Methionine regulates copper/hydrogen peroxide oxidation products of Abeta.

Metal-catalysed oxidation (MCO) may play a causative role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta peptide (Abeta), the major biomarker of AD, in the presence of copper ions reduces Cu(2+) to Cu(+) and catalyses the formation of H(2)O(2) that subsequently induces radicals through Fenton chemistry. Abeta is also subject to attack by free radicals, where the presence of Cu(2+) in conjunction with H(2)O(2) catalyses oxygenation, primarily at the methionine sulfur atom. This work investigates MCO of Abeta, to gain further insight into the role of oxidative stress in AD. By combining a fluorescence assay with gel electrophoresis to monitor MCO reactions of Abeta (1-28) in the presence and absence of methionine it was determined that methionine can both protect some residues against MCO and promote the oxidation of Tyr(10) specifically. Electrospray ionization mass spectrometric analysis of methionine MCO products indicated the formation of methionine sulfoxide, methionine sulfone and related hydroxylated products. Similar products could be formed from the oxidation of Met(35) of Abeta and may relate to changes in properties of the peptide following MCO.     

The metal-catalyzed oxidation of methionine in peptides by Fenton systems involves two consecutive one-electron oxidation processes.

The one-electron oxidation of methionine (Met) plays an important role in the redox reactions of Met in peptides and proteins under conditions of oxidative stress, e.g., during the metal-catalyzed oxidation of beta-amyloid peptide (beta A). However, little information is available with regard to mechanisms and product formation during the metal-catalyzed oxidation of Met. Here, we demonstrate that two-electron oxidation of Met in Fenton reactions, carried out aerobically by [Fe(II)(EDTA)](2-) and H(2)O(2) (EDTA = ethylenediaminetetra acetate) is the consequence of two consecutive one-electron transfer reactions carried out by either free or complexed hydroxyl radicals, followed by the reaction of an intermediary sulfur-nitrogen bonded radical cation (sulfuranyl radical) with O(2). The model peptide Met-Met represents an ideal substrate for these investigations as its one-electron oxidation, followed by reaction with molecular oxygen, produces unique intermediates, azasulfonium diastereomers, which can be chemically isolated before hydrolysis to sulfoxide occurs.     

Effect of methionine-35 oxidation on the aggregation of amyloid-β peptide

Aggregation of Aβ peptides into amyloid plaques is considered to trigger the Alzheimer’s disease (AD), however the mechanism behind the AD onset has remained elusive. It is assumed that the insoluble Aβ aggregates enhance oxidative stress (OS) by generating free radicals with the assistance of bound copper ions. The aim of our study was to establish the role of Met35 residue in the oxidation and peptide aggregation processes. Met35 can be readily oxidized by H₂O₂. The fibrillization of Aβ with Met35 oxidized to sulfoxide was three times slower compared to that of the regular peptide. The fibrils of regular and oxidized peptides looked similar under transmission electron microscopy. The relatively small inhibitory effect of methionine oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. The peptide oxidation pattern was more complex when copper ions were present: addition of one oxygen atom was still the fastest process, however, it was accompanied by multiple unspecific modifications of peptide residues. Addition of copper ions to the Aβ with oxidized Met35 in the presence of H₂O₂, resulted a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in the peptide molecule do not depend on the oxidation state of Met35 residue. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aβ–Cu(II) complex.     

Methionine oxidation by reactive oxygen species: reaction mechanisms and relevance to Alzheimer's disease

