Tandem mass spectrometry and infrared spectroscopy as a tool to identify peptide oxidized residues.

The final products obtained by the oxidation of small model peptides containing the thioether function, either methionine or S-methyl cysteine, have been characterized by tandem mass spectrometry and IR Multiple Photon Dissociation (IRMPD) spectroscopy. The modified positions have been clearly identified by the CID-MS(2) fragmentation mass spectra with or without loss of sulfenic acid, as well as by the vibrational signature of the sulfoxide bond at around 1000 cm(-1). The oxidation of the thioether function did not lead to the same products in these model peptides. The sulfoxide and sulfone (to a lesser extent) have been clearly identified as final products of the oxidation of S-methyl-glutathione (GS-Me). Decarboxylation or hydrogen loss are the major oxidation pathways in GS-Me, while they have not been observed in tryptophan-methionine and methionine-tryptophan (Trp-Met and Met-Trp). Interestingly, tryptophan is oxidized in the dipeptide Met-Trp, while that is not the case in the reverse sequence (Trp-Met).

Exploring oxidative modifications of tyrosine: an update on mechanisms of formation, advances in analysis and biological consequences.

Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidised derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.

One-electron oxidation and reduction of glycosaminoglycan chloramides: a kinetic study.

Hypochlorous acid and its acid-base counterpart, hypochlorite ions, produced under inflammatory conditions, may produce chloramides of glycosaminoglycans, these being significant components of the extracellular matrix (ECM). This may occur through the binding of myeloperoxidase directly to the glycosaminoglycans. The N-Cl group in the chloramides is a potential selective target for both reducing and oxidizing radicals, leading possibly to more efficient and damaging fragmentation of these biopolymers relative to the parent glycosaminoglycans. In this study, the fast reaction techniques of pulse radiolysis and nanosecond laser flash photolysis have been used to generate both oxidizing and reducing radicals to react with the chloramides of hyaluronan (HACl) and heparin (HepCl). The strong reducing formate radicals and hydrated electrons were found to react rapidly with both HACl and HepCl with rate constants of 1-1.7 × 10(8) and 0.7-1.2 × 10(8)M(-1)s(-1) for formate radicals and 2.2 × 10(9) and 7.2 × 10(8)M(-1)s(-1) for hydrated electrons, respectively. The spectral characteristics of the products of these reactions were identical and were consistent with initial attack at the N-Cl groups, followed by elimination of chloride ions to produce nitrogen-centered radicals, which rearrange subsequently and rapidly to produce C-2 radicals on the glucosamine moiety, supporting an earlier EPR study by M.D. Rees et al. (J. Am. Chem. Soc.125: 13719-13733; 2003). The oxidizing hydroxyl radicals also reacted rapidly with HACl and HepCl with rate constants of 2.2 × 10(8) and 1.6 × 10(8)M(-1)s(-1), with no evidence from these data for any degree of selective attack on the N-Cl group relative to the N-H groups and other sites of attack. The carbonate anion radicals were much slower with HACl and HepCl than hydroxyl radicals (1.0 × 10(5) and 8.0 × 10(4)M(-1)s(-1), respectively) but significantly faster than with the parent molecules (3.5 × 10(4) and 5.0 × 10(4)M(-1)s(-1), respectively). These findings suggest that these potential in vivo radicals may react in a site-specific manner with the N-Cl group in the glycosaminoglycan chloramides of the ECM, possibly to produce more efficient fragmentation. This is the first study therefore to conclusively demonstrate that reducing radicals react rapidly with glycosaminoglycan chloramides in a site-specific attack at the N-Cl group, probably to produce a 100% efficient biopolymer fragmentation process. Although less reactive, carbonate radicals, which may be produced in vivo via reactions of peroxynitrite with serum levels of carbon dioxide, also appear to react in a highly site-specific manner at the N-Cl group. It is not yet known if such site-specific attacks by this important in vivo species lead to a more efficient fragmentation of the biopolymers than would be expected for attack by the stronger oxidizing species, the hydroxyl radical. It is clear, however, that the N-Cl group formed under inflammatory conditions in the extracellular matrix does present a more likely target for both reactive oxygen species and reducing species than the N-H groups in the parent glycosaminoglycans.

trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components.

