Oxidative modification of human ceruloplasmin by peroxyl radicals.

Ceruloplasmin (CP), the blue oxidase present in all vertebrates, is the major copper-containing protein of plasma. We investigated oxidative modification of human CP by peroxyl radicals generated in a solution containing 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). When CP was incubated with AAPH, the aggregation of proteins was increased in a time- and dose-dependent manner. Incubation of CP with AAPH resulted in a loss of ferroxidase activity. Superoxide dismutase and catalase did not protect the aggregation of CP, whereas hydroxyl radical scavengers such as ethanol and mannitol protected the protein aggregation. The aggregation of proteins was significantly inhibited by the copper chelators, diethyldithiocarbamate and penicillamine. Exposure of CP to AAPH led to the release of copper ions from the enzyme and the generation of protein carbonyl derivatives. Subsequently, when the amino acid composition of CP reacted with AAPH was analyzed, cysteine, tryptophan, methionine, histidine, tyrosine, and lysine residues were particularly sensitive.     

Quantitation of eukaryotic topoisomerase I reactivity with DNA. Preferential cleavage of supercoiled DNA

A method has been used to quantitate the reaction between eukaryotic type I DNA topoisomerase and topological forms of DNA. This procedure (Trask, D.K., DiDonato, J.D. and Muller, M.T. (1984) Eur. Mol. Biol. Organ. J. 3, 671-676) measures the efficiency of DNA cleavage and concurrent formation of a covalent enzyme/DNA complex. Eukaryotic type I topoisomerases react preferentially by 5-10-fold with supercoiled DNA. The effect of supercoiling is clearly evident in that both the initial rate and final extent of the reaction is elevated. Because the dissociation rate is much lower than the association rate, it is possible to isolate native topoisomerase/DNA complexes. These complexes are comprised of enzyme molecules which are catalytically active when challenged with a second supercoiled DNA substrate. Collectively, the data support the conclusion that a functional intermediate in the reaction sequence is being detected and that the avian topoisomerase I preferentially cleaves supercoiled DNA.     

Mapping of peroxyl radical induced damage on genomic DNA

We have examined the DNA damage produced by reaction of peroxyl radicals with human fibroblast DNA. DNA damage consisted of both strand breaks and base modifications. The extent of strand breaks and base modifications induced as a function of peroxyl radical concentration was determined by quantitation of fragment size distributions using denaturing glyoxal-agarose gel electrophoresis. Both strand breaks and base modifications increased in a log linear fashion with respect to peroxyl radical concentration. Oxidative base modifications were observed to occur to a greater extent than strand breaks at every concentration measured. The sequence-specific distribution of peroxyl radical induced base damage was mapped for 803 nucleotide positions using the method of ligation mediated PCR. A total of 87% of all guanine positions in the examined sequences was found to be significantly oxidized. The order of reactivity of DNA bases toward oxidation by peroxyl radicals was found to be G > C > T. Adenine is essentially unreactive. The yield of oxidative base modifications at guanines and cytosines by peroxyl radicals depends on the exact specification of 5' and 3' flanking bases in a polarity dependent manner. Every guanine in the 5'XGC3' motif was found to be oxidized, where X is any 5' neighbor. In contrast, 5' and 3' purine flanks drastically reduced the extent of peroxyl radical G oxidation. The pattern of base modification and the influence of nearest neighbors differs substantially from that previously reported for hydrogen peroxide damage mediated by low valent transition metal ions for the identical DNA sequences.     

Quantitation of supercoiled DNA cleavage in nonradioactive DNA: application to ionizing radiation and synthetic endonuclease cleavage.

Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to "normalize" the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a protein-based, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and normalizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays.     

Reassignment of organic peroxyl radical adducts.

