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.     

Fluorescence decay and quantum yield characteristics of acridine orange and proflavine bound to DNA

Fluorescence properties (quantum yield, decay curve, lifetime and polarization) of acridine orange and proflavine bound to DNA were examined as a function of nucleotide to dye (P/D) ratio. First, mean fluoiescence lifetimes were determined by the phase-shift measurements. The lifetime and quantum yield of acridine orange increased in a parallel fashion with increasing P/D ratio. There was no parallel relation between the lifetime and quantum yield for proflavine; the lifetime showed a minimum around P/D = 10. Next, fluorescence decay curves were measured by the monophoton counting technique and analyzed with the aid of the method of moments and the Laplace transform method. The results showed that the fluorescence decay of bound acridine orange was exponential above P/D = 10. On the other hand, the decay of bound proflavine was exponential above P/D = 100, but markedly deviated from exponentiality with decreasing P/D ratio. The results of fluorescence polarization suggested that this phenomenon is the result of Förster energy transfer between proflavine molecules bound to the fluorescent site (AT pair) and bound to the quenching site (GC pair). Critical transfer distances were 26-4 and 37.0 Å, respectively, for bound proflavine and acridine orange.     

Inhibition of Pyrimidine Dimer Formation in DNA by Cationic Molecules: Role of Energy Transfer

In addition to the acridine dyes, acridine orange and proflavine, we find that three other cationic molecules which bind to DNA-ethidium bromide, chloroquine, and methyl green-inhibit the production of cyclobutyl pyrimidine dimers by ultraviolet radiation. Intercalation is not necessary for dimer inhibition. The long range nature of the inhibition implies that energy transfer is responsible. The transfer is between the lowest excited singlet state of DNA and the acceptor singlet, and seems to involve the F?rster mechanism.     

Circular dichroism of acridine orange bound to DNA

The circular dichroism (CD) spectra of DNA–acridine orange (DNA–AO) complex in the visible region were measured at DNA phosphate-to-dye ratios (P/D) from 1 to 550. The CD spectrum of DNA–AO complex in the P/D ratio between 1 and approximately 40 consists of four components, i.e., positive CD bands centered at 510 and 480 mμ, and negative CD bands at 497 and 468 mμ. The CD bands at 510 and 468 mμ are optimum at P/D = 4, and the change of ε₁ ? ε_r with P/D suggests that both of them are induced from the interaction between dye molecules bound to adjacent DNA binding sites, each of which is composed of four nucleotides. This is supported by the fact that the values of ε₁ ? ε_r for both decrease with increasing temperature or increasing methylene blue concentration added to the complex. The negative Cotton effect at, 497 mμ is most favored at larger P/D ratio (～8), and the suggested assignment is to the interaction between two dye molecules bound with an empty site between them. A positive Cotton effect at 480 mμ is observed at P/D ratio of less than 4 and is optimum at 1. Above P/D ratio of 40, the CD spectrum of the complex can not be resolved into its components and even at sufficiently high P/D ratio (550) the complex shows a small Cotton effect.     

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.     

Structure and optical activity of the DNA-aminoacridine complex

The electronic absorption and circular dichroism spectra of the DNA-acridine orange complex have been measured over a range of ionic strength, pH, and DNA phosphate to dye (P/D) ratios. Three circular dichroism bands associated with the long wavelength absorption band of acridine orange are induced on complex formation with DNA. Two of the dichroism bands, due mainly to dimeric dye molecules, are favored by low ionic strength, low pH (3.2), and a low P/D ratio (～3), while the third, deriving primarily from monomeric dye, is optimum at high ionic strength, neutral pH, and a larger P/D ratio (9). The data suggest that monomeric acridine orange binds to DNA in the form of a left-handed helical array with four dye molecules per turn, while the bound dimer has a skewed sandwich conformation which is itself dissymmetric. The stereochemical relations between the bound monomer dye and the DNA are consistent with a modified intercalation model for the DNA-acridine complex.     

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.     