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cytb(558)) and four cytosolic proteins p47(phox), p67(phox), Rac and p40(phox). The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural cis-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural cis geometry to the trans configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono-trans AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the cis-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono-trans AA which targets both the membrane fraction containing the cytb(558) and the cytosolic p67(phox). Our results suggest that the loss of the natural geometric feature (cis-AA) induces substantial structural modifications of p67(phox) that prevent its translocation to the complex.

Antiplasmodial activity of nitroaromatic and quinoidal compounds: redox potential vs. inhibition of erythrocyte glutathione reductase.

Prooxidant nitroaromatic and quinoidal compounds possess antimalarial activity, which might be attributed either to their formation of reactive oxygen species or to their inhibition of antioxidant enzyme glutathione reductase (GR, EC 1.6.4.2). We have examined the activity in vitro against Plasmodium falciparum of 24 prooxidant compounds of different structure (nitrobenzenes, nitrofurans, quinones, 1,1'-dibenzyl-4,4'-bipyridinium, and methylene blue), which possess a broad range of single-electron reduction potentials (E(1)(7)) and erythrocyte glutathione reductase inhibition constants (K(i(GR))). For a series of homologous derivatives of 2-(5'-nitrofurylvinyl)quinoline-4-carbonic acid, the relationship between compound K(i(GR)) and concentration causing 50% parasite growth inhibition (IC(50)) was absent. For all the compounds examined in this study, the dependence of IC(50) on their K(i(GR)) was insignificant. In contrast, IC(50) decreased with an increase in E(1)(7) and positive electrostatic charge of aromatic part of molecule (Z): log IC(50) (microM) = -(0.9846 +/- 0.3525) - (7.2850 +/- 1.2340) E(1)(7) (V) - (1.1034 +/- 0.1832) Z (r(2) = 0.8015). The redox cycling activity of nitroaromatic and quinoidal compounds in ferredoxin:NADP(+) reductase-catalyzed reaction and the rate of oxyhemoglobin oxidation in lysed erythrocytes increased with an increase in their E(1)(7) value. Our findings imply that the antiplasmodial activity of nitroaromatic and quinoidal compounds is mainly influenced by their ability to form reactive oxygen species, and much less significantly by the GR inhibition.

Pulse radiolysis studies on superoxide reductase from Treponema pallidum.

Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2*- to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi-molecular reaction of the ferrous center with O2, with a rate constant of 6 x 10 (8) M(-1) s(-1). A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s(-1). This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron-peroxide species.

Superoxide reductase from Desulfoarculus baarsii: reaction mechanism and role of glutamate 47 and lysine 48 in catalysis.

Superoxide reductase (SOR) is a small metalloenzyme that catalyzes reduction of O(2)(*)(-) to H(2)O(2) and thus provides an antioxidant mechanism against superoxide radicals. Its active site contains an unusual mononuclear ferrous center, which is very efficient during electron transfer to O(2)(*)(-) [Lombard, M., Fontecave, M., Touati, D., and Nivière, V. (2000) J. Biol. Chem. 275, 115-121]. The reaction of the enzyme from Desulfoarculus baarsii with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bimolecular reaction of superoxide reductase with superoxide, with a rate constant of (1.1 +/- 0.3) x 10(9) M(-1) s(-1). A first intermediate is formed which is converted to a second one at a much slower rate constant of 500 +/- 50 s(-1). Decay of the second intermediate occurs with a rate constant of 25 +/- 5 s(-1). These intermediates are suggested to be iron-superoxide and iron-peroxide species. Furthermore, the role of glutamate 47 and lysine 48, which are the closest charged residues to the vacant sixth iron coordination site, has been investigated by site-directed mutagenesis. Mutation of glutamate 47 into alanine has no effect on the rates of the reaction. On the contrary, mutation of lysine 48 into an isoleucine led to a 20-30-fold decrease of the rate constant of the bimolecular reaction, suggesting that lysine 48 plays an important role during guiding and binding of superoxide to the iron center II. In addition, we report that expression of the lysine 48 sor mutant gene hardly restored to a superoxide dismutase-deficient Escherichia coli mutant the ability to grow under aerobic conditions.