The study of the important role of peroxyl radicals in biological systems is limited by their difficult detection with direct electron spin resonance (ESR). Many ESR spectra were assigned to 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/peroxyl radical adducts based only on the close similarity of their ESR spectra to that of DMPO/superoxide radical adduct in conjunction with their insensitivity to superoxide dismutase, which distinguishes the radical adduct from DMPO/superoxide radical adduct. Later, the spin-trapping literature reported that DMPO/peroxyl radical adducts have virtually the same hyperfine coupling constants as synthesized alkoxyl radical adducts, raising the issue of the correct assignment of peroxyl radical adducts. However, using 17O-isotope labelling, the methylperoxyl and methoxyl radical adducts should be distinguishable. We have reinvestigated the spin trapping of the methylperoxyl radical. The methylperoxyl radical was generated in aerobic solution with 17O-molecular oxygen either in a Fenton system with dimethylsulfoxide or in a chloroperoxidase system with tert-butyl hydroperoxide. Two different spin traps, DMPO and 2,2,4-trimethyl-2H-imidazole-1-oxide (TMIO), were used to trap methylperoxyl radical. 17O-labelled methanol was used to synthesize methoxyl radical adducts by nucleophylic addition. It was shown that the 17O hyperfine coupling constants of radical adducts formed in methylperoxyl radical-generating systems are identical to that of the methoxyl radical adduct. Therefore, methylperoxyl radical-producing systems form detectable methoxyl radical adduct, but not detectable methylperoxyl radical adducts at room temperature. One of the possible mechanisms is the decomposition of peroxyl radical adduct with the formation of secondary alkoxyl radical adduct. These results allow us to reinterpret previously published data reporting detection of peroxyl radical adducts. We suggest that detection of 17O-alkoxyl radical adduct from 17O-labelled molecular oxygen can be used as indirect evidence for peroxyl radical generation.     

Kinetics of phycocyanine bilin groups destruction by peroxyl radicals

Bilin groups in c-phycocyanine are readily bleached by peroxyl radicals produced in the thermolysis of 2, 2'-azobis(2-amidinopropane). From an evaluation of the bilin groups destroyed per radical that interacts with the protein, it is concluded that the bilin moiety is the main target of the radicals. Kinetic expressions are derived that allows an estimation of the substrate reactivity from the analysis of the rate of bilin group modification as a function of the protein concentration. From this analysis it is concluded that micromolar concentrations of c-phycocyanine are able to reduce the steady state concentration of the peroxyl radicals by one half, indicating a high antioxidant activity for this compound. This conclusion is confirmed by measuring the capacity of the protein to protect 1-naphthol from modification by peroxyl radicals. The results obtained show that the bilin groups have, on a molar basis, an antioxidant activity similar to that of potent antioxidants such as catechin.     

Analysis of chemical and enzymatic cleavage frequencies in supercoiled DNA

Chemical and enzymatic probing methods are powerful techniques for examining details of sequence-dependent structure in DNA and RNA. Reagents that cleave nucleic acid molecules in a structure-specific, but relatively sequence-non-specific manner, such as hydroxyl radical or DNase I, have been used widely to probe helical geometry in nucleic acid structures, nucleic acid-drug complexes, and in nucleoprotein assemblies. Application of cleavage-based techniques to structures present in superhelical DNA has been hindered by the fact that the cleavage pattern attributable to supercoiling-dependent structures is heavily mixed with non-specific cleavage signals that are inevitable products of multiple cleavage events. We present a rigorous mathematical procedure for extracting the cleavage pattern specific to supercoiled DNA and use this method to investigate the hydroxyl radical cleavage pattern in a cruciform DNA structure formed by a 60 bp inverted repeat sequence embedded in a negatively supercoiled plasmid. Our results support the presence of a stem-loop structure in the expected location and suggest that the helical geometry of the cruciform stem differs from that of the normal duplex form.     

DNA cleavage by hydroxyl radicals generated in a vanadyl ion-hydrogen peroxide system.

Vanadyl ion (+4 oxidation state) has been shown to be an effective agent for chemoprotection of cancers in animals. For understanding the mechanism, distribution of vanadium was studied. More vanadium was found to accumulate in the nuclei of the liver of rats when it was given as vanadyl sulfate than when it was given as sodium vanadate (+5 oxidation state). The reactivity of vanadyl ion with DNA was investigated by the DNA cleavage technique and the reaction mechanism by ESR spectroscopy. Incubation of double-strand DNA with vanadyl ion and hydrogen peroxide resulted in marked concentration- and pH-dependent DNA cleavage. Studies by the ESR spin-trap method demonstrated that hydroxyl radicals are generated during the reactions of vanadyl ion with hydrogen peroxide. Thus the antineoplastic action of vanadyl ion is proposed to be due to DNA cleavage by hydroxyl radicals generated in the cells.     

