On the metabolic activation of the carcinogen N-hydroxy-N-2-acetylaminofluorene : III. Oxidation with horseradish peroxidase to yield 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene

Previous studies have shown that the carcinogen N-hydroxy-2-acetylaminofluorene is converted by one-electron oxidants to a free nitroxide radical which dismutates to N-acetoxy-2-acetylaminofluorene and 2-nitrosofluorene. The present study shows that the same oxidation can be achieved with horseradish peroxidase and H₂O₂. The free radical intermediate was detected by its ESR signal, and the yields of N-acetoxy-2-acetylaminofluorene and of 2-nitrosofluorene were determined under a number of conditions. Addition of tRNA to the reaction mixture containing N-acetoxy-N-2-acetyl[2′-³H]aminofluorene yielded tRNA-bound radioactivity; addition of guanosine yielded a reaction product which appears to be N-guanosin-8-yl)-2-acetylaminofluorene. The latter compound has previously been identified as a reaction product of N-acetoxy-2-acetylaminofluorene and guanosine. Preliminary attempts to demonstrate the formation of a nitroxide free radical or its dismutation products with rat liver mixed function oxidase systems were not successful.     

The Horseradish Peroxidase Catalysed Oxidation of Deoxyribose Sugars

The ability of horseradish peroxidase (E.C. 1.11.1.7. Donor: H₂O₂ oxidoreductase) to catalytically oxidize 2-deoxyribose sugars to a free radical species was investigated. The ESR spin-trapping technique was used to denionstrate that free radical species were formed. Results with the spin trap 3.5-dibronio-4-nitrosoben-zene sulphonic acid showed that horseradish peroxidase can catalyse the oxidation of 2-deoxyribose to produce an ESR spectrum characteristic of a nitroxide radical spectrum. This spectrum was shown to be a composite of spin adducts resulting from two carbon-centered species, one spin adduct being characterized by the hyperfine coupling constants a^N = 13.6Ganda^H_β = 11.0G, and the other by a^N = 13.4G and a^H = 5.8 G. When 2-deoxyribose-5-phosphate was used as the substrate, the spectrum produced was found to be primarily one species characterized by the hyperfine coupling constants a^N = 13.4G and a^H= 5.2. All the radical species produced were carbon-centered spin adducts with a β hydrogen, suggesting that oxidation occurred at the C(2) or C(5) moiety of the sugar. Interestingly, it was found that under the same experimental conditions, horseradish peroxidase apparently did not catalyze the oxidation of either 3-deoxyribose or D-ribose to a free radical since no spin adducts were found in these cases.

It can be readily seen that 2-deoxyribose and 2-deoxyribose-5-phosphate can be oxidized by HRP/H₂O₂ to form a free radical species that can be detected with the ESR spin-trapping technique. There are two probable sites for the formation of a CH type radical on the 2-deoxyribose sugar, these being the C(2) and the C(5) carbons. The fact that there is a species produced from 2-deoxy-ribose, but not 2-deoxy-ribose-5-phosphate, suggests that there is an involvement of the C(5) carbon in the species with the 1 1.0G β hydrogen. In the spectra formed from 2-deoxy-ribose, there is a big difference in the hyperfine splitting of the β hydrogens, suggesting that the radicals are formed at different carbon centers, while the addition of a phosphate group to the C(5) carbon seems to inhibit radical formation at one site. In related work, the chemiluminescence of monosaccharides in the presence of horseradish peroxidase was proposed to be the consequence of carbon-centered free radical formation (10).     

Author index to volume 154

Horseradish peroxidase-catalyzed N-demethylation of aminopyrine and dimethylaniline results in generation of free radical intermediates which can interact with glutathione (GSH) to form a glutathione radical. This can either dimerize to yield glutathione disulfide or react with O₂ to form oxygenated products of glutathione. Ethylmorphine is not a substrate in the peroxidase-mediated reaction, and free radical intermediates which react with GSH, are not formed from aminopyrine and dimethylaniline when the horseradish peroxidase/H₂O₂ system is replaced by liver microsomes and NADPH. Therefore, it appears unlikely that formation of free radical intermediates can be responsible for the depletion of GSH observed during N-demethylation of several drugs in isolated liver cells.     