Gamma radiation effects on alpha-lactalbumin: structural modifications.

Alpha-lactalbumin was irradiated in the lyophilized state in air at ambient temperature. The irradiated protein was examined by size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, circular dichroism, and microcalorimetry. Irradiation induced the loss of aromatic amino acids and of helicity so that fragmentation and aggregation products were obtained. The thermodynamic properties of the protein were also modified. The irradiated protein had lower stability, however, the temperature at which denaturation occurred process remained constant.

Gamma radiation effects on potassium pertechnetate in carbonate media.

Gamma radiation effects on the stability of the oxidation state of radioelements and diffusion within the framework of nuclear waste repositories are often neglected, although it may influence physicochemical processes and thus transfer to biosphere and geosphere. This knowledge is essential to model transport phenomena. This study reports on effects of gamma irradiation on technetium in carbonate media, which is representative of natural systems. Depending on media compositions, the gamma irradiation on pertechnetate leads to formation of Tc(IV) only in the absence of carbonate. CO3*- radicals are able to re-oxidise technetium intermediate oxidation states to the +7 state.

Redox properties of protein disulfide bond in oxidized thioredoxin and lysozyme: a pulse radiolysis study.

We have studied the one-electron reduction of oxidized Chlamydomonas reinhardtii thioredoxin and compared it to that of hen egg white lysozyme, using CO(2)(*) (-) free radicals as reductants. This comparison shows that the thioredoxin disulfide/thiol redox couple has different properties than that of lysozyme: the disulfide radical pK(a) is much lower (around 5 for small disulfides, 4.62 for lysozyme, <3 for thioredoxin). To get a better understanding of the modulation of the thioredoxin redox properties we have constructed the mutants W35A and D30A. Their reduction by pulse radiolysis indicates that W35 strongly controls both the disulfide radical acidity (the pK(a) in W35A is equal to ca. 4), and the thiol reactivity. Asp30 is also involved in the control of proton transfer to the disulfide free radical. In addition, its removal seems to increase the reduction potential of the thioredoxin thiyl/thiol couple. Overall, the reduction properties of thioredoxin confirm its nature as a unique reductant.

Protein gamma-radiolysis in frozen solutions is a macromolecular surface phenomenon: fragmentation of lysozyme, citrate synthase and alpha-lactalbumin in native or denatured states.

PURPOSE: To test whether radiolysis-induced fragmentation in frozen aqueous protein solution is dependent on solvent access to the surface of the protein or to the molecular mass of the polypeptide chain. MATERIALS AND METHODS: 60Co gamma-irradiation of three proteins at -78 degrees C: lysozyme, citrate synthase and alpha-lactalbumin in their native state, with or without bound substrate, or denatured (random coil in urea/acid-denatured state). RESULTS: By SDS-polyacrylamide gel electrophoresis/analysis of the protein-fragmentation process, it was found that for a given protein D37 values (dose to decrease the measured amount of protein, with an unaltered polypeptidic chain, to 37% of the initial amount) varied according to the state of the protein. D37 for denatured proteins was always much smaller than for native states, indicating a greater susceptibility to fragmentation. In urea, contrary to the native state, no well-defined fragments were observed. Radiolysis decay constants (K= 1/D37) increased with solvent-accessible surface area of these proteins estimated from their radii of gyration in the various states. This is shown also in previous data on native or SDS-denatured proteins. Denatured proteins which have a large surface area exposed to the solvent compared with native ones are more fragmented at equal doses. CONCLUSIONS: It is concluded that D37 is directly related to the exposed surface area and not to the molecular mass of the polypeptide chain.