The oxidation of methionine plays an important role in vivo, during biological conditions of oxidative stress, as well as for protein stability in vitro. Depending on the nature of the oxidizing species, methionine may undergo a two-electron oxidation to methionine sulfoxide or one-electron oxidation to methionine radical cations. Both reaction mechanisms derive catalytic support from neighboring groups, which stabilize electron-deficient reaction centers. In vivo, methionine sulfoxide is subject to reduction by the methionine sulfoxide reductase (Msr) system, suggesting that some methionine sulfoxide residues may only be transiently involved in the deactivation of proteins through reactive oxygen species (ROS). Other methionine sulfoxide residues may accumulate, depending on the accessibility to Msr. Moreover, methionine sulfoxide levels may increase as a result of a lower abundance of active Msr and/or the required cofactors as a consequence of pathologies and biological aging. On the other hand, methionine radical cations will enter predominantly irreversible reaction channels, which ultimately yield carbon-centered and/or peroxyl radicals. These may become starting points for chain reactions of protein oxidation. This review will provide detailed mechanistic schemes for the reactions of various prominent, biologically relevant ROS with methionine and organic model sulfides. Emphasis will be given on the one-electron oxidation pathway, characterizing the physico-chemical parameters, which control this mechanism, and its physiological relevance, specifically for the oxidation and neurotoxicity of the Alzheimer's disease beta-amyloid peptide (betaAP).     

Thioredoxin Ch1 of Chlamydomonas reinhardtii displays an unusual resistance toward one-electron oxidation.

To test thioredoxin resistance to oxidizing free radicals, we have studied the one-electron oxidation of wild-type thioredoxin and of two forms with the point mutations D30A and W35A, using azide radicals generated by gamma-ray or pulse radiolysis. The oxidation patterns of wild-type thioredoxin and D30A are similar. In these forms, Trp35 is the primary target and is 'repaired' by one-electron reduction; first by intramolecular electron transfer from tyrosine, and then from other residues. Conversely, during oxidation of W35A, Trp13 is poorly reactive. For all proteins, activity is conserved showing an unusual resistance toward oxidation.     

Abeta(31-35) and Abeta(25-35) fragments of amyloid beta-protein induce cellular death through apoptotic signals: Role of the redox state of methionine-35

In order to clarify the basis of neuronal toxicity exerted by the shortest active peptides of amyloid beta-protein (Abeta), the toxic effects of Abeta(31-35) and Abeta(25-35) peptides on isolated rat brain mitochondria were investigated. The results show that exposure of isolated rat brain mitochondria to Abeta(31-35) and Abeta(25-35) peptides determines: (i) release of cytochrome c; (ii) mitochondrial swelling and (iii) a significant reduction in mitochondrial oxygen consumption. In contrast, the amplitude of these events resulted attenuated in isolated brain mitochondria exposed to the Abeta(31-35)Met35(OX) in which methionine-35 was oxidized to methionine sulfoxide. The Abeta peptide derivative with norleucine substituting Met-35, i.e., Abeta(31-35)Nle-35, had not effect on any of the biochemical parameters tested. We have further characterized the action of Abeta(31-35) and Abeta(25-35) peptides on neuronal cells. Taken together our result indicate that Abeta(31-35) and Abeta(25-35) peptides in non-aggregated form, i.e., predominantly monomeric, are strongly neurotoxic, having the ability to enter within the cells, determining mitochondrial damage with an evident trigger of apoptotic signals. Such a mechanism of toxicity seems to be dependent by the redox state of methionine-35.     

Neurotoxic,redox-competent Alzheimer's beta-amyloid is released from lipid membrane by methionine oxidation

The amyloid beta peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism of this toxicity is contentious. Here we report that an Abeta peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Abeta) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Abeta. However, unlike the unoxidized peptide, Met(O)Abeta is unable to penetrate lipid membranes to form ion channel-like structures, and beta-sheet formation is inhibited, phenomena that are central to some theories for Abeta toxicity. Our results show that, like the unoxidized peptide, Met(O)Abeta will coordinate Cu2+ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Abeta production contributes to the elevation of soluble Abeta seen in the brain in Alzheimer's disease.     