Superoxide Dismutase Enhanced the Formation of Hydroxyl Radicals in a Reaction Mixture Containing Xanthone under UVA Irradiation

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 μM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O₂, H₂O₂, and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O₂ ^?·.     

Formation of the N,N′-Dialkylpyrazine Cation Radical from Glyoxal Dialkylimine Produced on Reaction of a Sugar with an Amine or Amino Acid

The formation of an intermediate product, which could easily give a radical product, in an early stage of the Maillard reaction was confirmed commonly occur in various sugar-amino compound systems, by detection of the N,N′-dialkylpyrazine cation radical generated on the addition of ascorbic acid (AsA) to the reaction mixtures. This intermediate was produced immediately after to glycosylamine formation, prior to Amadori rearrangement, and completely parallel to the formation of glyoxal dialkylimine, which was identified by TLC as a main component of the extract. Authentic glyoxal dialkylimine was shown to produce an identical radical on treatment with both reducing agents and acids instead of AsA. It was thus demonstrated that the intermediate is glyoxaldialkylimine and that acid hydrolysis followed by reduction is required for production of the free radical.     

Antioxidant properties of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK): scavenging of free radicals and prevention of protein destruction

Abstract

In numerous experimental systems, the neurohormone melatonin has been shown to protect against oxidative stress, an effect which appears to be the result of a combination of different actions. In this study, we have investigated the possible contribution to radical scavenging by substituted kynuramines formed from melatonin via pyrrole ring cleavage. N¹-Acetyl-5-methoxykynuramine (AMK), a metabolite deriving from melatonin by mechanisms involving free radicals, exhibits potent antioxidant properties exceeding those of its direct precursor N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and its analog N¹-acetylkynuramine (AK). Scavenging of hydroxyl radicals was demonstrated by competition with ABTS in a Fenton reaction system at pH 5 and by competition with DMSO in a hemin-catalyzed H₂O₂ system at pH 8. Under catalysis by hemin, oxidation of AMK was accompanied by the emission of chemiluminescence. AMK was a potent reductant of ABTS cation radicals, but, in the absence of catalysts, a poor scavenger of superoxide anions. In accordance with the latter observation, AMK was fairly stable in a pH 8 H₂O₂ system devoid of hemin. Contrary to AFMK, AMK was easily oxidized in a reaction mixture generating carbonate radicals. In an oxidative protein destruction assay based on peroxyl radical formation, AMK proved to be highly protective. No prooxidant properties of AMK were detected in a sensitive biological test system based on light emission by the bioluminescent dinoflagellate Lingulodinium polyedrum. AMK may contribute to the antioxidant properties of the indolic precursor melatonin.     

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Summary Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK _equil of 6 × 10^–17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.Abbreviations AFR ascorbate free radical - DHA dehydroascorbic acid - EDTA ethylenediaminetetraacetic acid - DTPA diethylenetri-aminepentaacetic acid - TEMPO 2,2,6,6-tetramethylpiperidinoxy     

Electron paramagnetic resonance study of the oxidation of N-methyl-substituted aromatic amines catalyzed by hemeproteins

The oxidation of N-mono- and dimethyl-substituted toluidines and aniline by H₂O₂, catalyzed by horseradish peroxidase or metmyoglobin, produces organic free radicals, detectable by electron paramagnetic resonance spectroscopy at room temperature. The radical cation of N,N-dimethyl-p-toluidine was conclusively identified, but the other resolved EPR signals were assigned to radical cations of radical dimerization products, e.g., N,N,N′,N′-tetramethylbenzidine formed from N,N-dimethylaniline. The N-demethylase activities of metmyoglobin were found to be uniformly smaller than those of horseradish peroxidase, consistent with the much faster reaction of the latter hemeprotein with H₂O₂. Detection of the monomeric radical cation of N,N-demethyl-p-toluidine correlated with the largest rate of N-demethylation among this class of compounds. These findings emphasize the importance of radical stability (provided, for example, by the para methyl substituent) on subsequent competing reactions of the radical cation of the N-methyl substrate, i.e., one-electron oxidation leading to formaldehyde release or radical dimerization, which becomes more probable for the less stable radical intermediates. Attempts were made to correlate these results with data obtained for the $\text{O}{}_2\text{NADPH}\text{-supported}$ oxidation of these same substrates by liver microsomal cytochrome P-450. However, pronounced differences in physical state and kinetic properties of this heterogeneous, membrane-associated microsomal hemeprotein and the soluble “model” hemeprotein systems precluded firm conclusions concerning a radical mechanism of N-demethylation monooxygenase activities of microsomal fractions.     