Oxidative dimerization of proteins: role of tyrosine accessibility.

PURPOSE: To investigate the importance of two possible mechanisms of tyrosine oxidation on the yield of protein dimerization. The model chosen is hen and turkey egg-white lysozymes, which differ by seven amino acids, among which one tyrosine is in the 3 position. MATERIALS AND METHODS: Aqueous solutions of proteins were oxidized by OH(*) or N(*)(3) free radicals produced by gamma or pulse irradiation in an atmosphere of N(2)O. Protein dimers were quantified by SDS-PAGE and reverse-phase HPLC. Dityrosines were identified by absorption and fluorescence. RESULTS: Using N(*)(3) free radicals, the initial yields of dimerization are equal to (8.6 +/- 0.7) x 10(-9) mol J(-1) for both proteins. Using OH(*) free radicals, they become equal to (1.23 +/- 0.1) x 10(-8) and (4.42 +/- 0.1) x 10(-8) mol J(-1) for hen and turkey egg-white lysozymes, respectively (gamma radiolysis). DISCUSSION. N(*)(3) radicals react primarily with tryptophan residues only. Tyrosine gets oxidized by intramolecular long-range electron migration, whereas OH(*) may react directly with tyrosines. We propose a low participation of Tyr3 in turkey protein in the intramolecular process, because Tyr3 is far from all tryptophans. On the other hand, Tyr3 is very accessible to solvent and in a flexible area; thus collisions with OH(*) could easily be followed by intermolecular dimerization.

Oxidation, nitrosation, and nitration of serotonin by nitric oxide-derived nitrogen oxides: biological implications in the rat vascular system.

Because NO is not very reactive in an oxygen-free buffer, a significant part of serotonin (5-HT) is transformed by NO in nondeaerated phosphate buffer, at pH 7.4, into (4-serotonyl)-4-serotonin, 4-nitrososerotonin, and 4-nitroserotonin. Dimerization and above all nitrosation occur through the HNO2 reaction in the pH 4-6 range, possibly via radical mechanism involving N2O3. 5-HT is readily a substrate for nitrosation by HNO2 or N2O3, whereas tyrosine was described as not very reactive under the same conditions. Peroxynitrite converts 5-HT to the (4-serotonyl)-4-serotonin and to the 4-nitro derivative. In order to evaluate whether such structural modifications could modulate the biological properties of 5-HT, arterial pressure was measured after i.v. bolus injection of these derivatives to anesthetized rats. Injections of the 4-nitroso- and 4-nitro-5-HT resulted in first a brief hypotensive response and did not give the subsequent hypertensive and hypotensive phases observed with 5-HT. Finally, when tested on some cloned rat 5-HT receptors stably transfected into LMTK- cells, both 4-nitroso and 4-nitro derivatives behaved as agonists and antagonists toward 5-HT1B and 5-HT2B receptors, respectively.

Inactivation of Cu,Zn-superoxide dismutase by free radicals derived from ethanol metabolism: a gamma radiolysis study.