The Structure of Tobacco Mosaic Virus and Its Components: Ultraviolet Optical Rotatory Dispersion

An investigation has been made of the optical rotatory dispersion in the region 226 to 366 mμ of tobacco mosaic virus (TMV), the protein subunits isolated therefrom, the rods synthesized from the protein subunits, and the ribonucleic acid (RNA) isolated from TMV. Both TMV and the protein rods show anomalous rotatory dispersion. The RNA shows a Cotton effect with an inflection point around 260 mμ, which is shifted to 272 mμ in concentrated urea solution. A suggested interpretation of the RNA rotatory dispersion is given. The rotatory dispersion of the protein subunits shows an incipient Cotton effect with an inflection point around 293 mμ and the beginnings of a large negative Cotton effect with a trough at 232 mμ. The dispersion data from the protein subunits can be interpreted to indicate that they contain between 25 and 35 per cent α-helix. On the basis of recent sequence investigations and the relationship between amino acid composition and polypeptide structure, the helical portion of the protein subunits can be located in the central section of the protein chain.     

Effect of gamma-irradiation on dye-DNA binding.

The effect of gamma-rays on the binding of proflavine and acridine orange to DNA was investigated by spectrophotometry. The effect of irradiation was observed on the buffered solutions of the free dye and free DNA. A dose of about 35 krad caused a hyperchromicity of 30-40 per cent to the DNA peak at 258 nm, while the same dose introduced a hypochromic effect to the monomer peaks of the dyes by 30 per cent. This implied that gamma-rays have an effect of decreasing the monomer concentration of free-day molecules in solution. From the results, we conclude that more dye is bound to the changed conformation of dye-bound DNA on irradiation. Scratchard-binding isotherms drawn for the unirradiated and irradiated complexes of Pf-DNA showed interesting differences. Similar isotherms could not be obtained for the acridine orange-DNA system.     

Conformation of horse heart ferricytochrome c. III. Comparative optical rotatory dispersion study of the protein with its derivative heme undecapeptide

The optical rotatory dispersion of horse heart ferricytochrome c and of a ferri heme undecapeptide have been determined under various conditions. Analysis of the Soret region makes it possible to characterize three different states of ferricytochrome c. the native state (superposition of a negative and a positive Cotton effect); an intermediate state (single positive Cotton effect whose magnitude Δ[M] is equal to 55,000); a denatured state (single positive Cotton effect whose magnitude Δ[M] is equal to 115,000) in which compared to both the native and intermediate states a more or less important decrease in helix content is observed. The optical rotatory dispersion spectra of the Soret region of the monomeric ferri heme undecapeptide is similar to that of denatured ferricytochrome c. The multiplicity of Cotton effects observed under certain conditions for the hemopeptide is a consequence, resulting from a polymerization, of intermolecular interactions. The comparison of the optical rotatory dispersion spectra of ferricytochrome c and the ferri heme undecapeptide indicates that in the intermediate state interactions remain between the heme group and the portion of the poly pep tide chain absent in the hemopeptide. These interactions disappear in the denatured state.     

Induced optical activity of acridine orange bound to poly-S-carboxymethyl-L-cysteine

The absorption and rotatory properties of acridine orange-poly-S-carboxymethyl-L -cysteine system in water and in 0.2 M NaCl have been measured at different pH and polymer-to-dye mixing ratios. The absorption spectra indicate that the dyes are bound to the polymer in dimeric or highly aggregated forms. At neutral pH where the polymer is randomly coiled, no optical activity is induced on the absorption bands of bound acridine orange. At acid pH where the polymer has the β-conformation, a pair of positive and negative circular dichroic bands occur at each of the absorption bands, centered around 458 and 261 mμ. The signs of those bands are opposite to those found for α-helical poly-L -glutamic acid. A model for the binding of dye to the β-form polymer is presented, in which dimeric dyes are attached to ionized carboxyl groups and slack one another to form linear arrays on both sides of an extended polypeptide chain. The observed circular dichroism spectra can be explained by the Tinoco's exciton mechanism, based on this model. Low molecular weight poly-S-carboxymethyl-L -cysteine induces quite a different circular dichroism on bound acridine orange.     