The production of oxygen-centered radicals by Bacillus-Calmette-Guerin-activated macrophages: An electron paramagnetic resonance study of the response to phorbol myristate acetate

The spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide of free radicals formed from Bacillus-Calmette-Guerin elicited peritoneal macrophages stimulated with phorbol myristate acetate resulted in the formation of a superoxide and hydroxyl spin adducts. The formation of both spin adducts was inhibited by copper/zinc superoxide dismutase. Only 70% of the hydroxyl spin adduct could be inhibited by catalase or the scavenger dimethyl sulfoxide. This suggests that the production of hydroxyl radicals involves prior formation of both superoxide radicals and hydrogen peroxide, implicating a Fenton catalysed Haber-Weiss reaction. The metal scavenger desferrioxamine also reduced the hydroxyl radical signal by 70%. The unaccounted 30% hydroxyl radical-like signals are probably due to carbon-centered free radicals formed by the lipoxygenase reaction. Spin trapping in the presence of the lipid-soluble spin trap, 5-octadecyl-5,3,3-trimethyl-1-pyrroline-N-oxide, resulted in a spectrum consistent with the presence of an oxaziridine nitroxide. This results from the free radical-induced cyclisation of a nitrone with an unsaturated fatty acid.     

Free radical involvement in long wavelength UV light activation of nitrosamines to mutagens

Nitrosamines are carcinogenic and mutagenic only after metabolic activation via endoplasmic reticulum bound mixed function oxidase enzyme systems. Rencently a new photochemical process has been discovered by which nitrosamines are converted into unknown mutagenic compounds by irradiation with long wavelength UV light (> 335 nm) in the presence of phosphate ion at neutral pH. The mutagenic activity is detected by Ames Salmonella Typhimurium strain TA100 in the absence of rat liver microsomes. We have shown that mutagen production with nitrosomorpholine is inhibited in the presence of light by various spin trapping agents (N-t-butyl-phenylnitrone, etc.). Concurrent with this inhibition a stable free radical signal has been detected whose kinetics of formation is similar to the time course of mutagen formation during irradiation in the absence of spin trap. The free radical signal is formed only when phosphate or similar ions are present in the reaction mixture. Monomethylphosphate and dimethylphosphate can substitute for phosphate ion but with small ESR signals and mutagen formation. Trimethylphosphate gives a weak, time independent ESR signal and does not cause mutagen formation. The ESR splitting constants (a^N and a^H) for signals generated with each of the different phosphate species show differences which suggest that these ions may be components of some intermediate free radical species that is involved in stable mutagen formation. Arsenate ion inhibits mutagen formation in the presence of phosphate but is able in the absence of phosphate to form a ESR signal similar to that observed with phosphate ion.     

Free radicals impair the anti-oxidative stress activity of DJ-1 through the formation of SDS-resistant dimer

DJ-1 is a causative gene for familial Parkinson’s disease (PD). Loss-of-function of DJ-1 protein is suggested to contribute to the onset of PD, but the causes of DJ-1 dysfunction remain insufficiently elucidated. In this study, we found that the SDS-resistant irreversible dimer of DJ-1 protein was formed in human dopaminergic neuroblastoma SH-SY5Y cells when the cells were exposed to massive superoxide inducers such as paraquat and diquat. The dimer was also formed in vitro by superoxide in PQ redox cycling system and hydroxyl radical produced in Fenton reaction. We, thus, found a novel phenomenon that free radicals directly affect DJ-1 to form SDS-resistant dimers. Moreover, the formation of the SDS-resistant dimer impaired anti-oxidative stress activity of DJ-1 both in cell viability assay and H₂O₂-elimination assay in vitro. Similar SDS-resistant dimers were steadily formed with several mutants of DJ-1 found in familial PD patients. These findings suggest that DJ-1 is impaired due to the formation of SDS-resistant dimer when the protein is directly attacked by free radicals yielded by external and internal stresses and that the DJ-1 impairment is one of the causes of sporadic PD.     