The reactions of free radicals derived from ethanol metabolism with Cu,Zn SOD were studied. 1-Hydroxyethyl radicals were generated by gamma radiolysis of a N2O-saturated ethanolic solution (10(-2) M) in phosphate buffer (10(-3) M, pH 7.4). To generate acetyl radicals by gamma radiolysis, we used ethylene glycol (10(-2) M) in phosphate buffer (10(-3) M, pH 7.4). This allows us to avoid the use of acetaldehyde, which may be toxic toward various cellular constituents. We have previously reported that HO. radicals reacting with either acetaldehyde or ethylene glycol produce the same free radicals (Santiard et al., 1991, J. Chim. Phys. 88, 967-976). the rate constant reaction of 1-hydroxyethyl free radicals with Cu,Zn-SOD was measured separately by competition kinetics with the spin trapping agent alpha-(4-pyridyl 1-oxide) N-terbutylnitrone (4-POBN), after having measured the rate constant of scavenging of 1-hydroxyethyl free radicals by 4-POBN in the absence of SOD. We found k1 (4-POBN + 1-hydroxyethyl radical) = 4.2 10(5) M-1 s-1 and kR (SOD + 1-hydroxyethyl radical) = 6.8 10(5) M-1 s-1). 1-Hydroxyethyl or acetyl radicals produced dose-dependent Cu,Zn-SOD inactivation. The inactivation rate constant of Cu,Zn-SOD by 1-hydroxyethyl radicals is ki = 1.13 10(4) M-1 s-1. Free radicals derived from ethanol metabolism can thus react SOD leading to enzyme inactivation, besides the fact that the reactivities of 1-hydroxyethyl radicals with 4-POBN and with proteins such as Cu,Zn SOD are of the same order of magnitude could explain the difficulties to trap in vivo these radicals.

Antioxidant action of sodium diethyldithiocarbamate: reaction with hydrogen peroxide and superoxide radical.

The oxidation of sodium diethyldithiocarbamate (DDC) by hydrogen peroxide or superoxide radicals has been investigated. Hydrogen peroxide oxidizes DDC, leading to the formation of a hydrated form of disulfiram, a dimer of DDC having a disulfide group. In equimolar conditions, the overall process appears as a first-order reaction (k = 0.025 +/- 0.005 s-1), the first step being a second-order reaction (k = 5.0 +/- 0.1 mol-1.1.s-1). No radical intermediate was observed in this process. In the presence of an excess of any of the reagents, the hydrated form of disulfiram transforms into different products corresponding to the fixation of oxygen by sulfur atoms or replacement of C = S group by ketone function, in the presence of an excess of hydrogen peroxide. Superoxide anions (produced by steady-state 60Co gamma-radiolysis) oxidize DDC, yielding similar products to those obtained with hydrogen peroxide with a maximum oxidation G-value of 0.3 mumol.J-1. The rate constant k(O2.- + DDC) is equal to 900 mol-1.1.s-1.

Intramolecular semiquinone disproportionation in DNA. Pulse radiolysis study of the one-electron reduction of daunorubicin intercalated in DNA.

The one-electron reduction of daunorubicin, a quinonic antitumor antibiotic, intercalated in DNA was studied by pulse radiolysis using carboxyl radicals as reductants. The reaction's first stage is the daunorubicin semiquinone formation (k = 1.9 x 10(8) mol-1.dm3.s-1) in a way entirely consistent with a simple competition between .COO- disproportionation and the drug reduction. The semiquinone drug disappears by a first-order reaction (k = 1340 s-1) producing the hydroquinone form. This reaction leads to an equilibrium similar to the one without DNA and the equilibrium constant is very close to its value free in water (Kc approximately 25). In addition, the stoichiometry of the first-order reaction is the one of a dismutation process. Therefore, it appears that the disproportionation occurs along an intramolecular path across DNA. This migration takes place under our experimental conditions, over a distance of ca. 100 base pairs, with a mobility of ca. 4.4 X 10(-11) m2.V-1.s-1, similar in magnitude to an excess electron mobility in doped organic polymers.

Reduction of daunorubicin in the presence of sulfur-containing peptides.

Daunorubicin, an anthracycline antitumor antibiotic, was reduced in the presence of reduced (GSH) or oxidized (GSSG) glutathione to evaluate the possibilities of detoxification or of potentiation of the drug by these compounds. The reductants were .COO- free radicals produced by gamma radiolysis. In both cases, the final product is 7-deoxydaunomycinone, i.e., the same as without glutathione. The reduction yield is also the same as without GSH or GSSG (0.23 mumol.J-1). No glutathione depletion was observed. Limits for the rate constants of some possible nonenzymatic detoxification reactions are given. To evaluate the possible interactions of daunorubicin with sulfur-containing proteins, the reduction of this drug by .COO- free radicals was also studied in the presence of a polypeptide containing two disulfide bridges, aponeocarzinostatine. The final product is also 7-deoxydaunomycinone. The yields of reduction of the drug and of a protein disulfide bridge are, respectively, 0.23 mumol.J-1 and less than or equal to 6 nmol.J-1. These values indicate that disulfide radical anions of the protein can reduce the drug, giving back the disulfide bridge, but that the drug transients neither oxidize nor reduce the protein.

