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PubMed1979

On the mechanism of nucleosome unfolding.

We have studied the relative stabilities to urea denaturation of histone-histone binding interactions as they occur both in chromatin and in histone complexes free in solution. We have used the two zero-length contact-site cross-linking agents, tetranitromethane and UV light, to measure the relative degree of H2B-H4 and H2A-H2B association under various conditions. The two interactions were disrupted coordinately when nuclei were treated with increasing concentrations of urea. In contrast, when histone complex in 2 M NaCl were treated with urea, the H2B-H4 interaction was found to be much less stable than the H2A-H2B interaction. We have shown previously that nucleosomes unfold at low ionic strengths such that the H2B-H4 but not the H2A-H2B interaction is broken in the process. We speculate that the preferential rupture of the H2B-H4 contact is of physiological significance.

Animals↗
PubMed1976

Chemical modification of carboxypeptidase A crystals. Nitration of tyrosine-248.

Nitration of bovine carboxypeptidase A crystals with tetranitromethane increases esterase activity, decreases peptidase activity, and modifies about one tyrosyl residue. Modification of enzyme crystals avoids the polymerization that occurs when the enzyme is nitrated in solution. Two procedures have been employed to identify the tyrosyl residues nitrated. The first involves cyanogen bromide cleavage and isolation of the fragment containing residues 104-301. After solubilization by succinylation, this fragment is digested with chymotrypsin, the peptides are fractionated by gel filtration, and the nitrotyrosyl peptides are purified by affinity chromatography on an antinitrotyrosyl antibody-Sepharose conjugate followed by ion-exchange chromatography. In the second, the nitroenzyme is heat denatured, digested by chymotrypsin, and fractionated on the affinity and ion-exchange columns. By both methods, the major mitropeptides, representing between 60 and 80% of the nitrotyrosyl label, are uniquely compatible with that segment of the sequence of carboxypeptidase containing Tyr-248. A nearby cation, either the active site zinc ion or Arg-145, would seem to be an important factor in determining the selective nitration of this residue.

Amino Acids↗
PubMed1975

Studies on NO2-Tyr82and NH2-Tyr82 derivatives of adrenodoxin. Effects of chemical modification on electron transferring activity.

Bovine apoadrenodoxin was treated with tetranitromethane to introduce a nitro group into the tyrosyl residue at position 82 of this protein. The degrees of nitration under the best conditions were estimated to be 90% and nearly 100% on the basis of amino acid analysis and the spectrophotometric method, respectively. An amino derivative was prepared by reducing the nitro group with sodium dithionite. The apoadrenodoxin derivatives could be reconstituted to have an iron-sulfur chromophore similar to the native adrenodoxin which contains a 1:1 molar ratio of labile sulfur to iron content and displays absorption peaks at 414 and 450 nm. The enzymatic acitivies of these reconstituted nitro and amino derivatives toward cytochrome c reduction in the presence of adrenodoxin reductase and NADPH were 19 and 7% of native adrenodoxin, respectively. We studied the kinetics of the direct reduction of the reconstituted amino derivative in the presence of NADPH and adrenodoxin reductase under anaerobic conditons. The initial rate of reduction for the amino derivative was 7% of the native adrenodoxin, which is in good agreement with its activity toward cytochrome c reduction. From these results, it is concluded that by modifying the tyrosyl residue at position 82 of the adrenodoxin polypeptide, the electron-transferring activity of the molecule is largely diminished.

Adrenodoxin↗
PubMed2002

Probing the topology of the glycine receptor by chemical modification coupled to mass spectrometry.

Tetranitromethane (TNM), a small aqueous reagent that specifically modifies solvent-accessible tyrosine residues to o-nitrotyrosine, was used to probe the topology of the GlyR. Homomers of human alpha1 GlyR were recombinantly expressed via a baculovirus system, affinity-purified, and reconstituted in lipid vesicles of defined composition. The native-like reconstituted receptors were then reacted with TNM, and GlyR reaction products were isolated by SDS-PAGE. After proteolytic digestion, TNM-labeled residues were identified using mass spectrometry by observing the mass shift corresponding to the nitrate moiety. In this manner, we have identified TNM modifications of tyrosine residues at positions 24, 75, 78, 161, 223, and 228 in the receptor. Of significance, nitrations at Tyr 223 and Tyr 228 occur within the first putative transmembrane helix (M1) of the receptor, and their labeling suggests a non-helical secondary structure for M1 for the glycine receptor. In a previously published report [Leite et al. (2000) J. Biol. Chem. 275, 13683], we also identified proteolytic cleavage sites within M1. Taken together, these studies support a topological model where the "historical" M1 segment cannot be entirely alpha-helical and may contain an extramembranous surface loop. Furthermore, we have also identified a tyrosine modification (Tyr 161) within a region of the N-terminal domain critical in agonist and antagonist binding.

