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Archives of Biochemistry and Biophysics Dec 2006Hydroethidine (HE) is a cell-permeable probe used for the intracellular detection of superoxide. Here, we report the direct measurement of the rate constant between...
Hydroethidine (HE) is a cell-permeable probe used for the intracellular detection of superoxide. Here, we report the direct measurement of the rate constant between hydroethidine and superoxide radical anion using the pulse radiolysis technique. This reaction rate constant was calculated to be ca. 2 x 10(6) M(-1) s(-1) in water:ethanol (1:1) mixture. The spectral characteristics of the intermediates indicated that the one-electron oxidation product of HE was different from the one-electron reduction product of ethidium (E+). The HPLC-electrochemical measurements of incubation mixtures containing HE and the oxygenated Fenton's reagent (Fe2+/DTPA/H2O2) in the presence of aliphatic alcohols or formate as a superoxide generating system revealed 2-OH-E+ as a major product. Formation of 2-OH-E+ by the Fenton's reagent without additives was shown to be superoxide dismutase-sensitive and we attribute the formation of superoxide radical anion to the one-electron reduction of oxygen by the DTPA-derived radical. Addition of tert-butanol, DMSO, and potassium bromide to the Fenton's system caused inhibition of 2-OH-E+ formation. Results indicate that reducing and oxidizing radicals have differential effects on the formation of 2-OH-E+.
Topics: Hydrogen Peroxide; Iron; Kinetics; Oxidants; Oxidation-Reduction; Phenanthridines; Pulse Radiolysis; Superoxides
PubMed: 17081495
DOI: 10.1016/j.abb.2006.09.031 -
FEBS Letters Apr 2022Flavohaemoglobins (FlavoHbs) function as nitric oxide dioxygenases, oxidizing nitric oxide with nitrite and shuttling electrons from NAD(P)H via FAD and O . Here, using...
Flavohaemoglobins (FlavoHbs) function as nitric oxide dioxygenases, oxidizing nitric oxide with nitrite and shuttling electrons from NAD(P)H via FAD and O . Here, using pulse radiolysis, we investigate intramolecular electron transfer between FAD and haem b in FlavoHbs. We found that reduction of FlavoHb with hydrated electrons proceeded via two phases: an initial fast phase and a second slower process. Absorbance measured at 600 nm revealed fast flavin reduction followed by a slower decrease corresponding to reoxidation of FAD. The slower process was partially lost in FlavoHbs lacking FAD. These results suggest that the slower phase is attributable to intramolecular electron transfer from FAD to the haem iron. The rate constant in the absence of azole compound (3.3 × 10 s ) was accelerated ~ 10-fold (2.7 × 10 s ) by the binding of econazole, reflecting a conformational change in the open-to-closed transition.
Topics: Anti-Bacterial Agents; Azoles; Candida; Electron Transport; Electrons; Flavin-Adenine Dinucleotide; Heme; Kinetics; NAD; Nitric Oxide; Oxidation-Reduction; Pichia
PubMed: 35253217
DOI: 10.1002/1873-3468.14327 -
Free Radical Biology & Medicine Jun 2016Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of...
Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3(•), CO3(•-),(•)NO2 and GS(•)). The rate constants for the Amplex® Red oxidation by N3(•) ((2)k=2.1·10(9)M(-1)s(-1), at pH=7.2) and CO3(•-) ((2)k=7.6·10(8)M(-1)s(-1), at pH=10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection.
Topics: Catalysis; Horseradish Peroxidase; Hydrogen Peroxide; Oxazines; Oxidants; Oxidation-Reduction; Peroxynitrous Acid; Pulse Radiolysis
PubMed: 27021961
DOI: 10.1016/j.freeradbiomed.2016.03.027 -
The Biochemical Journal Oct 1988The reactions between Trolox C, a water-soluble vitamin E analogue, and several oxidizing free radicals including the hydroxyl radical and various peroxy radicals were...
The reactions between Trolox C, a water-soluble vitamin E analogue, and several oxidizing free radicals including the hydroxyl radical and various peroxy radicals were examined by using the pulse-radiolysis technique. The results demonstrate that Trolox C may undergo rapid one-electron-transfer reactions as well as hydrogen-transfer processes; the resulting phenoxyl radical is shown to be relatively stable, in common with the phenoxyl radical derived from vitamin E. The reactions between the Trolox C phenoxyl radical and a variety of biologically relevant reducing compounds were examined by using both pulse radiolysis and e.s.r. The results demonstrate that the Trolox C phenoxyl radical is readily repaired by ascorbate (k = 8.3 x 10(6) dm3.mol-1.s-1) and certain thiols (k less than 10(5) dm3.mol-1.s-1) but not by urate, NADH or propyl gallate. Evidence from e.s.r. studies indicates that thiol-containing compounds may also enter into similar repair reactions with the alpha-tocopherol phenoxyl radical. Kinetic evidence is presented that suggests that Trolox C may 'repair' proteins that have been oxidized by free radicals.
