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PubMed1991

Structural analyses on yeast tRNA(Tyr) and its complex with tyrosyl-tRNA synthetase by the use of hydroxyl radical 'footprinting'.

Hydroxyl radical, generated by reduction of hydrogen peroxide by Fe(II)-EDTA, was used to investigate the contact sites of yeast tRNA(Tyr) with its cognate tyrosyl-tRNA synthetase (TyrRS). Exposure of free tRNA(Tyr) to this reagent gave cleavage patterns consistent with the tertiary structure of yeast tRNA(Phe) established by X-ray crystallography. When the probing reaction was performed under the conditions which stabilized complex formation between tRNA(Tyr) and TyrRS, aminoacyl-stem region of the tRNA was protected from cleavage. This result supports our earlier finding that the information for binding to TyrRS would reside mainly in the aminoacyl-stem of tRNA(Tyr).

Binding Sites↗
PubMed1982

Formation of hydroxyl radicals from the paraquat radical cation, demonstrated by a highly specific gas chromatographic technique. the role of superoxide radical anion, hydrogen peroxide, and glutathione reductase.

Yeast glutathione reductase catalyzes an NADPH-dependent reduction of the herbicide paraquat in vitro. The single-electron reduced paraquat radical reacts with O2 to generate the superoxide radical, O2.-. Hydroxyl radicals (OH.) can also be detected in this assay system by their reaction with phenol to form diphenols, as assayed quantitatively by a highly specific and sensitive method employing gas-liquid chromatography. Formation of hydroxyl radicals can be virtually completely suppressed by catalase and partially suppressed by superoxide dismutase. The role of hydroxyl radicals and superoxide in paraquat toxicity in vivo is discussed.

Chromatography, Gas↗
PubMed1991

The use of water-soluble radical scavengers to detect hydroxyl radical formation by polymorphonuclear leukocytes.

The polymorphonuclear leukocyte secretes both O2- and H2O2 when stimulated by various soluble or particulate stimuli. Since a reaction involving iron, O2-, and H2O2 could generate the hydroxyl radical (HO.) there has been speculation that the HO. may participate in the bactericidal activity of the neutrophil. A variety of water-soluble HO. scavengers have been used to test for the participation of HO. and the results imply that HO. might participate. However, other workers have not been able to detect the formation of significant amounts of HO. by the activated neutrophil. We have examined the effect of several commonly used HO. radical scavengers on the ability of the neutrophil to secrete O2- and H2O2. Several of these compounds actively inhibit secretion without affecting the viability of the neutrophil. After considering the various complications inherent in using water soluble radical scavengers, we suggest that they only be used with well defined experimental systems.

Allopurinol↗
PubMed1990

Spin trapping of ibuprofen radicals: evidence that ibuprofen is a hydroxyl radical scavenger.

The purpose of this study was to use electron paramagnetic resonance (EPR) spectroscopy to determine if ibuprofen, [2-(4-isobutylphenyl) propanoic acid], a potent nonsterodial anti-inflammatory agent, could modify hydroxyl radicals generation in vitro. Ibuprofen (IBU; 0.1-50 mM) in water or water alone was added to EPR tubes containing ferrous sulfate (0.5-2.0 mM), and either 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; 40 mM) or alpha-phenyl N-tert-butyl nitrone (PBN; 48 mM). Hydrogen peroxide (1 mM) was added to inititate the Fenton reaction, and the systems were then analyzed by EPR spectroscopy to determine the type and relative quantity of free radical(s) produced. IBU caused a dose-dependent decrease of signal intensity of the hydroxyl radical adduct of DMPO (DMPO-OH) which is an indication that IBU either scavenges the hydroxyl radical and/or chelates iron. In addition, other radicals (presumably IBU radicals) produced in these systems were trapped by both DMPO (aN = 16.1 G, aH beta = 24.0 G) and PBN (aN = 15.7 G, aH beta = 4.4 G and aN = 17.0 G, aH beta = 2.1 G). The signal height of these IBU radicals increased in systems containing ferrous sulfate (1 mM), hydrogen peroxide (1 mM), PBN (48 mM), and increasing IBU concentrations. Therefore, we conclude that IBU scavenges the hydroxyl radical. If IBU chelated iron, then less hyroxyl radicals would be generated, less IBU radical formed and the signal height of IBU radicals trapped by PBN would have decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Antioxidants↗
PubMed1996

Detection of thiyl radical adducts formed during hydroxyl radical- and peroxynitrite-mediated oxidation of thiols--a high resolution ESR spin-trapping study at Q-band (35 GHz).

