A pulse radiolytic study of the interaction of nitroxyls with free-radical adducts of purines: consequences for radiosensitization

Using the technique of pulse radiolysis, it has been demonstrated that the radicals, produced on interaction of the hydroxyl radical with purine nucleotides/nucleosides, interact with the nitroxyls, TMPN and NPPN. It has been possible to discern the various interactions in terms of the known redox properties of the various OH-radical adducts of the purines based upon spectral and kinetic data. It has been confirmed that the properties of the radical produced on interaction of Br2-. with dGMP, based upon its subsequent interactions with nitroxyls, are quantitatively the same as those for the .OH-radical adduct of dGMP with oxidizing properties. The implications of these findings are presented in terms of the potential competition between nitroxyls and cellular radiation modifiers for the various DNA radicals with different redox potentials, and thereby assess the potential importance of the reactivity of the oxidizing-purine radicals towards nitroxyls in radiobiological studies.     

Pulse radiolysis studies of some aza analogues of nucleic acid components

The technique of pulse radiolysis has been utilized to study the reactions of some aza analogues of nucleic acid components with hydrated electrons and OH radicals. The absorption spectra of the transient free radical adducts which result from these reactions and their decay kinetics were determined. The 5-aza analogues gave similar results to those of pyrimidine bases. The 6-aza analogues also showed similar kinetics, however, transient spectra were different. The presence of the sugar moiety in these aza analogues changed the rate law of the OH adduct transient decay from second order to first order kinetics. This finding may have implications for the understanding of the radiation chemistry of DNA.     

Reaction of reducing hydroxyl radical adducts of pyrimidine nucleotides with riboflavin and flavin adenine dinucleotide (FAD) via electron transfer: a pulse radiolysis study

Using the techniques of pulse radiolysis with time-resolved spectrophotometric detection, it has been demonstrated that the interaction of reducing OH radical adducts of dCMP, TMP and UMP with riboflavin (RF) and flavin adenine dinucleotide (FAD) does proceed via an electron transfer reaction. From buildup kinetics of radical species, the rate constants of electron transfer from reducing OH adducts of pyrimidines to RF and FAD have been determined, respectively. It could be deduced that RF and FAD would reduce the probability of repair of the damaged DNA in the presence of enzymes and antioxidants, accordingly RF and FAD might have a radiosensitization effect on DNA damage.     

One-electron reduction of the oxyform of 2,4-diacetyldeuterocytochrome P-450cam

The reaction of the hydrated electron with a ferrous oxygenated form of modified cytochrome P-450cam, containing 2,4-diacetyldeuteroheme, was investigated by the use of pulse radiolysis. The ferrous oxygenated form of this enzyme was reduced by hydrated electrons to form the product, which exhibits absorption maximum at 470 and 370 nm. From the spectrum obtained, the oxidation state of the product is discussed in relation to the higher oxidation states of chloroperoxidase.     

The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals

Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical "scavengers" compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good's buffers with hydroxyl radicals have been determined by this method.     

One-electron reduction of flavodoxin. A fast kinetic study.

The reaction kinetics of fully oxidized flavodoxin from Clostridium MP with the hydrated electron have been investigated by the pulse radiolysis method. Four spectrally distinct processes have been observed with the ultimate formation of the singly reduced flavin form of the protein. The last two species obtained in the reaction sequence are spectrally similar, and are connected through a reaction which is first order. It is proposed that this reaction involves a protein conformational alteration.     