Behavior of supercoiled DNA.

We study DNA supercoiling in a quantitative fashion by micromanipulating single linear DNA molecules with a magnetic field gradient. By anchoring one end of the DNA to multiple sites on a magnetic bead and the other end to multiple sites on a glass surface, we were able to exert torsional control on the DNA. A rotating magnetic field was used to induce rotation of the magnetic bead, and reversibly over- and underwind the molecule. The magnetic field was also used to increase or decrease the stretching force exerted by the magnetic bead on the DNA. The molecule's degree of supercoiling could therefore be quantitatively controlled and monitored, and tethered-particle motion analysis allowed us to measure the stretching force acting on the DNA. Experimental results indicate that this is a very powerful technique for measuring forces at the picoscale. We studied the effect of stretching forces ranging from 0.01 pN to 100 pN on supercoiled DNA (-0.1 < sigma < 0.2) in a variety of ionic conditions. Other effects, such as stretching-relaxing hysteresis and the braiding of two DNA molecules, are discussed.     

Contribution of haemoglobin and membrane constituents modification to human erythrocyte damage promoted by peroxyl radicals of different charge and hydrophobicity

We have investigated the influence of the free radical initiator characteristics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2′-Azobis(2-amidinopropane) (AAPH) and 4,4′-azobis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilicity, as evaluated from octanol/water partition constants. At physiological pH, they are a di-cation and a di-anion, respectively.

AAPH and ACV readily oxidise purified oxyhemoglobin in a very efficient free radical-mediated process, particularly for ACV-derived radicals, where nearly one heme moiety was modified per radical introduced into the system, suggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-compounds lead to different oxidation products. Methemoglobin, hemichromes and choleglobin were produced in AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominantly form hemichromes, with very low production of choleglobin.

Red cell damage was evaluated at the level of hemoglobin and membrane constituents modification, and was expressed in terms of free radical doses. Before the onset of the lytic process, ACV leads to more lipid peroxidation than AAPH, and induces a moderate oxidation of intracellular Hb. This intracellular oxidation is markedly increased if ACV hydrophilicity is decreased by lowering the pH. On the other hand, AAPH-derived radicals are considerable more efficient in promoting protein band 3 modification and cell lysis, without significant intracellular hemoglobin oxidation. These results show that the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant factor leading to red blood cell lysis.     

Contribution of haemoglobin and membrane constituents modification to human erythrocyte damage promoted by peroxyl radicals of different charge and hydrophobicity

We have investigated the influence of the free radical initiator characteristics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2'-Azobis(2-amidinopropane) (AAPH) and 4,4'-azobis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilicity, as evaluated from octanol/water partition constants. At physiological pH, they are a di-cation and a di-anion, respectively.

AAPH and ACV readily oxidise purified oxyhemoglobin in a very efficient free radical-mediated process, particularly for ACV-derived radicals, where nearly one heme moiety was modified per radical introduced into the system, suggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-compounds lead to different oxidation products. Methemoglobin, hemichromes and choleglobin were produced in AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominantly form hemichromes, with very low production of choleglobin.

Red cell damage was evaluated at the level of hemoglobin and membrane constituents modification, and was expressed in terms of free radical doses. Before the onset of the lytic process, ACV leads to more lipid peroxidation than AAPH, and induces a moderate oxidation of intracellular Hb. This intracellular oxidation is markedly increased if ACV hydrophilicity is decreased by lowering the pH. On the other hand, AAPH-derived radicals are considerable more efficient in promoting protein band 3 modification and cell lysis, without significant intracellular hemoglobin oxidation. These results show that the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant factor leading to red blood cell lysis.     