Amino Acid Sequence↗
PubMed2005

Time course and site(s) of cytochrome c tyrosine nitration by peroxynitrite.

Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.

Amino Acid Sequence↗
PubMed1997

Analysis of hydroperoxide-induced tyrosyl radicals and lipoxygenase activity in aspirin-treated human prostaglandin H synthase-2.

A hydroperoxide-induced tyrosyl radical has been proposed as a key cyclooxygenase intermediate for the "basal" isoform of prostaglandin H synthase (PGHS-1). In the present study with the "inducible" isoform (PGHS-2), hydroperoxide was also found to generate a radical in high yield, a wide singlet at g = 2.0058 (29 G peak to trough). Reaction of PGHS-2 with a tyrosine-modifying reagent, tetranitromethane (TNM), resulted in cyclooxygenase inactivation and a much narrower radical EPR signal (22 G peak to trough). Addition of a cyclooxygenase inhibitor, nimesulide, similarly resulted in a narrow PGHS-2 radical. In PGHS-1, cyclooxygenase inhibition by tyrosine nitration with TNM or by active site ligands leads to generation of a narrow EPR instead of a wide EPR, with both signals originating from authentic tyrosyl radicals, indicating that the hydroperoxide-induced radicals in PGHS-2 are also tyrosyl radicals. Treatment of PGHS-2 with aspirin (acetyl salicylic acid, ASA) was previously shown to result in acetylation of a specific serine residue, cyclooxygenase inhibition, and increased lipoxygenase activity. Acetylation of PGHS-1 by ASA, in contrast, inhibited both lipoxygenase and cyclooxygenase activity. We now have found the ASA-treated PGHS-2 radical to be indistinguishable from that in control PGHS-2. Addition of nimesulide to ASA-treated PGHS-2 inhibited the lipoxygenase and resulted in a narrow radical EPR like that seen in PGHS-2 treated with TNM or nimesulide alone. Retention of PGHS-2 oxygenase activity was thus associated with retention of the native radical, and loss of activity was associated with alteration of the radical. Both native and ASA-treated PGHS-2 produced only the R stereoisomer of 11- and 15-HETE, demonstrating that the lipoxygenase stereochemistry was not changed by ASA. Native and ASA-treated PGHS-2 had lipoxygenase K(m) values considerably higher than that of the control PGHS-2 cyclooxygenase. Taken together, these results suggest that the same PGHS-2 tyrosyl radical serves as the oxidant for both cyclooxygenase and lipoxygenase catalysis and that acetylation of PGHS-2 by ASA favors arachidonate binding in an altered conformation which results in abstraction of the pro-R hydrogen from C13 and formation of 11(R)- and 15(R)-HETE.

Arachidonic Acid↗
PubMed2000

Critical role of tyrosine 277 in the ligand-binding and transactivating properties of retinoic acid receptor alpha.