CO2.- radical induced cleavage of disulfide bonds in proteins. A gamma-ray and pulse radiolysis mechanistic investigation.

Disulfide bond reduction by the CO2.- radical was investigated in aponeocarzinostatin, aporiboflavin-binding protein, and bovine immunoglobulin. Protein-bound cysteine free thiols were formed under gamma-ray irradiation in the course of a pH-dependent and protein concentration dependent chain reaction. The chain efficiency increased upon acidification of the medium, with an apparent pKa around 5, and decreased abruptly below pH 3.6. It decreased also at neutral pH as cysteine accumulated. From pulse radiolysis analysis, CO2.- proved able to induce rapid one-electron oxidation of thiols and of tyrosine phenolic groups in addition to one-electron donation to exposed disulfide bonds. The bulk rate constant of CO2.- uptake by the native proteins was 5- to 10-fold faster at pH 3 than at pH 8, and the protonated form of the disulfide radical anion, [symbol: see text], appeared to be the major protein radical species formed under acidic conditions. The main decay path of [symbol: see text] consisted of the rapid formation of a thiyl radical intermediate [symbol: see text] in equilibrium with the closed, cyclic form. The thiyl radical was subsequently reduced to the sulfhydryl level [symbol: see text] on reaction with formate, generating 1 mol of the CO2.- radical, thus propagating the chain reaction. The disulfide radical anion [symbol: see text] at pH 8 decayed through competing intramolecular and/or intermolecular routes including disproportionation, protein-protein cross-linking, electron transfer with tyrosine residues, and reaction with sulfhydryl groups in prereduced systems. Disproportionation and cross-linking were observed with the riboflavin-binding protein solely. Formation of the disulfide radical cation [symbol: see text], phenoxyl radical Tyr-O. disproportionation, and phenoxyl radical induced oxidation of preformed thiol groups should also be taken into consideration to explain the fate of the oxygen-centered phenoxyl radical.

One-electron reduction of daunorubicin intercalated in DNA or in a protein: a gamma radiolysis study.

The one-electron reduction of daunorubicin, an anthracycline antibiotic, intercalated in DNA or in the apoprotein of the riboflavin binding protein, was studied by gamma radiolysis. The two reduction mechanisms appear very similar to the one found for the non-intercalated drug. Hydrogen peroxide, which oxidizes non-intercalated hydroquinone daunorubicin with two electrons in one step (C. Houée-Levin, M. Gardès-Albert and C. Ferradin, FEBS lett., 173, 27-30, (1984], reacts with daunorubicin hydroquinone in DNA but not in the protein. It appears thus that the site accessibility to hydrogen peroxide in DNA is better than in the protein. Biological consequences are discussed.

One-electron reduction of daunorubicin intercalated in DNA or in a protein: a gamma radiolysis study.

The one-electron reduction of daunorubicin, an anthracycline antibiotic, intercalated in DNA or in the apoprotein of the riboflavin binding protein, was studied by gamma radiolysis. The two reduction mechanisms appear very similar to the one found for the non-intercalated drug. Hydrogen peroxide, which oxidizes non-intercalated hydroquinone daunorubicin with two electrons in one step (C. Houée-Levin, M. Gardès-Albert and C. Ferradin, FEBS lett., 173, 27-30, (1984)), reacts with daunorubicin hydroquinone in DNA but not in the protein. It appears thus that the site accessibility to hydrogen peroxide in DNA is better than in the protein. Biological consequences are discussed.