Thiyl radicals (RS.) formed during peroxynitrite- or hydroxyl radical-dependent oxidation of thiols, i.e., glutathione (GSH) and L-cysteine (CySH) were trapped with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and analyzed by X-band and Q-band electron spin resonance (ESR) spectroscopy. At X-band, the ESR parameters of DMPO-glutathionyl radical adduct (DMPO/.SG) and DMPO-hydroxyl radical adduct (DMPO/.OH) are nearly similar in aqueous solutions and as a result, except for the lowfield spectral line, the remaining spectral lines of DMPO/ .SG virtually over-lap with those of the DMPO/.OH adduct. In contrast, at Q-band, most of the spectral lines due to the DMPO/.SG were separated from the DMPO/ .OH. Inclusion of a superoxide dismutase (SOD) mimic completely abolished the formation of the DMPO/.OH adduct and not the DMPO/.SG adduct during ONOO(-)-mediated oxidation of GSH and DMPO. In the presence of formate, the DMPO/.SG spectrum was replaced by the DMPO/.CO2- spectrum which was monitored by Q-band ESR spectroscopy. Thus, spin-trapping at Q-band provides unambiguous proof for the glutathionyl radical-dependent oxidation of formate by peroxynitrite. High resolution Q-band ESR spectra of DMPO/.Scys were also obtained. Biological applications of the Q-band spin-trapping technique to detect thiyl radicals in cellular systems are discussed.

Biological Assay↗
PubMed1993

Use of aromatic hydroxylation of phenylalanine to measure production of hydroxyl radicals after myocardial ischemia in vivo. Direct evidence for a pathogenetic role of the hydroxyl radical in myocardial stunning.

A pathogenetic role of .OH in myocardial stunning has been inferred from the protective effects of .OH scavengers and iron chelators. However, conclusive demonstration of the .OH radical hypothesis of myocardial stunning requires direct verification of three major, but still unproven, assumptions: (1) .OH is produced in the stunned myocardium in vivo; (2) antioxidant therapy inhibits .OH production; and (3) such inhibition results in enhanced recovery of contractility (ie, .OH is necessary for the development of myocardial stunning). Since phenylalanine (Phe) reacts with .OH to form the hydroxylated products ortho-, meta-, and para-tyrosines (o-, m-, and p-tyr), we used aromatic hydroxylation of Phe to detect .OH formation in the stunned myocardium. Open-chest dogs undergoing a 15-minute coronary occlusion followed by reperfusion received an intravenous infusion of Phe (54.3 mg/kg for 11.5 minutes beginning 90 seconds before reperfusion); these animals were given either no antioxidant therapy (group I, n = 15), N-2-mercaptopropionyl glycine (MPG) (group II, n = 11), or MPG combined with superoxide dismutase, catalase, and desferrioxamine (group III, n = 12). In addition, group IV (nonischemic control group, n = 6) received Phe but did not undergo coronary occlusion, whereas group V (ischemic control group, n = 16) underwent a 15-minute occlusion but did not receive Phe or antioxidants. The plasma concentrations of tyrosines in the local venous effluent and in the arterial blood were measured with high-performance liquid chromatography. In group I, production of o- and m-tyr, which are specific markers of .OH formation, began during coronary occlusion but increased dramatically immediately after reperfusion, peaking at 1 minute and continuing up to 10 minutes of reperfusion. In group II, the production of o- and m-tyr was markedly decreased throughout the first 10 minutes of reperfusion. In group III, the production of m-tyr was decreased to levels similar to those in group II, whereas the production of o-tyr was almost completely abolished. There was no appreciable production of o- or m-tyr in group IV. Recovery of contractile function (assessed as systolic wall thickening) was increased in group I vs group V. Recovery of function was further enhanced in group II, with only a slight additional improvement in group III.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Effects of the superoxide radical scavenger superoxide dismutase, and of the hydroxyl radical scavenger mannitol, on reperfusion injury in isolated rabbit hearts.

