Photosensitization of consitutents of nucleic acids by proflavine. Mechanism of formation of hydrogen addition radicals in frozen aquenous solutions (author's transl)]

It is shown that the insertion of nucleotides between proflavine molecules is favourable to photosensitization. Furthermore (1) each molecule of proflavine gives at the most one free radical in the substrate, (2) the chromophore is largely restored when oxygen is not present, (3) superoxide radicals are observed in the presence of oxygen, and (4) formyl radicals are detected. The scheme elaborated for the mechanism gives an explanation for all these observations.     

Photodynamic activity of acridine orange: peroxide radical induction in DNA and synthetic polynucleotides

As shown by electron paramagnetic resonance, acridine orange induces the formation of peroxide radicals in DNA when dye-DNA mixtures frozen at 77 K are irradiated with visible light. The reaction is oxygen dependent and strongly reduced by the addition of an electron scavenger. Factors of the medium can modulate the reaction: an ionic strength increased up to 0.3 greatly enhances the dye efficiency whereas the presence of phosphate ions has an inhibiting influence. Acridine orange, which is slightly less efficient than proflavine on native DNA, induces an important peroxide radical formation in poly(dG).poly(dC) but has no action on the poly(dA).poly(dT)polymer.     

Visible-light-induced OH radicals in DNA-proflavine complexes: an e.p.r. and spin trapping study

Using the spin-trapping technique, irradiation with visible light of complexes between DNA and proflavine was shown to generate OH radicals. The characteristic spectra were not obtained when proflavine or DNA were irradiated alone, nor when oxygen was absent. Using DMPO as a spin trap we found that the intensity of the DMPO-OH e.p.r. signal was enhanced when the molar ratio between bound proflavine and the DNA phosphorus increased up to a value of 0.15 demonstrating the efficiency of the intercalated dye molecules. A strong decrease of the e.p.r. signal was observed in the presence of various OH. scavengers like t-butanol, isopropanol or sodium benzoate. The OH. production was also decreased when the irradiation was made in the presence of SOD, ceruloplasmin or catalase and after addition of Chelex 100 resin.     

Stabilization of the T-state of human hemoglobin by proflavine, an antiseptic drug.

The effect of proflavine (3,6-diaminoacridine), an antiseptic drug, on the spectroscopic and oxygen binding properties of ferrous human adult hemoglobin (Hb) has been investigated. Upon binding of proflavine to the nitric oxide derivative of ferrous human adult hemoglobin (HbNO), the X-band EPR spectrum displays the characteristics which have been attributed to the T-state of the ligated tetramer. In parallel, oxygen affinity for the deoxygenated derivative of ferrous human adult Hb decreases in the presence of proflavine. The effect of proflavine on the spectroscopic and ligand binding properties of ferrous human adult Hb is reminiscent that of 2,3-D-glycerate bisphosphate, the physiological modulator of Hb action.     

Superoxide dismutase protects against paraquat-mediated dioxygen toxicity and mutagenicity: studies in Salmonella typhimurium

Paraquat is univalently reduced to the relatively stable, but oxygen-sensitive, paraquat radical (PQ.+). This PQ.+ can react with dioxygen to generate the superoxide radical, which can further generate other more deleterious species of oxygen free radicals (i.e., hydroxyl radical, OH.). These oxygen free radicals are known to cause chromosomal breaks; therefore, it was logical to postulate that paraquat is a mutagen. This proved to be the case when tested in a modified Ames test using a liquid incubation assay. Salmonella typhimurium strains TA98 and TA100 were grown in the presence of various concentrations of PQ, as well as in the presence of known mutagenic compounds: mitomycin C, azide, and proflavine. Paraquat was much more toxic and mutagenic in a simple nutritionally restricted medium than in a rich complex medium and these toxic and mutagenic effects were oxygen dependent. Furthermore, cells containing high levels of superoxide dismutase were more resistant to the toxic and mutagenic effects of paraquat than were cells containing a normal level of this enzyme.     