Strand specificity of the topoisomerase II mediated double-stranded DNA cleavage reaction

The strand specificity of topoisomerase II mediated DNA cleavage was analyzed at the nucleotide level by characterizing the enzyme's interaction with a strong DNA recognition site. This site was isolated from the promoter region of the extrachromosomal rRNA genes of Tetrahymena thermophila and was recognized by type II topoisomerases from a variety of phylogenetically diverse eukaryotic organisms, including Drosophila, Tetrahymena, and calf thymus. When incubated with this site, topoisomerase II was found to introduce single-stranded breaks (i.e., nicks) in addition to double-stranded breaks in the nucleic acid backbone. Although the nucleotide position of cleavage on both the noncoding and coding strands of the rDNA remained unchanged, the relative ratios of single- and double-stranded DNA breaks could be varied by altering reaction conditions. Under all conditions which promoted topoisomerase II mediated DNA nicking, the enzyme displayed a 3-10-fold specificity for cleavage at the noncoding strand of its recognition site. To determine whether this specificity of topoisomerase II was due to a faster forward rate of cleavage of the noncoding strand or a slower rate of its religation, a DNA religation assay was performed. Results indicated that both the noncoding and coding strands were religated by the enzyme at approximately the same rate. Therefore, the DNA strand preference of topoisomerase II appears to be embodied in the enzyme's forward cleavage reaction.     

Bactericidal activity of alkyl peroxyl radicals generated by heme-iron-catalyzed decomposition of organic peroxides.

To clarify the nature of cytocidal molecular species among the radicals generated in the iron-catalyzed reactions of peroxides (ROOH), we examined the cytocidal effects of these radicals against gram-positive and gram-negative bacteria in the presence or absence of various radical scavengers. Three organic peroxides, t-butyl hydroperoxide (t-BuOOH), methyl ethyl ketone peroxide (MEKOOH), and cumene hydroperoxide, were used. Each radical generated from these peroxides was identified and quantitated by electron spin resonance (ESR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The major cytotoxic radical species generated in the mixtures of various peroxides and heme iron, especially methemoglobin, metmyoglobin, or hemin, was the alkyl peroxyl radical (ROO.). Strong bactericidal action against gram-positive bacteria was observed in the peroxide-heme iron system, especially in the case of t-BuOOH and MEKOOH. Killing curves for gram-positive bacteria showed an initial lag period, which may indicate the multihit/multitarget kinetics of cell killing. When the diethylenetriamine pentaacetic acid (DTPA)-Fe2+ complex was used as a catalyst for decomposition of various peroxides, alkyl, alkoxyl, and alkyl peroxyl radicals were identified by spin-trapping analysis. However, study of the time course of alkyl peroxyl radical production in the DTPA-Fe2+ complex system revealed that radical species generated in this system were very short lived: a maximal level was achieved within 1 min and then declined sharply, and no bactericidal activity was observed after 10 min. In contrast, the alkyl peroxyl radical level generated by the organic peroxide-heme iron system remained high for 30 min or longer. The generation of alkyl peroxyl radicals quantified by ESR correlated quite well with the bactericidal effect of the system of peroxide plus iron. In addition, bactericidal activity was completely inhibited by the addition of the spin trap DMPO, as well as of other various radical scavengers (alpha-tocopherol and L-ascorbic acid), into the peroxide-heme iron system, but this effect was not observed with superoxide dismutase, beta-carotene, dimethyl sulfoxide, diphenylamine, or butylated hydroxyltoluene. In view of these results, it is assumed that alkyl peroxyl radicals are the potent molecular species that are cytotoxic against bacteria, whereas alkoxyl radicals (RO.) generated in this system do not affect bacterial viability.     

Condensation of supercoiled DNA induced by MnCl2.

Multivalent cations condense DNA in vitro, but it had been thought that a valence of at least + 3 was required in aqueous solution. We have found that Mn2+ can produce toroidal condensates of supercoiled plasmid DNA, but not of linearized plasmid. Mg2+ does not cause condensation, and neither MgCl2 nor NaCl can negate the effect of MnCl2, indicating that the condensation mechanism with Mn is not primarily electrostatic. Supercoiled MnDNA is more extensively digested than the linear form by S1 nuclease. Supercoiling appears to cooperate with Mn2+ in stabilizing helix distortions and also provides a "pressure" that enhances lateral association.     