Hydroxyl radical formation, secondary to superoxide radical generation, has been advocated as the actual mechanism of oxygen radical-mediated damage in biological systems. The present study was designed to compare the efficacy of administration of the hydroxyl radical scavenger mannitol vs. that of the superoxide radical scavenger superoxide dismutase (SOD) in reducing myocardial reperfusion injury, and to test whether combined treatment with both agents would confer better tissue protection compared with either intervention alone. Rabbit hearts perfused within a 31P nuclear magnetic resonance (31P-NMR) spectrometer were subjected to 30 minutes of total global ischemia at 37 degrees C. At reflow, 12 hearts in each group received either (a) a bolus of standard perfusion buffer, followed by 45 minutes of reperfusion (controls); (b) the superoxide radical scavenger recombinant human SOD (h-SOD, as a 60,000 U bolus followed by a 100 U/ml infusion for 15 minutes); (c) the hydroxyl radical scavenger mannitol (50 mM bolus followed by 15 minutes of 50 mM infusion; or (d) a combination of both agents. All treated hearts were switched to standard buffer for the remaining 30 minutes of reperfusion. Treatment with h-SOD alone was associated with a significant improvement in the recovery of cardiac contractility and coronary flow, as well as of ATP content, compared to control hearts. In contrast, mannitol treatment resulted in a small, nonsignificant improvement in these parameters. The addition of mannitol to h-SOD did not result in further significant improvement of contractility and ATP recovery compared to h-SOD alone. These data demonstrate that under our experimental conditions significant protection against reperfusion injury can be achieved by the administration of h-SOD alone, without the need for additional hydroxyl radical scavenger therapy with mannitol. These results do not exclude that significant tissue protection may be achieved by different doses of mannitol or by other agents. However, they suggest that under definite experimental conditions prevention of hydroxyl radical formation, rather than attempts to minimize hydroxyl radical toxicity, might be a more efficient method to prevent oxygen radical-mediated reperfusion injury in isolated hearts.

Adenosine Triphosphate↗
PubMed1987

Superoxide-dependent and ascorbate-dependent formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Are lactoferrin and transferrin promoters of hydroxyl-radical generation?

Apo-lactoferrin and apo-transferrin protect against iron-ion-dependent hydroxyl-radical (.OH) generation from H2O2 in the presence of superoxide radicals or ascorbic acid at pH 7.4, whether the necessary iron is added as ionic iron or as ferritin. Iron-loaded transferrin and lactoferrin [2 mol of Fe(III)/mol] show no protective ability, but do not themselves accelerate .OH production unless chelating agents are present in the reaction mixture, especially if the proteins are incorrectly loaded with iron. At acidic pH values, the protective ability of the apoproteins is diminished, and the fully iron-loaded proteins can release some iron in a form able to accelerate .OH generation. The physiological significance of these observations is discussed.

Apoproteins↗
PubMed1988

Generation of superoxide radical, hydrogen peroxide and hydroxyl radical during the autoxidation of N,N,N',N'-tetramethyl-p-phenylenediamine.

The autoxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) at neutral pH has been shown to generate superoxide radical and hydrogen peroxide. The rate of formation of these species was increased in the presence of certain iron and copper compounds; in the presence of iron complexed with EDTA, hydroxyl radical was also produced. Hydrogen peroxide was detected in erythrocytes incubated with TMPD and these cells suffered oxidative damage as reflected by methaemoglobin formation and glutathione depletion; the one-electron oxidation product of TMPD, Wurster's Blue, was equally effective in producing such changes in erythrocytes. N-Methylated p-phenylenediamines are known to be mutagenic and myotoxic, and it is suggested that 'active oxygen' species may be involved in the initiation of these harmful effects.

Animals↗
PubMed2004

High-level ab initio studies of the electronic excited states of the hydroxyl radical and water-hydroxyl complex.

The lowest-energy electronic transitions in the hydroxyl radical and the hydrogen bound complex H(2)O.HO have been studied using ab initio methods. We have used the complete active-space self-consistent field and multireference configuration interaction (MRCI) methods to calculate vertical excitation energies and oscillator strengths. At the MRCI level the lowest-lying (2)Sigma(+)<--(2)Pi electronic transition is redshifted by about 2500 cm(-1) upon formation of the H(2)O.HO complex. We propose that this transition could be used to identify the complex in the gas phase, which in turn could be used to examine the role of H(2)O.HO in atmospheric reactions.

Journal Article↗
PubMed1978

Hydroxyl radical production by free and DNA-bound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of hydroxyl radicals by spin trapping.