Topics: Ascorbic Acid; Benzopyrans; Benzothiazoles; Binding, Competitive; Chromans; Electron Spin Resonance Spectroscopy; Electron Transport; Free Radicals; Hydrogen-Ion Concentration; Indicators and Reagents; Oxidation-Reduction; Pulse Radiolysis; Sulfhydryl Compounds; Sulfonic Acids
PubMed: 2849418
DOI: No ID Found -
Saudi Journal of Biological Sciences Feb 2018The degradation process of acephate in aqueous solution with OH and [Formula: see text] produced by Co-γ irradiation and electron pulse radiolysis was studied in the...
The degradation process of acephate in aqueous solution with OH and [Formula: see text] produced by Co-γ irradiation and electron pulse radiolysis was studied in the present paper. In the aqueous solution, acephate reacted with [Formula: see text] and transformed to transient species which can absorb weakly in the wavelength range of 300-400 nm and decay very fast. According to the decay of hydrated electron, the reaction rate constant of [Formula: see text] and acephate is (3.51 ± 0.076) × 10 dm·mol·s. The transient species produced in the reaction of OH and acephate do not distinctly absorb the light in the wavelength range of 300-700 nm, so the decay and kinetics of the transient species cannot determinedirectly. The competing reaction of KSCN oracephate with OH were studied to obtain the reaction rate constant of OH and acephate, which is (9.1 ± 0.11) × 10 dm·mol·s. Although acetylamide and inorganic ions were determined in the products of the reaction of acephate with OH or [Formula: see text], the concentration of inorganic ions in the products of the reaction of acephate with OH is higher than that in the product of the reaction of acephate with [Formula: see text]. Moreover, there were sulfide in the products of the reaction of acephatewith [Formula: see text]. The degradation pathways of acephate by OH and [Formula: see text] were also proposed based on the products from GC-MS.
PubMed: 29472769
DOI: 10.1016/j.sjbs.2017.10.022 -
Biomolecules Jun 2023Numerous chemical probes have been used to measure or image oxidative, nitrosative and related stress induced by free radicals in biology and biochemistry. In many... (Review)
Review
Numerous chemical probes have been used to measure or image oxidative, nitrosative and related stress induced by free radicals in biology and biochemistry. In many instances, the chemical pathways involved are reasonably well understood. However, the rate constants for key reactions involved are often not yet characterized, and thus it is difficult to ensure the measurements reflect the flux of oxidant/radical species and are not influenced by competing factors. Key questions frequently unanswered are whether the reagents are used under 'saturating' conditions, how specific probes are for particular radicals or oxidants and the extent of the involvement of competing reactions (e.g., with thiols, ascorbate and other antioxidants). The commonest-used probe for 'reactive oxygen species' in biology actually generates superoxide radicals in producing the measured product in aerobic systems. This review emphasizes the need to understand reaction pathways and in particular to quantify the kinetic parameters of key reactions, as well as measure the intracellular levels and localization of probes, if such reagents are to be used with confidence.
Topics: Reactive Oxygen Species; Oxidation-Reduction; Free Radicals; Superoxides; Oxidants; Antioxidants; Coloring Agents; Oxidative Stress
PubMed: 37509077
DOI: 10.3390/biom13071041 -
International Journal of Molecular... Jun 2022Hydroxyl radicals (HO) have long been regarded as a major source of cellular damage. The reaction of HO with methionine residues (Met) in peptides and proteins is a...
Hydroxyl radicals (HO) have long been regarded as a major source of cellular damage. The reaction of HO with methionine residues (Met) in peptides and proteins is a complex multistep process. Although the reaction mechanism has been intensively studied, some essential parts remain unsolved. In the present study we examined the reaction of HO generated by ionizing radiation in aqueous solutions under anoxic conditions with two compounds representing the simplest model peptide backbone CHC(O)NHCHXC(O)NHCH, where X = CHCHSCH or CHSCH, i.e., the Met derivative in comparison with the cysteine-methylated derivative. We performed the identification and quantification of transient species by pulse radiolysis and final products by LC-MS and high-resolution MS/MS after γ-radiolysis. The results allowed us to draw for each compound a mechanistic scheme. The fate of the initial one-electron oxidation at the sulfur atom depends on its distance from the peptide backbone and involves transient species of five-membered and/or six-membered ring formations with different heteroatoms present in the backbone as well as quite different rates of deprotonation in forming α-(alkylthio)alkyl radicals.
Topics: Cysteine; Hydroxyl Radical; Methionine; Oxidation-Reduction; Peptides; Pulse Radiolysis; Sulfides; Tandem Mass Spectrometry
PubMed: 35742994
DOI: 10.3390/ijms23126550 -
Molecules (Basel, Switzerland) Sep 2021The conversion of ribonucleosides to 2'-deoxyribonucleosides is catalyzed by ribonucleoside reductase enzymes in nature. One of the key steps in this complex radical...