Factors influencing transduction of genetic determinants of penicillinase activity and pathogenicity in staphylococcus aureua. II. Antiphage activity of acridine derivatives]

Acridine dyes examined earlier (acrichine, acridine orange, proflavine and rivanol) and newly-synthesized preparations (acridines No. No. 37--40) were studied in the capacity of nonspecific agents influencing the lytic cycle in development of staphylococcus phages. Acrichine and acridine No. 37 failed to prevent lysis of the indicator staphylococcus cultures (strains 16/160 and 8325) by bacteriophages; proflavine, rivanol, acridines No. No. 39--40 produced a marked inhibitory effect; acridine orange and acridine No. 38 inhibited the staphylococcus lysis completely. Some preparations could be used to investigate the transduction phenomenon.     

Ultraviolet spectra of the complexes of acridine orange with deoxyribonucleic acid and polyphosphates

Summary Two kinds of changes were found in ultraviolet spectrum of acridine orange bound to polyphosphate and native or denatured DNA: (a) changes similar to those caused by aggregation in the solutions of pure acridine orange (i.e. blue shifts of the bands at 37300 cm^–1 and 43670 cm^–1, a decrease of absorbance of the band at 34600 cm^–1 and an increase of absorbance of the band at 43670 cm^–1), which were observed at those ratiosP/D, when the dye formed aggregates on the surface of the polyanion; (b) a decrease of absorbance in the whole near ultraviolet region, which had high value even when isolated dye molecules were bound to the polyanion. While the first kind of changes is due to mutual interactions between the aggregated acridine orange molecules, the second kind can be explained as due to interaction of the dye molecules with adjacent chromophores of the polyanion and/or solvent. The maximum value of the hypochromic effect in the near ultraviolet maximum was higher for complexes of denatured DNA than for complexes of native DNA.     

Dielectric behavior of DNA--dye complex. II

The dielectric relaxation of native DNA and the effect of aminoacridine dyes, such as acridine orange (AO), proflavine (PF), and ethidium bromide (EB) have been investigated at different molar DNA phosphate (P) to dye (D) ratios in the frequency range 100 Hz–100 kHz. The static dielectric constant was observed to decrease with increasing binding of aminoacridines. This was interpreted as arising from the neutralization of the surface changes of the DNA molecules as a result of dye binding. At any P/D ratio the extent of charge neutralization was greatest for AO and least for the EB–DNA complex. The relaxation time (τ) for dye-bound DNA was greater compared to that for native DNA. This increase in τ was ascribed to the increase in the length of the dye-bound DNA. The maximum value of τ occurred at P/D = 20, 10, and 2 for AO-, PF-, and EB-treated DNA, respectively. The variation of τ at various levels of binding gave a qualitative idea about the conformational changes of DNA due to its binding with the dyes.     

Photodynamic mutagenic action of acridine compounds on yeast Saccharomyces cerevisiae

The photodynamically produced mutagenicity and toxicity of 8 acridine compounds were compared in Saccharomyces cerevisiae under resting and growing conditions. Without irradiation none of the acridines induced respiratory-deficient ('petite') colonies, indicative of mitochondrial DNA damage, in resting cells; and only acriflavine and proflavine induced 'petites' in growing cells. Also, without irradiation none of the acridines were significantly toxic or mutagenic for nuclear DNA under resting or growing conditions. However, with irradiation, acriflavine, proflavine, acridine yellow and rivanol became effective 'petite'-inducing mutagens and highly toxic for resting cells, while acriflavine, proflavine, and acridine orange became effective nuclear mutagens for resting cells. Acridine, quinacrine and 9-aminoacridine were not at all biologically effective with irradiation for resting cells. The results presented here indicate that singlet oxygen is generated by a photodynamic mechanism when acriflavine is irradiated, and further, that acridine, quinacrine and 9-aminoacridine are ineffective photosensitizers, because they are incapable of generating singlet oxygen with irradiation.     