Pulse radiolysis and spectral studies of the interaction of cetylpyridinium chloride and Methylene Blue with connective-tissue glycosaminoglycans and related compounds

1. Hydrated electrons produced by pulse radiolysis were used to study the interaction of polyanionic glycosaminoglycans and related compounds with the counterions Methylene Blue and cetylpyridinium chloride. 2. The effect of added salt (potassium chloride) on the interaction indicates that the relative binding affinities, with respect to the types of anionic site present, increases for both counterions in the order CO(2) (-)相似文献   

Reactions of reducing radicals with ribonuclease

Radiation-induced reactions of hydrated electrons, formate- and ethanol radicals with ribonuclease were studied by pulse radiolysis and by electrophoresis. Initially formate radicals react rapidly and very specifically with the disulphide bonds of ribonuclease. This reaction leads to aggregation by intermolecular S-S-interchange, the process being more effective at pH 4, since formation and decay of S-S-.-radical anions increases with decreasing pH. With high doses additional unreducible aggregates are formed. Radical formation at the positively charged histidine residues seems to be involved. Hydrated electrons do not react as selectively as the formate radicals, but with several sites in native ribonuclease. Thus with low doses unreducible aggregates are formed. Electrophoresis shows that reaction of the electrons causes fragmentation of the peptide chain, when OH-radicals are scavenged. Very weak transient spectra and very little degradation result on reaction of ethanol radicals with ribonuclease.     

Radiation chemists look at damage in redox proteins induced by X‐rays

The three‐dimensional structure of proteins, especially as determined by X‐ray crystallography, is critical to the understanding of their function. However, the X‐ray exposure may lead to damage that must be recognized and understood to interpret the crystallographic results. This is especially relevant for proteins with transition metal ions that can be oxidized or reduced. The detailed study of proteins in aqueous solution by the technique of pulse radiolysis has provided a wealth of information on the production and fate of radicals that are the same as those produced by X‐ray exposure. The results reviewed here illustrate how the products of the interaction of radiation with water or with solutes added to the crystallization medium, and with proteins themselves, are formed, and about their fate. Of particular focus is how electrons are produced and transferred through the polypeptide matrix to redox centers such as metal ions or to specific amino acid residues, for example, disulfides, and how the hydroxyl radicals formed may be converted to reducing equivalents or scavenged.     

Mechanism of reduction of bleomycin-Cu(II) by CO2- and oxidation of bleomycin-Cu(I) by H2O2 in the absence and presence of DNA

The reduction reaction of bleomycin-Cu(II) by CO2- has been studied by gamma and pulse radiolysis at pH7. The CO2- radical reduces bleomycin-Cu(II) at a rate of (6.7 +/- 0.7) X 10(8) dm3 mol-1 s-1. In the presence of calf thymus DNA the rate of the reduction decreased as the concentration of DNA increased, indicating that the reduction reaction proceeds through free bleomycin-Cu(II). The stoichiometry and the kinetics of the oxidation of bleomycin-Cu(I) by H2O2 in the presence and absence of DNA have been studied. Our observations suggest that the OH. radical is not produced during this reaction and the degradation of the drug occurs in the absence and presence of DNA. We assume that bleomycin-Cu(II) in the presence of a reducing agent and molecular oxygen or H2O2 does not cleave DNA since the oxidizing species, which are formed during the oxidation reaction by H2O2, attack the drug even in the presence of DNA.     

A kinetic study on the interaction of deprotonated purine radical cations with amino acids and model peptides

By use of pulse radiolysis techniques, the radical cations of purine nucleotides have been successfully produced by the SO4- ion oxidation. Time-resolved spectroscopic evidence is provided that the one-electron-oxidized radicals of dAMP and dGMP can be efficiently repaired by aromatic amino acids (including tyrosine and tryptophan) via electron transfer reaction. As a model peptide, Arg-Tyr-AcOH was also investigated with regard to its interaction with deprotonated purine radical cations. The rate constants of the electron transfer reactions were determined to be (1 approximately 5) x 10(8) dm(3) mol(-1) s(-1). These results suggest that the aromatic amino acids in DNA-associated proteins may play some role in electron transfer reactions through DNA.     