Retinoic acid receptors specifically bind all-trans-retinoic acid (RA) and function as RA-inducible transcriptional regulatory factors. Binding of RA to RARalpha, beta, and gamma is sensitive to nitration with tetranitromethane, a tyrosine-specific modifying reagent. To identify tyrosine residue(s) that are important for RA binding, we carried out chemical modification experiments with purified RARalpha ligand-binding domain (RARalpha-LBD) subjected to partial acid hydrolysis and selective proteolysis. The chemically modified peptides containing each of the three Tyr residues present in the RARalpha-LBD sequence were then analyzed and identified by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC/ESI-MS). We found that RA binding to RARalpha-LBD protected Tyr(277)-containing peptides from nitration. Protection of Tyr(277) could result either from direct masking by the bound ligand or from ligand-induced changes in receptor conformation and tyrosine accessibility. The role of Tyr residues was further documented by site directed mutagenesis using three site-specific RARalpha mutants: Y208A, Y277A, and Y362A. The affinity for RA of these mutant receptors was in the range of that of the wild-type protein, except for the Y277A receptor mutant, which displays a 15-20-fold reduction in affinity and transactivation activity for RA. Whereas mutation of Tyr(277) into alanine had a variable effect on different agonists and antagonists binding, it caused a dramatic decrease of retinoid-dependent transactivation activity. This later effect was also observed with mutation of Tyr(277) into phenylalanine. It is unlikely that major conformational changes are responsible for the lower affinity of RA binding and RA-dependent transactivation since these mutants displayed wild-type dimerization and DNA-binding activities. Limited proteolysis revealed that upon ligand binding, the Y277A mutant induced a conformational change slightly different from that obtained with the wild-type protein. These data could suggest that Tyr(277) play a critical role in the ligand-induced conformational changes required for the activation of RARalpha.

Alanine↗
PubMed1999

Chemical modification of xylanase from Trichosporon cutaneum shows the presence of carboxyl groups and cysteine residues essential for enzyme activity.

The endo-beta-1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine.

Amino Acids↗
PubMed1998

Active site of brain Zn2+-glycerophosphocholine cholinephosphodiesterase and regulation of enzyme activity.

Properties of active site of Zn2+-glycerophosphocholine cholinephosphodiesterase from ox brain were examined using substrates and inhibitors of the phosphodiesterase. The anionic binding site expressed a selectivity for a positively-charged group. Meanwhile, the glyceryl moiety-binding site appeared to be a narrow crevice of a limited size, excluding the entry of acylglycerophospholipids containing long acyl chains. While endogenous quaternary ammonium compounds such as phosphocholine, choline or carnitine inhibited the enzyme, divalent metal ions such as Co2+, Mn2+ or Zn2+ enhanced the activity by 1.5 to 2-folds. The pH dependence for the inhibition by phosphocholine or the hydrolysis of substrate implies the involvement of a basic amino acid residue with a pK value of 9.6-9.7, probably lysine, in the binding of phosphoryl group. In further support, the lysine modifiers such as trinitrobenzene sulfonic acid or diethylpyrocarbonate expressed some inactivation. The pH-rate profile indicates that an amino acid residue with a pK value of 10.2, presumably tyrosine, may participate as a nucleophile in the catalysis. This might be further supported by the inactivation of the enzyme by tyrosine modifiers such as tetranitromethane or HOI-generating system. Separately, the phosphodiesterase was observed to be susceptible to the action of hydrogen peroxide or peroxynitrite-generating system. From these results, it is implied that the phosphodiesterase may be affected by endogenous sources.

Allosteric Regulation↗
PubMed2000

Differential contribution of the conserved tyrosine residues to activity and structural stability of Ophiophagus hannah alpha-neurotoxins.

Two alpha-neurotoxins, Oh-4 and Oh-7, from king cobra (Ophiophagus hannah) venom were subjected to Tyr modification with tetranitromethane. Selective nitration of Tyr4 in Oh-4 caused a slight decrease in lethal toxicity of 11% and a decrease in nicotinic acetylcholine receptor (nAchR)-binding activity of 28%, whereas nitration of Tyr4 in Oh-7 resulted in an approximately equal 60% decrease in lethality and nAchR-binding activity. When the Tyr23 in Oh-4 or Tyr22 in Oh-7 appears to be 'buried' in the toxin following further modification, the toxins lost their biological activity and conformational change concurrently. Nevertheless, the dinitrated Oh-4 retained a beta-sheet structure as revealed by CD spectra and exhibited a precipitin reaction with anti-Oh-4 sera. These results indicate that both Tyr4 and Tyr22 play a crucial role in the neurotoxicity of Oh-7, whereas intact Tyr23 is involved in the manifestation of the toxicity of Oh-4 to a greater extent. In contrast to Oh-4, the conformational stability of Oh-7 depends heavily upon the integrity of Tyr22.