Reactivity of ebselen and related selenoorganic compounds with 1,2-dichloroethane radical cations and halogenated peroxyl radicals

The reactivity of ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)one, and structurally related analogues was studied by pulse radiolysis. The rate constant for the reaction of ebselen with trichloromethylperoxyl radicals was determined to be 2.9 X 10(8) M-1 s-1, while its sulfur analogue, 2-phenyl-1,2-benzisothiazol-3(2H)one, was oxidized at much lower rates, k less than or equal to 10(7) M-1 s-1. Among several derivatives studied, the only other compound that exhibited a high rate constant was 2-(methylseleno)-benzoic acid-N-phenylamide. Oxidation of ebselen by other halogenated peroxyl radicals was also carried out and revealed a direct relationship between rate constant and the degree of halogenation of the oxidant. The transient radicals generated during oxidation of ebselen and the analogues were characterized by optical absorption and conductivity measurements and were attributed to one-electron-oxidized radical cations. The oxidation potentials were determined by cyclic voltammetry. Comparative evaluation of the in vitro behavior during microsomal lipid peroxidation revealed ebselen to be the most potent antioxidant of the compounds investigated, 2-(Methylseleno)-benzoic acid-N-phenylamide, despite its high rate constant for oxidation by halogenated peroxyl radicals, was found to be a poor antioxidant. The rate constant of oxidation of ebselen by trichloromethylperoxyl radicals is comparable to that of alpha-tocopherol under similar conditions, underscoring the potential pharmacological interest of ebselen as an antioxidant.     

Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide.

A method for achieving strand specific nicking of DNA has been developed. Phosphorothioate groups were incorporated enzymatically into the (-)strand of M13 RF IV DNA. When such DNA is reacted with restriction endonucleases in the presence of ethidium bromide nicked DNA (RF II) is produced. All of the restriction enzymes tested linearised phosphorothioate-containing DNA in the absence of this dye. The strand specificity of the reaction was investigated by employing the ethidium bromide mediated nicking reaction in the phosphorothioate-based oligonucleotide-directed mutagenesis method. The mutational efficiencies obtained were in the region of 64-89%, indicating that these restriction enzymes hydrolyse the phosphodiester bond at the cleavage site of the unsubstituted (+)strand.     

Electrode potential-modulated cleavage of surface-confined DNA by hydroxyl radicals detected by an electrochemical biosensor

Damage to DNA frequently involves interruption of DNA sugar-phosphate strands (strand breaks, sb). Under aerobic conditions, transition metal ions cause DNA damage through production of reactive oxygen species (frequently via Fenton-type reactions). Formation of sb in covalently closed supercoiled (sc) DNA can be detected using an electrochemical biosensor based on a scDNA-modified mercury electrode. By controlling the potential of the electrode, this technique can be employed in studies of redox reactions involved in formation of DNA strand breaks, and to detect species involved in these reactions. ScDNA anchored at HMDE was cleaved by catalytic amounts of iron/EDTA ions in the absence of chemical reductants when appropriate electrode potential (sufficiently negative to reduce [Fe(EDTA)]- to [Fe(EDTA)]2-) was applied. The process required oxygen or hydrogen peroxide. The extent of DNA damage increased with the shift of the electrode potential to negative values, displaying a sharp inflection point matching the potential of [Fe(EDTA)]2-/[Fe(EDTA)]- redox pair. In the absence of transition metal ions, significant DNA damage was observed at potentials sufficiently negative for reduction of dioxygen at the mercury electrode. This observation suggests cleavage of the surface-attached scDNA by radical intermediates of oxygen reduction at HMDE.     

Photochemical DNA cleavage by novel water-soluble sulfonated dihydroxy phosphorus(V) tetrabenzotriazacorrole

The photosensitizing properties of a novel water-soluble phthalocyanine-like photosensitizer, sulfonated dihydroxy phosphorus(V) tetrabenzotriazacorrole {P(OH)(2)TBCS(n)}, have been reported. It is relevant for the use of this dye as photodynamic sensitizer. The compound exhibited significant photocleavage of supercoiled (SC) pUC19 DNA. The photooxygenation and photocleavage to DNA showed high efficiency in generating singlet oxygen.