Oxidation-Reduction Reactions of Iron Bleomycin in the Absence and Presence of DNA

Using the pulse radiolysis technique, we have demonstrated that bleomycin-Fe(III) is stoichiometrically reduced by CO₂^- to bleomycin-Fe(II) with a rate of (1.9 ± 0.2) × 10⁸M^-1s^-1. In the presence of calf thymus DNA, the reduction proceeds through free bleomycin-Fe(III) and the binding constant of bleomycin-Fe(III) to DNA has been determined to be (3.8 ± 0.5) x 10⁴ M^-1. It has also been demonstrated that in the absence of DNA O₂^-1 reacts with bleomycin-Fe(III) to yield bleomycin-Fe(II)O₂, which is in rapid equilibrium with molecular oxygen, and decomposes at room temperature with a rate of (700 ± 200) s^-1. The resulting product of the decomposition reaction is Fe(III) which is bound to a modified bleomycin molecule. We have demonstrated that during the reaction of bleomycin-Fe(II) with O₂, modification or self-destruction of the drug occurs, while in the presence of DNA no destruction occurs, possibly because the reaction causes degradation of DNA.     

Oxygen inhibition of nitroreductase: electron transfer from nitro radical-anions to oxygen.

The inhibition of many nitroreductases by oxygen has been explained by Mason and Holtzman in terms of electron transfer to oxygen from the nitro radical-anions, which have been identified as the first intermediate in some reductase systems. We have used the pulse radiolysis technique to measure the bimolecular rate constants of this electron-transfer reaction for over 20 nitro compounds, including substituted 2- and 5-nitroimidazoles of interest as antiprotozoal drugs and radiosensitizers, nitrofurans in use as antibacterial agents, and substituted nitrobenzenes previously used as model substrates for nitroreductases. The logarithm of the rate constant for the reaction of the nitro radical-anion with oxygen is linearly related to the one-electron reduction potential of the nitro compound.     

Thiyl free radicals and the oxidation of ferrocytochrome c. Direct observation of coupled hydrogen-atom- and electron-transfer reactions.

Absolute rate constants for the reaction of ferrocytochrome c with the thiyl radicals derived from cysteine, GSH, penicillamine and N-(2-mercaptopropionyl)glycine were measured by using the technique of pulse radiolysis. The reaction is believed to occur through a one-electron-transfer process, in agreement with the hypothesis that thiols may act as catalysts linking hydrogen-atom- and electron-transfer reactions.     

Repair effect of thymine radical anion by echinocoside using pulse radiolysis

Repair activities of thymine radical anion by echinocoside, isolated from Pedicularis plicata. were studied using pulse radiolysis technique. The thymine radical anion was produced by the reaction of hydrated electron with thymine. Echinocoside. one of the polyphenols of phenylpropanoid glycoside, was added to the thymine aqueous solution saturated with N2. Kinetic analysis by transient absorption spectrum showed that thymine radical anion was formed at first, and then after several decades of microseconds of pulse radiolysis. the spectrum of thymine radical anion was changed to that of echinocoside radical anion. The evidence indicated that thymine radical anion was repaired through one-electron-transfer between the DNA base radical anion and echinocoside. The rate constant of electron transfer by echinocoside was 1.45× 109 dm3 · mol1 · s 1.     

Mechanism of radiation-induced reactions in aqueous solution of coumarin-3-carboxylic acid: effects of concentration, gas and additive on fluorescent product yield

The radiation-induced reactions of a water-soluble coumarin derivative, coumarin-3-carboxyl acid (C3CA), have been investigated in aqueous solutions by pulse radiolysis with a 35 MeV electron beam, final product analysis following (60)Co γ-irradiations and deterministic model simulations. Pulse radiolysis revealed that C3CA reacted with both hydroxyl radicals ((?)OH) and hydrated electrons (e(-) (aq)) with near diffusion-controlled rate constants of 6.8 × 10(9) and 2.1 × 10(10) M(-1) s(-1), respectively. The reactivity of C3CA towards O(2)(? -) was not confirmed by pulse radiolysis. Production of the fluorescent molecule, 7-hydroxy-coumarin-3-carboxylic acid (7OH-C3CA), was confirmed by final product analysis with a fluorescence spectrometer coupled to a high performance liquid chromatography (HPLC) system. Production yields of 7OH-C3CA following (60)Co γ-irradiations depended on the irradiation conditions and ranged from 0.025 to 0.18 (100 eV) (-1). Yield varied with saturating gas, additive and C3CA concentration, implying the presence of at least two pathways capable of providing 7OH-C3CA as a stable product following the scavenging reaction of C3CA with (?)OH, including a peroxidation/elimination sequence and a disproportionation pathway. A reaction mechanism for the two pathways was proposed and incorporated into a deterministic simulation, showing that the mechanism can explain experimentally measured 7OH-C3CA yields with a constant conversion factor of 4.7% from (?)OH scavenging to 7OH-C3CA production, unless t-BuOH was added.     