Obligatory free radical intermediate in the oxidative activation of the carcinogen N-hydroxy-2-acetylaminofluorene

We have demonstrated that the nitroxyl free radical form of the carcinogen N-hydroxy-2-acetylaminofluorene (OH-AAF) is an obligatory intermediate in the cumene hydroperoxide-hematin-induced oxidative activation of this carcinogen into 2-nitrosofluorene and N-acetoxy-2-acetylaminofluorene. Both the rate of N-OH-2-acetylaminofluorene oxidation and the amount of its nitroxyl free radical were experimently observed as a function of reaction time. Rate equations were derived for a model in which the nitroxyl free radical form of OH-AAF was an obligatory intermediate in the reaction. Using this theory it was possible to compute one experimental variable, the rate of OH-AAF oxidation, utilizing the other experimental variable, the amount of nitroxyl free radical present at any time during the reaction. The theory also predicts a linear relationship between the rate of OH-AAF oxidation and the square of the free radical content; and this was found to be true experimentally. The dismutation rate of constant of the nitroxyl free radical of OH-AAF was found to be 2.7 · 10⁵ M^?1 · s^?1.     

Kinetics of ubiquinone reduction by the resolved succinate: Ubiquinone reductase

A significant lag in the thenoyltrifluoroacetone (TTFA)-sensitive succinate: ubiquinone reductase activity was observed when a ubiquinone-deficient resolved preparation of the enzyme was assayed in the presence of exogenous ubiquinone-2 (Q2) and 2,6-dichlorophenolindophenol. No such lag was seen when the free radical of N,N,N′,N′-tetramethyl-p-phenylenediamine (Wurster's Blue) was used as the terminal electron acceptor, or when the reduction of Q2 was directly measured. The apparent Km value for exogenous Q2 was determined in the Q2-mediated TTFA-sensitive succinate: Wurster's Blue reductase reaction. When the enzyme activity was measured directly by monitoring Q2 reduction without terminal acceptors, the time course of the reaction deviated from zero-order kinetics at Q2 concentrations which were much higher than those expected from the KQ2m value determined in the presence of Wurster's Blue. The time course of Q2 reduction fits a curve describing a competitive interrelationship between oxidized and reduced Q2 at the specific binding site. The data obtained are in agreement with the Q-pool behavior of ubiquinone in mitochondrial membranes and suggest that the rate of ubiquinone reduction by succinate is dependent on the ratio.     

Contribution of 3-Deoxyglucosone as an Intermediate to Browning of Saké

The behaviour of 3-deoxyglucosone (3-D-G) was investigated in the process of browning of Saké, and it was concluded that the browning of Saké involved at least three kinds of browning reactions which were the amino-carbonyl reaction, the caramelization and some other browning reaction by components other than glucose in Saké. The active dicarbonyl compound of 3-D-G was confirmed to act as an important intermediate in both the amino-carbonyl reaction and caramelization. The caramelization was the browning of glucose by itself via 3-D-G, while the amino-carbonyl reaction was the browning of glucose via 3-D-G by the interaction with the amino compounds in Saké. The remainder was, however, independent of glucose and 3-D-G. The temperature coefficient for the rate of the overall browning of Saké was estimated to be 2.8 to 2.9 of Q₁₀ value.     

Screening for glucose‐triggered modifications of glutathione

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. These structures may undergo further Amadori rearrangement and free radical‐mediated oxidation to finally generate irreversible advanced glycation end products (AGEs). One of the factors known to modulate the glycation of proteins is glutathione, the most abundant nonprotein thiol tripeptide with the γ‐linkage, H‐Glu(Cys‐Gly‐OH)‐OH (GSH). Screening for products formed by GSH with D ‐glucose is an essential step in understanding the participation of GSH in glycation (the Maillard) reaction. Under the conditions used in these studies we observed N‐(1‐deoxy‐D ‐fructos‐1‐yl)‐pyroglutamic acid as the major glycation product formed in the mixtures of GSH and glucose in vitro. A RP HPLC/MS and tandem MS analyses of the GSH/glucose mixtures revealed that cleavage of the N‐terminal glutamic acid and the formation of pyroglutamic acid‐related Amadori product were accompanied by generation of Cys‐Gly‐derived Amadori and thiazolidine compounds. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