The conversion of ribonucleosides to 2'-deoxyribonucleosides is catalyzed by ribonucleoside reductase enzymes in nature. One of the key steps in this complex radical mechanism is the reduction of the 3'-ketodeoxynucleotide by a pair of cysteine residues, providing the electrons via a disulfide radical anion (RSSR) in the active site of the enzyme. In the present study, the bioinspired conversion of ketones to corresponding alcohols was achieved by the intermediacy of disulfide radical anion of cysteine (CysSSCys) in water. High concentration of cysteine and pH 10.6 are necessary for high-yielding reactions. The photoinitiated radical chain reaction includes the one-electron reduction of carbonyl moiety by disulfide radical anion, protonation of the resulting ketyl radical anion by water, and H-atom abstraction from CysSH. The (CysSSCys) transient species generated by ionizing radiation in aqueous solutions allowed the measurement of kinetic data with ketones by pulse radiolysis. By measuring the rate of the decay of (CysSSCys) at λ = 420 nm at various concentrations of ketones, we found the rate constants of three cyclic ketones to be in the range of 10-10 Ms at ~22 °C.
Topics: Biomimetics; Disulfides; Hydroxyl Radical; Ketones; Kinetics; Water
PubMed: 34576900
DOI: 10.3390/molecules26185429 -
The Journal of Physical Chemistry. B Jan 2022This work shows that S atom substitution in phosphate controls the directionality of hole transfer processes between the base and sugar-phosphate backbone in DNA...
This work shows that S atom substitution in phosphate controls the directionality of hole transfer processes between the base and sugar-phosphate backbone in DNA systems. The investigation combines synthesis, electron spin resonance (ESR) studies in supercooled homogeneous solution, pulse radiolysis in aqueous solution at ambient temperature, and density functional theory (DFT) calculations of in-house synthesized model compound dimethylphosphorothioate (DMTP(O)═S) and nucleotide (5'--methoxyphosphorothioyl-2'-deoxyguanosine (G-P(O)═S)). ESR investigations show that DMTP(O)═S reacts with Cl to form the σσ* adduct radical -P-S[Formula: see text]Cl, which subsequently reacts with DMTP(O)═S to produce [-P-S[Formula: see text]S-P-]. -P-S[Formula: see text]Cl in G-P(O)═S undergoes hole transfer to Gua, forming the cation radical (G) via thermally activated hopping. However, pulse radiolysis measurements show that DMTP(O)═S forms the thiyl radical (-P-S) by one-electron oxidation, which did not produce [-P-S[Formula: see text]S-P-]. Gua in G-P(O)═S is oxidized unimolecularly by the -P-S intermediate in the sub-picosecond range. DFT thermochemical calculations explain the differences in ESR and pulse radiolysis results obtained at different temperatures.
Topics: DNA; Phosphates; Pulse Radiolysis; Sugars; Sulfur
PubMed: 34990129
DOI: 10.1021/acs.jpcb.1c09068 -
Journal of Radiation Research 2011A comparative study using the pulse radiolysis technique was carried out to investigate transient absorption spectra and rate constants for the reactions of (•)OH and... (Comparative Study)
Comparative Study
A comparative study using the pulse radiolysis technique was carried out to investigate transient absorption spectra and rate constants for the reactions of (•)OH and N(3)(•) with edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) and its four analogue compounds, 1,3-dimethyl-2-pyrazolin-5-one, 3-methyl-1-(pyridin-2-yl)-2-pyrazolin-5-one, 1-phenyl-3-trifluoromethyl-2-pyrazolin-5-one and 1-(4-chlorophenyl)-3-methyl-2-pyrazolin-5-one. The results showed that, unlike reaction mechanisms previously proposed, the phenyl group of edaravone played an important role in the reaction with (•)OH and OH adducts to the phenyl group were formed. Quantum chemical calculations also strongly supported this attribution and suggested that the most favorable site for attacks by (•)OH is the ortho position of the phenyl group. Moreover, the rate constants for the reactions of edaravone and its analogues towards (•)OH and N(3)(•) were about 8.0 × 10(9), and 4.0 × 10(9) dm(3) mol(-1) s(-1), respectively. Edaravone displayed higher reactivity compared to the others, in contrast to a previous report in which 3-methyl-1-(pyridin-2-yl)-2-pyrazolin-5-one showed the highest reactivity towards (•)OH.
Topics: Antipyrine; Computer Simulation; Edaravone; Free Radical Scavengers; Hydroxyl Radical; Models, Chemical; Nitrogen; Pulse Radiolysis
PubMed: 21139328
DOI: 10.1269/jrr.10060