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.     

Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli

Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.     

Influence of the DNA structure on the free radical induction due to proflavine and light treatment

Summary Induction of peroxide free radicals (detected by Electron Paramagnetic Resonance at 77 K) due to the photodynamic activity of proflavine was measured on bacteriophage X174 DNA either single-stranded (ss) as isolated from the virion, or double-stranded supercoiled (RFI) as isolated from the infected bacteria. Comparison was made with calf thymus DNA photosensitization.In order to use equivalent DNA-proflavine complexes, binding of the dye to the three DNA's was first determined under those conditions of high ionic strength favourable to the photodynamic reaction.Free radical induction was maximal for definite amounts of bound proflavine (which varied depending upon the DNA substrate) and at an ionic strength value of 0.5. The level of the maximal reaction increased in the following order: from Xss DNA to calf thymus DNA and finally to XRFI DNA. The conformation of the proflavine-DNA complex was thus a determinant for the efficiency of the photodynamic process. The ionic strength effect could not be explained by the evolution of the proflavine triplet state in irradiated proflavine-calf thymus DNA complexes.     

Electron transfer between protein and DNA in gamma-irradiated deoxyribonucleoprotein.

When deoxyribonucleoprotein-proflavine complexes were studied by electron spin-resonance spectroscopy following gamma-irradiation, it was found that stable free radicals were not formed at random on the complex but were preferentially located on proflavine. Since proflavine intercalalated to DNA bases serves as a final acceptor of electrons liberated by ionization, the result of our experiment was regarded as suggesting that the electron transfer from the protein moiety to the DNA moiety occurred in the irradiated deoxyribonucleoprotein.     

Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1

Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB. Direct and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (K(D) approximately 0.1 +/- 0.05 microM) and a weaker binding site(s) (K(D) approximately 1.1 +/- 0.05 microM). Titrations of RRE-IIB with proflavine, monitored using (1)H NMR, demonstrate that the high-affinity proflavine binding interaction occurs with a 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1 proflavine.RRE-IIB complex indicate that the two proflavine molecules bind specifically and close to each other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine.RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIB in a manner analogous to what has been observed in the Rev peptide.RRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.     

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.     

Influence de l'intensité et de la longueur d'onde de la lumière sur le rendement des radicaux libres dans les semences de tomate et de carotte

Influence of light intensity and wavelength on the yield of free radicals in seeds of tomato and carrot.
Seeds of tomato ( Lycopersicum esculentum L. cv. Gribovsky-1180) and carrot ( Daucus carota L. cv. Santanet) were irradiated by continuous light of different wavelengths (white, blue, green, red, far-red and He-Ne laser) and different power density. Free radical output was measured using Electron Spin Resonance spectra (ESR). The dependence of the content of free radicals (FR) on the light power density was established. The yield of FR in the seeds irradiated with monochromatic light of the same power density was inversely proportional to the wavelength. The ESR spectra showed maximum absolute number of FR for white light. Successive irradiations gave cumulative effects on the output of free radicals in both directions: increase by the action of white light and decrease by the action of red and far-red light. The contact of irradiated seeds with steam decreased the amount of FR.     

活性氧、自由基与植物的衰老

介绍近 1 0年来有关活性氧、自由基的产生 ,对植物的伤害及植物对活性氧、自由基清除的研究进展。     

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.     

