions. Of the two methods of numerical analysis, the planimetric technique was more consistant. Although highly consistant G1/CRBC ratios were obtained for any one stain, independently of staining procedures, variations between stains were noted. It is suggested that the detergent treatment in combination with DNA-specific stains provide optimal results.     

Binding of ethidium and bis(methidium)spermine to Z DNA by intercalation

The interaction of ethidium bromide, a DNA intercalating drug, and bis( methidium )spermine, a DNA bis-intercalating compound, with the left-handed Z form of poly(dG-dC) has been studied in 4.4 M NaCl. Spectrophotometric analysis using absorption, fluorescence and circular dichroism indicates that the complex formed between ethidium and Z DNA resembles very closely that formed with B DNA. This suggests that ethidium binds to Z DNA by intercalation. 31P NMR spectra are presented showing both the conversion of the Z form to the B form with increasing amounts of drug and the typical Z form spectrum at low binding densities. Data are also presented which show that the bifunctional intercalator bis( methidium )spermine binds to Z DNA in a manner similar to its binding to B DNA, i.e., by bis-intercalation. These results are important for our understanding the behavior of Z DNA and its biological significance.     

Initial studies of a phytoestrogen-deoxyribonucleic acid interaction

Molecular modeling studies show that estrogens such as estradiol complement the topography of spaces between base pairs in unwound DNA and simultaneously hydrogen bond phosphate moieties on opposite strands. We demonstrate here that the phytoestrogen coumestrol has this capability, in addition to its documented properties of UV absorbance at lambda greater than 300 nm and fluorescence. The latter properties enable spectroscopic examination of interactions with DNA by methods not possible with estrogenic steroids. On exposure to calf thymus DNA, the UV spectrum of coumestrol displays a bathochromic shift and simultaneous hypochromic effect with an isosbestic point at 370 nm, suggesting a shift between coexisting free and bound states. Similar results are observed with the intercalating agents adriamycin, ethidium bromide, and acridine. The fluorescence spectrum of coumestrol is quenched on exposure to DNA as are those of adriamycin and acridine. Coumestrol differs from the intercalators in that denatured DNA does not affect its UV spectrum or alter its relative fluorescence yield. Unlike classical intercalators, coumestrol has no influence on the thermal stability of calf thymus DNA. Preliminary electrophoretic analysis of DNA plasmid conformers indicates that coumestrol is incapable of significantly altering DNA superhelical density, in contrast to ethidium bromide. These initial physicochemical data provide evidence for the DNA base-estrogen electronic and/or hydrophobic interactions suggested by modeling studies, yet tend to rule out classical intercalation as an explanation for these phenomena.     

A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.     

A comparison of fluorochromes for direct viable counts by image analysis

Total direct and direct viable counts of fresh and injured cultures of Escherichia coli were determined by image analysis in preparations stained with acridine orange, ethidium bromide and 4',6-diamidino-2-phenyl indole (DAPI). Cells stained with DAPI were not detected by image analysis. Fresh cultures stained with acridine orange or ethidium bromide gave comparable counts. Injured E. coli stained with ethidium bromide gave higher counts that with acridine orange. Injured cultures stained with acridine orange contain high proportions of green cells which are less easily detected than red cells in image analysis. In certain cases it may be better to use ethidium bromide, which stains all cells red, for direct viable counts by image analysis.     

An evaluation of the conformational changes in the superhelical DNA of eukaryotic cells by direct nucleoid fluorometry. II. The characteristics of the change in acridine orange fluorescence in studying the superhelical DNA of rat thymocytes

Comparative studies of acridine orange (AO) and ethidium bromide interactions with supercoiled DNA (scDNA) of thymocytes were performed in which various conformational changes were induced. AO may be efficiently used for evaluation of conformational alterations of scDNA. Moreover, employing the maximum values of AO fluorescence allows to determine the maximum levels of scDNA relaxation.     

Diffusion-enhanced lanthanide energy-transfer study of DNA-bound cobalt(III) bleomycins: comparisons of accessibility and electrostatic potential with DNA complexes of ethidium and acridine orange