In Vitro Nonenzymatic Glycation Enhances the Role of Myoglobin as a Source of Oxidative Stress

Metmyoglobin (Mb) was glycated by glucose in a nonenzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO² and GMbO², respectively. H²O²-induced iron release was significantly higher from GMbO² than that from MbO². This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H²O² was studied. GMbO² degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO². Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H²O² with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO². Compared to MbO², GMbO² was rapidly auto-oxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO². GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H²O². These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients.     

Oxidative Cleavages of 1,2-Diaminosugars and their Significance in the Mechanism of the Amino-carbonyl Reactions

N-Benzyl-(l-deoxy-l-p-toluidino)-D-fructosylamine gives extensive browning on heating in methanol or ethyl acetate especially in the present acid, and from the browned solutions N-benzyl-D-arabinonamide has been isolated. Under stream of oxygen the browning is less extensive but the yield of D-arabinonamide is increased; under nitrogen the browning is slight and unchanged diaminosugar has been recovered. In the presence of an excess of benzyl-amine, benzylammonium D-arabinonate has been isolated. N-Cyelohexyl- (2-cyclohexylamino-2-deoxy) -D-glucosylamine on heating with acid in stream of oxygen gives small amounts of N, N-bis (cyclohexyl) -oxaldiamide, dicyclohexyliminoglyoxal and cyclohexylammonium D-arabinonate as the oxidation products. The mechanism of the oxidative cleavages has been explained in terms of the high reducing ability of the enediamine structures which are formed by rearrangement of 1,2-diaminosugars; in the case of N-cyclohexyl- (2-cyclohexylamino-2-deoxy) -D-glucosylamine dehydrogenation of the enediamine structure to diiminoglucosone should be involved. The findings are applied to explain the fragmentation of sugar molecules in the amino-carbonyl reactions.     

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals.     

Possibility of the Nonenzymatic Browning (Maillard) Reaction in the ISM

The possibility of the occurrence of the nonenzymatic browning reaction in the gaseous phase in the interstellar medium has been investigated by using Density Functional Theory computations. Mechanisms for the reactions between formaldehyde (Fald) + glycine (Gly), Fald + NH ₃ and Fald + methylamine (MeAm) have been proposed, and the possibility of the formation of different compounds in the proposed mechanisms has been evaluated through calculating the Gibb's free energy changes for different steps of the reaction, by following the total mass balance. The Fald + Gly reaction under basic conditions is found as the most favorable for producing 1-methyl-amino methene or 1-methyl-amino methelene (MAM). The reaction under acidic conditions is found to be the least favorable for producing MAM. The Fald + NH ₃ reaction is found to be plausible for the production of MeAm, which can participate by reaction with Fald, resulting in the formation of MAM.     

Inhibition of the Fenton reaction by nitrogen monoxide

The toxicity of iron is believed to originate from the Fenton reaction which produces the hydroxyl radical and/or oxoiron(2+). The effect of nitrogen monoxide on the kinetics of the reaction of iron(II) bound to citrate, ethylenediamine-N,N′-diacetate (edda), ethylenediamine-N,N,N′,N′-tetraacetate (edta), (N-hydroxyethyl)amine-N,N′,N′-triacetate (hedta), and nitrilotriacetate (nta) with hydrogen peroxide was studied by stopped-flow spectrophotometry. Nitrogen monoxide inhibits the Fenton reaction to a large extent. For instance, hydrogen peroxide oxidizes iron(II) citrate with a rate constant of 5.8×10³ M⁻¹ s⁻¹, but in the presence of nitrogen monoxide, the rate constant is 2.9×10² M⁻¹ s⁻¹ . Similar to hydrogen peroxide, the reaction of tert-butyl hydroperoxide with iron(II) complexes is also efficiently inhibited by nitrogen monoxide. Generally, nitrogen monoxide binds rapidly to a coordination site of iron(II) occupied by water. The rate of oxidation is influenced by the rate of dissociation of the nitrogen monoxide from iron(II). Electronic Supplementary Material Supplementary material is available for this article at