Circular Dichroism↗
PubMed1980

Differential effects of nitrated ricin and nitrated and dithionite-reduced ricin on protein-synthesis inhibition and transmembrane tramsport in eukaryotic cells.

Ricin, was nitrated with tetranitromethane and reduced with sodium dithionite. Of the 8.0 nitro groups incorporated, 3.2 were on the A chain and 5.1 were on the B chain. Nitrated ricin1 was somewhat less active than nitrated and reduced ricin1 in inhibiting protein synthesis in vitro, but both were highly inhibitory. However, the modified toxins were less than 1% as active as ricin in inhibiting protein synthesis in cultured cells. Indirect immunofluorescence assays demonstrated tha both modified toxins were specifically bound to the cell surface and could be displaced by galactose.

Animals↗
PubMed1982

Does superoxide anion participate in 2-oxoglutarate-dependent hydroxylation?

The possible role of superoxide anion in 2-oxoglutarate-coupled dioxygenase reactions has been investigated. gamma-Butyrobetaine hydroxylase (EC 1.14.11.1) was inhibited by human erythrocyte superoxide dismutase (EC 1.15.1.1), probably due to release of Cu(2+) or Zn(2+), as the inhibition was more pronounced after heat-inactivation of the dismutase and as Cu(2+) was a potent inhibitor. Bovine superoxide dismutase and the Mn(2+)-containing superoxide dismutase from Escherichia coli were not inhibitory. Superoxide anion generated from xanthine/xanthine oxidase was not stimulatory and could not replace ascorbate. Thymine 7-hydroxylase (EC 1.14.11.6) and thymidine 2'-hydroxylase (EC 1.14.11.3) were not inhibited by erythrocyte superoxide dismutase or stimulated by superoxide anion. gamma-Butyrobetaine hydroxylase was inhibited by a number of low-molecular-weight compounds, such as tetranitromethane, Nitro Blue Tetrazolium, adrenaline and Tiron, which may act as scavengers of superoxide anion. Involvement of this radical in other oxygenase reactions has been inferred from the findings that they were inhibitory for the respective enzymes. Several of these compounds also inhibited gamma-butyrobetaine hydroxylase. It could be concluded from these experiments, however, that mechanisms other than disposal of superoxide anion might equally well be operative, such as hydrophobic interaction with the enzyme protein and interaction with compounds required for full enzymic activity, e.g. iron and ascorbate. The results appear to rule out a requirement for superoxide anion generated in free solution, and have not yielded evidence for participation of enzyme-bound superoxide anion in 2-oxoglutarate-dependent hydroxylations.

Copper↗
PubMed1986

Oxidation of reduced Cu,Zn superoxide dismutase by molecular oxygen. A kinetic study.

The rate of oxidation of reduced bovine Cu,Zn superoxide dismutase [ECu(I)] by molecular O2 was studied by magnetic-resonance techniques and was found to be low under physiological conditions. The analysis of the kinetic data and of the experiments carried out in the presence of tetranitromethane confirms that O2.- is a product of the oxidation process. At [ECu(I)]/([ECu(I)] + [ECu(II)]) greater than 0.5 the O2.- produced reacts mainly with ECu(I), increasing the oxidation rate of the enzyme, whereas at [ECu(I)]/([ECu(I)] + [ECu(II)]) less than 0.5 it reacts mainly with ECu(II), decreasing the oxidation rate, the kinetics, at constant O2 concentration, being an apparent second-order process. The oxidation rate increased linearly with both O2 and OH- concentration, indicating that only a deprotonated form of the ECu(I) reacts with O2.-.

Hydrogen-Ion Concentration↗
PubMed1999

Peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Coincident nitration of enzyme tyrosyl residues has minimal impact on catalytic activity.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.

Catalysis↗
PubMed2002

Evidence that light modulates protein nitration in rat retina.

As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4-33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology.

Albumins↗
PubMedDate not supplied

Predominant K-ras codon 12 G --> A transition in chemically induced lung neoplasms in B6C3F1 mice.