Energy transfer in the "rapid-diffusion" limit reflects the equilibrium properties of a donor-acceptor system. Rates of energy transfer from freely diffusing terbium chelates to DNA-binding chromophores change dramatically when DNA is added; energy transfer from an electrically neutral chelate is reduced because the energy acceptor becomes partially buried in DNA, while energy transfer from a positive chelate is increased because of electrostatic attraction. The rate constants for energy transfer to DNA-bound chromophores from a positively charged terbium chelate, relative to those from a neutral chelate, were used to estimate the following values for the electrostatic potential near the surface of each DNA-bound acceptor at 298 K in the presence of 1.0 mM added salt (in units of -e/kT): acridine orange, 4.54 +/- 0.11; ethidium, 4.66 +/- 0.07; green Co(III) bleomycin A2, 4.06 +/- 0.11; orange Co(III) bleomycin A2, 3.11 +/- 0.10. Smaller numbers indicate less negative potentials; these can be due to a combination of (1) positive charge on the chromophore, (2) location of the chromophore [particularly Co(III) bleomycin] away from the DNA phosphates, and/or (3) separation of DNA phosphate negative charges by an intercalator. The magnitudes of the individual rate constants indicate that all the DNA-bound chromophores can be directly encountered by the terbium probes. Energy-transfer rate constants from a neutral terbium chelate to DNA-bound and free acceptors can provide a measure of the accessibility of the terbium probe to each bound chromophore. The ratios of these rate constants were as follows: acridine orange, 0.17 +/- 0.01; ethidium, 0.27 +/- 0.02; green form of Co(III) bleomycin A2, 0.48 +/- 0.06; orange form of Co(III) bleomycin A2, 0.71 +/- 0.06. These results are consistent with the probable differences in binding mechanisms for the intercalating chromophores (ethidium and acridine orange) as compared to the Co(III) bleomycins (in which the relevant chromophores are nonintercalating metal centers). In addition, all the results imply that the green Co(III) bleomycin chromophore binds closer to DNA than the orange; this provides a first step toward understanding the structural basis for the different biological properties of these metallobleomycins. Control experiments and theoretical considerations necessary to establish the validity of the results are also presented.     

The "interceptor" properties of chlorophyllin measured within the three-component system: intercalator-DNA-chlorophyllin

In aqueous solutions, in the presence of double-stranded DNA, chlorophyllin (CHL) forms complexes with each of the three DNA intercalators: acridine orange (AO), quinacrine mustard (QM), and doxorubicin (DOX). The evidence for these interactions was obtained by measurement changes in the absorption and fluorescence spectra of the mixtures containing DNA and intercalators during titration with CHL. A model of simple competition between DNA and CHL for the intercalator was used to define the measured interactions. The concentrations of the complexes estimated based on this model were consistent with the concentrations obtained by actual measurement of the absorption spectra. The present data provide further support for the role of chlorophyllin as an "interceptor" that may neutralize biological activity of aromatic compounds including mutagens and antitumor drugs.     

1H-nuclear magnetic resonance of intercalators and rGCA: a potential mutagenicity probe

Variable temperature 1H-nuclear magnetic resonance (NMR) has been used to study the interaction of the RNA trimer, GpCpA, with the intercalators ethidium bromide and the acridine derivatives; proflavin, 9-amino-acridine, acridine orange, acridine yellow and acriflavin. The complexes formed were studied at nucleic acid to drug ratios of 1:1 and 5:1, the latter being useful in defining the effects of structural variation in the acridine series and in determining the site of intercalation. All the intercalators greatly stabilized the oligonucleotide duplex, the average melting temperature (Tm) increasing by up to 30 degrees C. Significant changes in individual Tms and chemical shifts were observed for all the GpCpA protons. 9-Amino-acridine and acriflavin did not stabilize the GpCpA duplex as substantially as the other acridine derivatives. It is suggested that this intercalator:GpCpA system, and its associated NMR-derived Tm, is a useful physical probe for potential mutagens.     

Linear dichroism and polarized fluorescence of dye-complexed DNA fibers

Summary A simple method to obtain well orientated DNA fibers for studying the ordered binding of dyes and fluorochromes by linear dichroism and polarized fluorescence is described. The metachromatic dye toluidine blue and the intercalating fluorochromes ethidium bromide and acridine orange showed a perpendicular alignement to DNA; the minor groove binding fluorochromes 33258 Hoechst and DAPI appeared parallel. Thus, DNA fibers represent a suitable cytochemical test substrate for studying the orientation of bound dyes by polarization methods.     

In Pseudomonas aeruginosa ethidium bromide does not induce its own degradation or the assembly of pumps involved in its efflux

Xu et al. [Biochem. Biophys. Res. Commun. 305 (2003) 941] reported that, when a mutant strain of Pseudomonas aeruginosa lacking its major multidrug efflux pump complex, MexAB-OprM, was incubated with 100 μM ethidium bromide, the fluorescence, caused by its binding to DNA following its entry into cells, decreased gradually. The authors concluded that the intracellular ethidium bromide “induced” either its degradation or its efflux through the assembly of unknown efflux pumps. We found, through quantitation of ethidium bromide by absorption spectroscopy, that the total amount of ethidium bromide in the system remained constant under these conditions, indicating the absence of its degradation. Furthermore, intracellular ethidium bromide kept increasing during the experiment, showing that the decrease of fluorescence was due to self-quenching, and that ethidium bromide is not pumped out by a newly assembled efflux system.     