Scavenging effects on active oxygen radicals by schizandrins with different structures and configurations

We have studied the scavenging effects of different structures and configurations of schizandrins isolated from Fructus Schizandrae, a traditional Chinese herb, on active oxygen radicals with the method of spin-trapping technique. The active oxygen radicals were produced from human polymorphonuclear leukocytes (PMN) stimulated with phorbol myristate acetate (PMA). In addition, the scavenging effects of schizandrins on hydroxyl radicals (.OH) in Fenton's reaction and the scavenging effects on superoxide anions (O2-.) in both riboflavin/EDTA and xanthine/xanthine oxidase systems have also been studied. They are compared with the scavenging effects of both Vitamin C (Vc) and Vitamin E (VE). The experimental results have shown that the scavenging effect of schizandrin B (Sin B) on the active oxygen radicals is stronger than that of S(-) Sin B and R(+) Sin B. For schizandrins of the same molecular structures with different stereoconfigurations the scavenging effects of S type of the benzene ring on active oxygen radicals are stronger than those of R type and for schizandrins of the same stereoconfigurations with different structures the scavenging effects of schizandrin C (Sin C) on the active oxygen radicals are stronger than those of Sin B.     

The reactivity of carotenoid radicals with oxygen

The possibility that carotenoid radicals react with oxygen to form chain-carrying peroxyl radicals has been postulated to account for the reduction in antioxidant effetiveness displayed by some carotenoids at high oxygen concentrations. The primary objective of the work described in this paper was to measure the rate constants for oxygen addition to a series of carotenoid radicals and to examine any influence of carotenoid structural features on these rate constants. Laser flash photolysis has been used to generate long-lived carotenoid radicals (PhS-CAR^√) derived from radical addition reactions with phenylthiyl radicals (PhS^√) in benzene. The PhS-CAR^√ radicals are scavenged by oxygen at rates that display a moderate dependence on the number of conjugated double bonds (n_db) in the carotenoid. The rate constants range from ∼10³ to ∼10⁴ M^- 1 s^- 1 for n_db = 7-11. The data also suggest that the presence of terminal cyclic groups may cause an increase in the rate constant for oxygen addition.     

New sensitive agents for detecting singlet oxygen by electron spin resonance spectroscopy.

Free radicals are well-established transient intermediates in chemical and biological processes. Singlet oxygen, though not a free radical, is also a fairly common reactive chemical species. It is rare that singlet oxygen is studied with the electron spin resonance (ESR) technique in biological systems, because there are few suitable detecting agents. We have recently researched some semiquinone radicals. Specifically, our focus has been on bipyrazole derivatives, which slowly convert to semiquinone radicals in DMSO solution in the presence of potassium tert-butoxide and oxygen. These bipyrazole derivatives are dimers of 3-methyl-1-phenyl-2-pyrazolin-5-one and have anti-ischemic activities and free radical scavenging properties. In this work, we synthesized a new bipyrazole derivative, 4,4'-bis(1p-carboxyphenyl-3-methyl-5-hydroxyl)-pyrazole, DRD156. The resulting semiquinone radical, formed by reaction with singlet oxygen, was characterized by ESR spectroscopy. DRD156 gave no ESR signals from hydroxyl radical, superoxide, and hydrogen peroxide. DRD156, though, gives an ESR response with hypochlorite. This agent, nevertheless, has a much higher ability to detect singlet oxygen than traditional agents with the ESR technique.     

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.     

Measurements in vivo of parameters pertinent to ROS/RNS using EPR spectroscopy

The technique of in vivo EPR spectroscopy can provide useful and even unique information pertinent to the study of oxygen/ nitrogen radicals and related processes. The parameters that can be measured include: (a) Oxygen centered radicals (by spin trapping); (b) carbon centered radicals (by spin trapping and sometimes by direct observation); (c) sulfur centered radicals (by spin trapping and sometimes by direct observation); (d) nitric oxide (by spin trapping); (e) oxygen (using oxygen sensitive paramagnetic materials); (f) redox state (using metabolism of nitroxides); (g) thiol groups (using special nitroxides); (h) pH (using special nitroxides); (h) perfusion (using washout of paramagnetic tracers); (i) some redox active metal ions (chromium, manganese). The current state of the art for these and other measurements is discussed, especially in relationship to experiments that are likely to be useful for studies of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS).     

Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.