Based on long-term toxicity and carcinogenicity studies in B6C3F1 mice conducted by the National Toxicology Program, 2,2-Bis(bromomethyl)-1,3-propanediol (BMP) and tetranitromethane (TNM) have been identified as carcinogens. Following 2 yr of exposure to 312, 625, or 1,250 ppm BMP in feed, or exposure to 0.5 or 2 ppm TNM by inhalation, increased incidences of lung neoplasms were observed in B6C3F1 mice at all exposure concentrations compared to unexposed mice. The present study characterizes genetic alterations in the K-ras protooncogene in BMP- and TNM-induced lung neoplasms, respectively, and compares the findings to spontaneous lung neoplasms from corresponding control mice. The frequencies of the K-ras mutations were 57% (29/51) in BMP-induced lung neoplasms compared to 15% (3/20) in lung neoplasms from dosed feed control mice, and 54% (14/26) in TNM-induced lung neoplasms compared to 60% (3/5) in lung neoplasms from inhalation control mice. G --> A transitions at the second base of the K-ras codon 12 (GGT --> GAT) were the most frequent pattern of K-ras mutations identified in BMP-induced (20/29) and TNM-induced lung neoplasms (13/14), which differed from the mutational patterns identified in the lung neoplasms from unexposed control mice. These results indicate that mutations in the K-ras gene are involved in B6C3F1 lung carcinogenesis following BMP- and TNM-exposure, and the high frequency and specificity of the ras mutation profile in lung neoplasms (G --> A transition) may be due to in vivo genotoxicity by the parent compounds or their metabolites.

Animals↗
PubMed1984

Site of OH radical attack on dihydrouracil and some of its methyl derivatives.

The site of attack of OH radicals on dihydrouracil and five of its methylated derivatives was determined by pulse radiolysis using N,N,N',N'-tetramethylphenylenediamine (TMPD) to detect oxidizing radicals and tetranitromethane (TNM) as well as K3Fe(CN)6 to detect reducing radicals. In the case of dihydrouracil OH radicals abstract preferentially an H atom at C(6) to give the 6-yl radical (greater than or equal to 90 per cent) which at pH approximately 6.5 reduces TNM and K3Fe(CN)6 at almost diffusion-controlled rates. Only a small fraction of OH radicals abstract the H atom at C(5) (less than or equal to 10 per cent). The resulting 5-yl radical oxidizes TMPD to TMPD+ at pH 7-8. With the methylated derivatives of dihydrouracil, OH radicals react less selectively, especially in the case of N(1)-methyl derivatives. This methyl group is activated to a similar degree as the methylene group at C(6). In 1-Medihydrouracil the yield of N(1)-CH2 radicals is about 29 per cent, which has been deduced from the yield of formaldehyde formed after oxidation of this radical by TNM at pH approximately 6.5 and the subsequent hydrolysis. Radicals at the other methyl substituents are generated to a lesser extent (less than or equal to 10 per cent) and are relatively unreactive towards oxidizing agents such as TNM and K3Fe(CN)6 as well as towards the reducing agent TMPD. Although methyl substitution opens new routes for OH attack the preferred site of H abstraction remains C(6) (greater than 60 per cent).

Ferricyanides↗
PubMed1984

Gamma-radiolysis of poly(U) in aqueous solution. The role of primary sugar and base radicals in the release of undamaged uracil.

p6e release of undamaged uracil has been measured after gamma-irradiation of aqueous solutions of poly(U). By varying the experimental conditions, the respective efficiencies with which OH, H and e-aq release uracil were found to be 51, 28 and about 3 per cent. From the determination of G(H2) in acid solutions it is concluded that greater than or equal to 95 per cent of the H atoms add to the base moiety. It is proposed that the H-adduct radicals (and similarly the OH-adduct radicals) react with the sugar moiety of a different nucleotide. Subsequent reactions of the sugar radicals so formed lead to the release of undamaged uracil. In N2O-saturated solutions the addition of tetranitromethane (TNM) causes a sharp drop in released uracil from G = 2.9 to G = 0.19. The strong suppression of uracil release by TNM is presumably due to its scavenging of the major base radical with the remaining G(uracil release) being due to a direct attack of OH at the sugar moiety. In contrast, G(uracil release) from 5'-UMP gamma-irradiated under N2O is only 0.39, dropping to 0.31 in the presence of TNM. This indicates the important role of the polymeric structure in the mechanism leading to efficient base release. These results on base release are compared with reported data on strand breakage.

Cobalt Radioisotopes↗