The reduced antitumor antibiotic mitomycin C in aqueous solution exposed to air gives a 36-line electron spin resonance spectrum of the semiquinone identified by computer simulation. Incubation of this radical with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) gives the PBN.OH nitroxide radical identified by independent generation. This nitroxide radical is also formed from similar treatment of a DNA to which mitomycin C is covalently attached. Incubation of the semiquinone from mitomycin C, mitomycin B, or streptonigrin (SN) with catalase or with superoxide dismutase inhibits the generation of OH, implying the intermediacy of H2O2 and O2 in its formation. The formation of the spin-trapped nitroxide radical is similarly inhibited by EDTA, suggesting the intermediacy of trace metal ions in the generation of hydroxyl radicals from SN. The results are consistent with the generation by the aminoquinone antibiotics in vivo of OH. already implicated in the degradation of DNA.

Catalase↗
PubMed1998

Effect of lu-duo-wei on scavenging superoxide and hydroxyl radicals in vitro.

Irradiation of a riboflavin-containing system by ultraviolet light was used to produce superoxide radical (.O2-). Hydroxyl radical (.OH) was generated by the system of Fe(II)-H2O2. Using electron spin resonance and spin trapping techniques, the effect of Lu-Duo-Wei, a capsule consisting extracts of green tea, Fructus Lycii and Semen Ziziphi Spinosae, on scavenging .O2- and .OH was observed. The results showed that the efficiency of scavenging .O2- and .OH by Lu-Duo-Wei was much higher than that by tea polyphenol alone. It is suggested that the synergistic action of tea polyphenol and other components are responsible for the scavenging effect of Lu-Duo-Wei on .O2- and .OH.

Drugs, Chinese Herbal↗
PubMed1997

Sarcoplasmic reticulum Ca(2+)-pump dysfunction in rat cardiomyocytes briefly exposed to hydroxyl radicals.

The effects of hydroxyl radical exposure of intact cardiomyocytes on sarcoplasmic reticulum (SR) function were investigated. For this purpose, isolated rat heart myocytes were exposed briefly (1 min) to the hydroxyl radical generating system (H2O2/FeCl2 or FeSO4) or 5-5'-dithiobis-nitrobenzoic acid (DTNB), a sulfhydryl oxidizing reagent, and following this a SR-enriched fraction was isolated. Marked decreases in the SR calcium uptake activities were seen in the myocytes exposed to either the hydroxyl radical-generating system or DTNB. The exposure of myocytes to the hydroxyl radical, but not DTNB, markedly increased the amount of malonyldialdehyde (MDA) in the subsequently isolated SR. Total sulfhydryl group content in SR was decreased by exposure of myocytes to DTNB. Further, there was a significant decrease in [3H]-NEM binding to SR isolated from the hydoxyl radical-treated myocytes indicating that sulfhydryl groups are affected (oxidized). Both mannitol and catalase were found to offer complete protection against the inhibitory effect of peroxide +/- iron on calcium uptake. Also the above-mentioned alterations in both MDA and sulfhydryl group content were prevented by mannitol and catalase. Exogenously added cyclic AMP-dependent protein kinase (A-PK) or calmodulin (CAM) increased SR calcium uptake activity. In the SR isolated from the treated myocytes, the stimulatory effects of A-PK and CAM were also seen, although under all assay conditions calcium uptakes were of lower magnitude. The findings are consistent with the view that the damaging effect of the hydroxyl radical and DTNB on the functioning of SR occurs rapidly in the intact cardiomyocytes. The hydroxyl radical-provoked damage involves both protein sulfhydryl and lipid oxidation.

Animals↗
PubMed1995

Spin trapping endogenous radicals in MC-1010 cells: evidence for hydroxyl radical and carbon-centered ascorbyl radical adducts.