Interaction of intercalating and non-intercalating agents with DNA: use of hydroxyapatite chromatography and S1 nuclease

We have used hydroxyapatite (HA) chromatography and S1 nuclease hydrolysis to study the modification in the secondary structure of DNA caused by certain intercalating and non-intercalating ligands. The principal conclusions of HA experiments were as follows: (1) when native DNA, complexed with drugs believed to bind to DNA by intercalation (ethidium bromide, acridine orange, actinomycin D and acriflavin), is chromatographed on HA a lower affinity of DNA for HA is observed; also, the DNA elutes from HA columns as a drug-DNA complex; (ii) ligands that are known to interact with DNA by surface interactions do not show these effects; (iii) it may be possible to quantitate the binding of the intercalating drug to DNA and to determine its degree of binding by HA chromatography. Possibly, intercalation causes a change in the configuration of the sugarphosphate backbone of DNA, resulting in an altered steric orientation or 'burial' of phosphate groups with reduced availability for surface interactions with HA. S1 nuclease was used to determine the thermal melting profiles of DNA complexed with ethidium bromide and acridine orange. The melting profile in both cases was found to be biphasic with considerably reduced denaturation even at 95 degrees C. This is accounted for by the property of intercalating agents of stabilizing the secondary structure of DNA and the reported preference in binding to G-C base pairs.     

A comparison by ultracentrifugation of the effects on DNA of ethidium bromide and of acridine orange at low ionic strength

The application of scaled particle theory to the gels formed by DNA in the ultracentrifuge has provided values for the effective length and the effective radius of the DNA particle. Ethidium bromide has been shown to cause extensive lengthening of the DNA in dilute salt. Acridine orange interaction with DNA resulted in modest changes in DNA dimensions. These results are explained in terms of binding for acridine orange and of denaturation of DNA by ethidium bromide.     

The effects of grafting of 2-pyridyl to [Ru(bpy)(2)(Hpip)](2+) on acid-base and DNA-binding properties: experimental and DFT studies

A new Ru(II) complex of [Ru(bpy)(2)(Hpip)](2+) {bpy = 2,2'bipyridine; Hppip = 2-(4-(pyridin-2-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} has been synthesized by grafting of 2-pyridyl to parent complex [Ru(bpy)(2)(Hpip)](2+) {Hppip = 2-(4-phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline}. The acid-base properties of [Ru(bpy)(2)(Hpip)](2+) studied by UV-visible and luminescence spectrophotometric pH titrations, revealed off-on-off luminescence switching of [Ru(bpy)(2)(Hpip)](2+) that was driven by the protonation/deprotonation of the imidazolyl and the pyridyl moieties. The complex was demonstrated to be a DNA intercalator with an intrinsic DNA binding constant of (5.56 ± 0.2) x 10(5) M-1 in buffered 50 mM NaCl, as evidenced by UV-visible and luminescence titrations, reverse salt effect, DNA competitive binding with ethidium bromide, steady-state emission quenching by [Fe(CN)6]4-, DNA melting experiments and viscosity measurements. The density functional theory method was also used to calculate geometric/electronic structures of the complex in an effort to understand the DNA binding properties. All the studies indicated that the introduction of 2-pyridyl onto Hpip ligand is more favorable for extension of conjugate plane of the main ligand than that of phenyl, and for greatly enhanced ct-DNA binding affinity accordingly.     

The binding of cytochrome c peroxidase and ferricytochrome c. A spectrophotometric determination of the equilibrium association constant as a function of ionic strength

Complex formation between cytochrome c peroxidase and ferricytochrome c perturbs the optical absorption spectrum in the Soret band by about 2%. This perturbation can be utilized as a measure of the complex formed in solution and permits the determination of the stoichiometry and the equilibrium association constant for this reaction. At pH 6, in cacodylate/KNO3 buffers, only a 1:1 complex between cytochrome c peroxidase and ferricytochrome c is detected. The equilibrium association constant for the complex has been determined as a function of ionic strength and varies between (6.0 +/- 3.6) x 10(6) M-1 and (2.2 +/- 1.9) x 10(6) M-1 over the ionic strength range 0.01 M to 0.20 M.