Identification of methionine sulfoxide diastereomers in immunoglobulin gamma antibodies using methionine sulfoxide reductase enzymes

Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met[O]) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping, we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized, as well as those in an IgG1 oxidized with peroxide. The rapid identification of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers, which previously has been indistinguishable using traditional techniques, but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of new formulation strategies to stabilize protein therapeutics.Key words: immunoglobulin gamma antibody, methionine sulfoxide, oxidation, photo-oxidation, methionine sulfoxide reductase     

Role of methionine 35 in the intracellular Ca2+ homeostasis dysregulation and Ca2+-dependent apoptosis induced by amyloid beta-peptide in human neuroblastoma IMR32 cells

Amyloid beta-peptide (Abeta) plays a fundamental role in the pathogenesis of Alzheimer's disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid beta-peptide (Abeta) 1-42 (Met35) strongly affects the peptide's ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca(2+) homeostasis resulting in the activation of pro-apoptotic pathways has been proposed as a mechanism underlying Abeta toxicity. Therefore, we investigated correlations between the Met35 redox state, Abeta toxicity, and altered intracellular Ca(2+) signaling in human neuroblastoma IMR32 cells. Cells incubated for 6-24 h with 10 microM Abeta1-42 exhibited significantly increased KCl-induced Ca(2+) transient amplitudes and resting free Ca(2+) concentrations. Nifedipine-sensitive Ca(2+) current densities and Ca(v)1 channel expression were markedly enhanced by Abeta1-42. None of these effects were observed when cells were exposed to Abeta containing oxidized Met35 (Abeta1-42(Met35-Ox)). Cell pre-treatment with the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (1 microM) or the Ca(v)1 channel blocker nifedipine (5 microM) significantly attenuated Abeta1-42-induced apoptosis but had no effect on Abeta1-42(Met35-Ox) toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Abeta1-42 toxicity by rendering the peptide capable of disrupting intracellular Ca(2+) homeostasis and thereby provoking apoptotic cell death.     

Redox processes of methionine relevant to beta-amyloid oxidation and Alzheimer's disease.

This minireview gives an overview over the oxidation mechanisms of methionine (Met) relevant for analogous processes which may lead to the oxidation of beta-amyloid (betaA) peptides. The Cu(II)-catalyzed oxidation of a C-terminal Met(35) residue in betaA peptides may be a key to the known propensities of these peptides to form H2O2 and free radicals. Though the reduction potentials of Cu(II) and Met would seem unfavorable, there are several structural features of betaA, which may promote a one-electron oxidation of Met. The potentially close association of the Met sulfur with the C=O group C-terminal of Ile(31) in the C-terminus of betaA may support the formation of an S-O bonded radical cation intermediate. Evidence for such S-O bond formation has recently been obtained for a model, N-acetylmethionine amide. Additional support for a potential catalytic role of an oxygen-containing functional group comes from numerous studies with organic model sulfides.     

Identification of methionine sulfoxide diastereomers in immunoglobulin gamma antibodies using methionine sulfoxide reductase enzymes

Light-induced formation of singlet oxygen selectively oxidizes methionines in the heavy chain of IgG2 antibodies. Peptide mapping has indicated the following sensitivities to oxidation: M252 > M428 > M397. Irrespective of the light source, formulating proteins with the free amino acid methionine limits oxidative damage. Conventional peptide mapping cannot distinguish between the S- and R-diastereomers of methionine sulfoxide (Met(O)) formed in the photo-oxidized protein because of their identical polarities and masses. We have developed a method for identification and quantification of these diastereomers by taking advantage of the complementary stereospecificities of the methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which promote the selective reduction of S- and R-diastereomers of Met(O), respectively. In addition, an MsrBA fusion protein that contains both Msr enzyme activities permitted the quantitative reduction of all Met(O) diastereomers. Using these Msr enzymes in combination with peptide mapping, we were able to detect and differentiate diastereomers of methionine sulfoxide within the highly conserved heavy chain of an IgG2 that had been photo-oxidized, as well as those in an IgG1 oxidized with peroxide. The rapid identification of the stereospecificity of methionine oxidation by Msr enzymes not only definitively differentiates Met(O) diastereomers, which previously has been indistinguishable using traditional techniques, but also provides an important tool that may contribute to understanding of the mechanisms of protein oxidation and development of new formulation strategies to stabilize protein therapeutics.     