Incubation of MC-1010 cells with the spin-trapping agent 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) followed by brief treatment with the solid oxidant lead dioxide (PbO2) yielded, after filtration, a cell-free solution that contained two nitroxyl adducts. The first was the hydroxyl radical adduct, 5,5-dimethyl-2-hydroxypyrrolidine-1-oxyl (DMPO-OH), which formed immediately upon PbO2 oxidation. The second had a 6-line EPR spectrum typical of a carbon-centered radical (AN = 15.9 G; AH = 22.4 G) and formed more slowly. No radical signals were detected in the absence of either cells or PbO2 treatment. The 6-line spectrum could be duplicated in model systems that contained ascorbate, DMPO and DMPO-OH, where the latter was formed from hydroxyl radicals generated by sonolysis or the cleavage of hydrogen peroxide with Fe2+ (Fenton reaction). In addition, enrichment of MC-1010 cells with ascorbate prior to spin trapping yielded the 6-line EPR spectrum as the principal adduct following PbO2 oxidation and filtration. These results suggest that ascorbate reacted with DMPO-OH to form a carbon-centered ascorbyl radical that was subsequently trapped by DMPO. The requirement for mild oxidation to detect the hydroxyl radical adduct suggests that DMPO-OH formed in the cells was reduced to an EPR-silent form (i.e., the hydroxylamine derivative). Alternatively, the hydroxylamine derivative was the species initially formed. The evidence for endogenous hydroxyl radical formation in unstimulated leukocytes may be relevant to the leukemic nature of the MC-1010 cell line. The spin trapping of the ascorbyl radical is the first report of formation of the carbon-centered ascorbyl radical by means other than pulse radiolysis. Unless it is spin trapped, the carbon-centered ascorbyl radical immediately rearranges to the more stable oxygen-centered species that is passive to spin trapping and characterized by the well-known EPR doublet of AH4 = 1.8 G.

Ascorbic Acid↗
PubMed2004

Mapping protein-ligand interactions by hydroxyl-radical protein footprinting.

Hydroxyl-radical protein footprinting is a direct method to map protein sites involved in macromolecular interactions. The first step is to radioactively end-label the protein. Using hydroxyl radicals as a peptide backbone cleavage reagent, the protein is then cleaved in the absence and presence of ligand. Cleavage products are separated by high-resolution gel electrophoresis. The digital image of the footprinting gel can be subjected to quantitative analysis to identify changes in the sensitivity of the protein to hydroxyl-radical cleavage. Molecular weight markers are electrophoresed on the same gel and hydroxyl-radical cleavage sites assigned by interpolation between the known cleavage sites of the markers. The results are presented in the form of a difference plot that show regions of the protein that change their susceptibility to cleavage while bound to a ligand.

Amino Acid Sequence↗
PubMed1991

Cytocidal and filamentous response of Escherichia coli cells exposed to low concentrations of hydrogen peroxide and hydroxyl radical scavengers.

The hydroxyl radical scavengers thiourea, ethanol, and dimethyl sulfoxide significantly reduced the killing of Escherichia coli elicited by low concentrations of H2O2 (resulting in mode 1 killing) when treatments with the oxidant and the scavengers were performed in a complete growth medium (K medium) but not in M9 salts. In addition, thiourea efficiently prevented the toxic response to high concentrations of H2O2 (resulting in mode 2 killing) under both exposure conditions. Sod A cells, which do not respond with a bimodal pattern of toxicity when challenged with increasing concentrations of H2O2, were markedly protected by thiourea against the lethal action of various levels of the oxidant (which in the wild-type strain result in either mode 1 or mode 2 killing) under conditions of treatments in both K medium and M9 salts. In some experiments, wild-type cells were challenged with a low concentration of H2O2 (in the absence or presence of hydroxyl radical scavengers) and then postincubated in fresh K medium for various time intervals. It was found that scavengers were able to inhibit the filamentous response generated by exposure to the oxidant in K medium. Both the length and the number of filaments were markedly reduced. Treatment in M9 salts resulted in a limited number of very short filaments, and this response was slightly reduced by the scavengers.

Dimethyl Sulfoxide↗
PubMed1966

Addition radicals formed by hydroxyl radical bombardment of uracil.

Direct addition of O-H radicals at room temperature to the carbon No.5 of the uracil ring has been proved by measurement of the proton hyperfine structure in the electron spin resonance of the resulting uracil + O-H radicals. Upon addition of the OH, the H originally bound to carbon No. 5 shifts to No. 6, thus forming a methylene group at carbon No. 6, with the two protons having equivalent coupling of 28 gauss. The spin density on carbon No. 5 is O.64. The radicals were produced when powdered samples of uracil were subjected to a low-velocity beam of O-H radicals coming from either hydrogen peroxide or water vapor under reduced pressure and subjected to an electric discharge.

Chemical Phenomena↗