Optical studies on the structure of hemoglobin messenger ribonucleic acid

The secondary structure of hemoglobin mRNA (HbmRNA has been investigated by optical rotatory dispersion (ORD), circular dichroism (CD) and ultraviolet absorbance spectrophotometry. The dependence on temperature or reaction with formaldehyde of the CD and absorbance are characteristic of a structure with substantial base pairings at 20°C. The presence of Cotton effects in one of the major ultraviolet absorption bands indicates a highly ordered secondary structure. The UV hyperchromism on thermal denaturation is consistent with a value of 58% double helical content.     

Extension of the Ring Oxygen Helicity Rule to Phenyl and p-Nitrophenyl Glycopyranosides

The Cotton effect below 200 nm was first studied in aromatic glycopyranosides with our new technique of optical rotatory dispersion (ORD). The phenyl and p-nitrophenyl α- and β-d-glycopyranosides showed a strong Cotton effect at around 180 nm which practically governed the optical rotations in the visible wavelength region. The rotational strength and sign of this Cotton effect were shown to reflect the anomeric configurations and conformations, which led to an extension of the ring oxygen helicity rule for alkyl and alkyl 1-thioglycosides to aromatic glycopyranosides.     

The Inhibition of Bacteriophage DNA Injection by Dyes Bound to the DNA

A delay (~10 min) in the appearance of intracellular phage is caused by preincubating the infecting phage T4o₁ in proflavine, acridine orange, or ethidium, but not polyamines. No significant delay in attachment is observed. Apparently the presence of the dye is required inside the permeability barrier of the phage at the time of infection. The effect of proflavine is reduced in the presence of polyamines, suggesting that the active site is on DNA. The phage-host complex is sensitive to shear if the infecting phage have been incubated in proflavine or ethidium, indicating that the completion of DNA injection is delayed. Finally no partially injected complexes could be detected after shearing, which suggests that most of the delay occurs near the beginning of the injection process.     

Circular dichroism and structure of the complex of acridine orange with poly(L-glutamic acid)

Circular dichroism and absorption spectra have been measured on solutions of acridine orange and poly(L -glutamic acid) mixed at two molar ratios of carboxyl group-to-dye, P/D 25 and 0.8, and at different pH's. Characteristic circular dichroism is induced at the absorption bands of acridine orange when the P/D is 25 and the solution is acidic. Another type of circular dichroism is manifest at neutral and alkaline pH when P/D is 0.8. For the induction of the former type of circular dichroism, a helical array of acridine-orange dimers bound to the α-helix is postulated, in which the dye molecular planes are almost perpendicular to the helical axis. Assuming the helical geometry and optical parameters, combined with the observed magnitude of transition electric moment, the rotatory strength of the complex is calculated to the zeroth order approximation, and the observed circular dichroism spectra have been reproduced.     

Effect of Deoxyribonucleic Acid Ligands on Deoxyribonucleases and Deoxyribonucleic Acid Polymerase I of Escherichia coli K-12

Endonuclease I, exonuclease I, and exonuclease II-deoxyribonucleic acid (DNA) polymerase I activities are not vital functions in Escherichia coli, although the latter two enzymes have been indirectly shown to be involved in DNA repair processes. Acridines such as acridine orange and proflavine interfere with repair in vivo, and we find that such compounds inhibit the in vitro activity of exonuclease I and DNA polymerase I but stimulate endonuclease I activity and hydrolysis of p-nitrophenyl thymidine-5′-phosphate by exonuclease II. Another acridine, 10-methylacridinium chloride, binds strongly to DNA but is relatively inert both in vivo and in vitro. These experiments suggest that acridines affect enzyme activity by interacting with the enzyme directly as well as with DNA. Resulting conformational changes in the DNA-dependent enzymes might explain why similar acridines which form similar DNA complexes have such a wide range of physiological effects. Differential sensitivity of exonuclease I and DNA polymerase I to acridine inhibition relative to other DNA-dependent enzymes may contribute to the acridine sensitivity of DNA repair.