Stereospecific oxidation of calmodulin by methionine sulfoxide reductase A

Methionine sulfoxide reductase A has long been known to reduce S-methionine sulfoxide, both as a free amino acid and within proteins. Recently the enzyme was shown to be bidirectional, capable of oxidizing free methionine and methionine in proteins to S-methionine sulfoxide. A feasible mechanism for controlling the directionality has been proposed, raising the possibility that reversible oxidation and reduction of methionine residues within proteins is a redox-based mechanism for cellular regulation. We undertook studies aimed at identifying proteins that are subject to site-specific, stereospecific oxidation and reduction of methionine residues. We found that calmodulin, which has nine methionine residues, is such a substrate for methionine sulfoxide reductase A. When calmodulin is in its calcium-bound form, Met77 is oxidized to S-methionine sulfoxide by methionine sulfoxide reductase A. When methionine sulfoxide reductase A operates in the reducing direction, the oxidized calmodulin is fully reduced back to its native form. We conclude that reversible covalent modification of Met77 may regulate the interaction of calmodulin with one or more of its many targets.     

Calorimetry and mass spectrometry study of oxidized calmodulin interaction with target and differential repair by methionine sulfoxide reductases

Calmodulin is known to be a target for oxidation, which leads to conversion of methionine residues to methionine sulfoxides. Previously, we reported that both methionine sulfoxide reductases MsrA and MsrB were able to reduce methionine sulfoxide residues in oxidized calmodulin. In the present study, we have made use of the interaction between calmodulin and RS20, a peptide model for calmodulin targets, to probe the structural consequences of oxidation and mode of repair both by MsrA and MsrB. Isothermal titration calorimetry and differential scanning calorimetry showed that oxidized calmodulin interacts with RS20 via its C-terminal domain only, resulting in a non-productive complex. As shown by spectrofluorometry, oxidized calmodulin treated with MsrA exhibited native binding affinity for RS20. In contrast, MsrB-treatment of oxidized calmodulin resulted in 10-fold reduced affinity. Mass spectrometry revealed that the sulfoxide derivative of methionine residue 124 was differentially repaired by MsrA and MsrB. This provided a basis for rationalizing the difference in binding affinities of oxidized calmodulin reported above, since Met124 residue had been shown to be critical for interaction with some targets. This study provides the first evidence that in an oxidized polypeptide chain MetSO residues might be differentially repaired by the two Msr enzymes.     

Amyloid beta peptides do not form peptide-derived free radicals spontaneously, but can enhance metal-catalyzed oxidation of hydroxylamines to nitroxides

Amyloid beta (Abeta) peptides play an important role in the pathogenesis of Alzheimer's disease. Free radical generation by Abeta peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from Abeta-(1-40) and Abeta-(25-35) peptides react with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) to form a PBN/.Abeta peptide radical adduct with a specific triplet ESR signal assert that the peptide itself was the source of free radicals. We now report that three Abeta peptides, Abeta-(1-40), Abeta-(25-35), and Abeta-(40-1), do not yield radical adducts with PBN from the Oklahoma Medical Research Foundation (OMRF). In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without Abeta peptides produced a three-line ESR spectrum. It was shown that this nitroxide is di-tert-butylnitroxide and is formed in the Sigma PBN solution as a result of transition metal-catalyzed auto-oxidation of the respective hydroxylamine present as an impurity in the Sigma PBN. Under some conditions, incubation of PBN from Sigma with Abeta-(1-40) or Abeta-(25-35) can stimulate the formation of di-tert-butylnitroxide. It was shown that Abeta peptides enhanced oxidation of cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6, 6-tetramethylpiperidine (TEMPONE-H), which was strongly inhibited by the treatment of phosphate buffer with Chelex-100. It was shown that ferric and cupric ions are effective oxidants of TEMPONE-H. The data obtained allow us to conclude that under some conditions toxic Abeta peptides Abeta-(1-40) and Abeta-(25-35) enhance metal-catalyzed oxidation of hydroxylamine derivatives, but do not spontaneously form peptide-derived free radicals.     