Effect of solvent viscosity,polarity and pH on the charge transfer between tryptophan radical and tyrosine in bovine serum albumin: a pulse radiolysis study

The effect of viscosity, solvent polarity and pH of the medium on the reaction of a protein, bovine serum albumin (BSA), with organohalo-peroxyl radical in aqueous solution has been studied using pulse radiolysis technique. Unlike in dilute aqueous solution, electron transfer from tyrosine to tryptophan radical in BSA has been clearly observed at a viscosity of 7.7 centiPoise (cP). The oxidation of BSA, tryptophan and tyrosine in different media has also been compared with those taking place in dilute aqueous solution. The effect of solvent characteristics on the observed charge transfer has been discussed.     

Simulation of the passage of fast electrons and the early stage of water radiolysis by the Monte Carlo method

A numerical computer simulation of the processes of the interaction of electrons with liquid water and vapor was performed, beginning with the absorption of the energy of ionizing radiation and including the chemical changes in the medium. The specific features of the liquid phase compared with the gaseous phase were taken into account. Among them are the decrease of the ionization potential and collective excitations of the plasmon type. The mass stopping powers and ranges of electrons in liquid water and vapor were calculated. Within the frames of the stochastic model the kinetics of water radiolysis in the picosecond range of radiolysis was calculated by the Monte Carlo method. The mechanism of water radiolysis was found with the electron-ion recombination and the reactions of quasi-free and solvated electrons taken into account.     

Adsorptive stripping voltammetry of human chorionic gonadotropin and two of its specific antibodies

The electrochemical behaviour of human chorionic gonadotropin (hCG), as well as rat anti-βhCG and rabbit anti-βhCG antibodies, has been studied using cyclic voltammetry and adsorptive stripping voltammetry. Conditions have been optimised for the determination of these species by differential pulse adsorptive stripping voltammetry with respect to accumulation potential, accumulation time, scan rate, pulse amplitude and drop size. This technique has also been used to investigate the interaction of hCG with each of its specific antibodies in solution.     

Pulse radiolytic study of the interaction of SO4.- with deoxynucleosides. Possible implications for direct energy deposition

Using the technique of pulse radiolysis it has been demonstrated that the interaction of SO4.- with deoxynucleosides (k approximately less than 2 X 10(8)-2.3 X 10(9) dm3 mol-1 s-1) in aqueous solution at pH 7.0 results in the formation of the corresponding one-electron oxidized radicals which either deprotonate or hydrate to yield OH adducts. Based upon the ease of oxidation of the deoxynucleosides, dG, dA, dC, dT, by SO4.-, the apparent redox potentials are in the order dG much greater than dA approximately equal to dC greater than dT. With the exception of deoxyuridine, the deoxynucleoside radicals produced on interaction with SO4.- have been shown to have oxidizing properties based upon the interactions with tetranitromethane and the nitroxyls, TMPN and NPPN. The deoxynucleoside radicals (dG, dA and dC) do not interact with oxygen (k less than 10(6) dm3 mol-1 s-1) in contrast to the interaction observed with the thymidine radical (k = 2.5 X 10(7) dm3 mol-1 s-1). The implications of these findings are presented in terms of the properties of the discussed radicals as relating to those of potential DNA base radicals (positive centres) produced by direct energy deposition within DNA. The use of SO4.- to mimic, to some extent, the effects of direct energy deposition in DNA may assist in our understanding of the resulting molecular processes relevant to radiobiological studies.