Methionine 35 oxidation reduces fibril assembly of the amyloid abeta-(1-42) peptide of Alzheimer's disease

The major component of amyloid plaques in Alzheimer's disease (AD) is Abeta, a small peptide that has high propensity to assemble as aggregated beta-sheet structures. Using three well established techniques for studying amyloid structure, namely circular dichroism, thioflavin-T fluorescence, and atomic force microscopy, we demonstrate that oxidation of the Met-35 side chain to a methionine sulfoxide (Met-35(ox)) significantly hinders the rate of fibril formation for the 42-residue Abeta-(1-42) at physiological pH. Met-35(ox) also alters the characteristic Abeta fibril morphology and prevents formation of the protofibril, which is a key intermediate in beta-amyloidosis and the associated neurotoxicity. The implications of these results for the biological function and role of Abeta with oxidative stress in AD are discussed.     

Role of N-terminal methionine residues in the redox activity of copper bound to alpha-synuclein

Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson’s disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1–6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1–6) fragment, and in the redox activity of the Cu–AS(1–6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)–AS(1–6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu–AS(1–6) complex by ~80 mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu–AS complex.     

Free-energy simulations of the oxidation of c-terminal methionines in calmodulin

In the course of aging or under conditions of oxidative stress, methionine residues of calmodulin undergo oxidation, leading to loss of biological activity of the protein. We have performed free-energy simulations of the effects of C-terminal methionine side-chain oxidation on the properties of calmodulin. The simulation results indicate that oxidation should have a destabilizing effect on all three protein functional states: calcium free, calcium loaded, and with both calcium and target peptide bound. Because the different states are destabilized by different amounts, this leads to a more complex pattern in the observable effects on protein thermal stability, calcium affinity, and binding of a target peptide. The influence of oxidation on the free energy of CaM unfolding is estimated by comparing the free-energy cost of oxidizing a Met residue in a Gly-Met-Gly peptide and in the protein. The protein thermal stability of the oxidized forms is lowered by a moderate amount 1-3 kcal/mol, in qualitative agreement with experimental results of 0.3 kcal/mol. The calculated changes in affinity for calcium and for the target peptide show opposing trends. Oxidation at position 144 is predicted to enhance peptide binding and weaken calcium binding, whereas oxidation at 145 weakens peptide binding and enhances affinity for calcium. The lower affinity of Met 145-oxidized calmodulin toward the target peptide correlates with experimentally observed lowering of calmodulin-activated Ca-ATPase activity when oxidized calmodulin from aged rat brains is used. Thus, our simulations suggest that Met 145 is the oxidation site in the C-terminal fragment of calmodulin. The microscopic mechanism behind the calculated free energy changes appears to be a greater affinity for water of the oxidized Met side-chain relative to normal Met. Structures with Met exposed to solvent had consistently lower free energies than those with buried Met sidechains.     

Marked difference in cytochrome c oxidation mediated by HO(*) and/or O(2)(*-) free radicals in vitro

Cytochrome c (cyt c) is an electron carrier involved in the mitochondrial respiratory chain and a critical protein in apoptosis. The oxidation of cytochrome c can therefore be relevant biologically. We studied whether cytochrome c underwent the attack of reactive oxygen species (ROS) during ionizing irradiation-induced oxidative stress. ROS were generated via water radiolysis under ionizing radiation (IR) in vitro. Characterization of oxidation was performed by mass spectrometry, after tryptic digestion, and UV-visible spectrophotometry. When both hydroxyl and superoxide free radicals were generated during water radiolysis, only five tryptic peptides of cyt c were reproducibly identified as oxidized according to a relation that was dependent of the dose of ionizing radiation. The same behavior was observed when hydroxyl free radicals were specifically generated (N(2)O-saturated solutions). Specific oxidation of cyt c by superoxide free radicals was performed and has shown that only one oxidized peptide (MIFAGIK+16), corresponding to the oxidation of Met80 into methionine sulfoxide, exhibited a radiation dose-dependent formation. In addition, the enzymatic site of cytochrome c was sensitive to the attack of both superoxide and hydroxyl radicals as observed through the reduction of Fe(3+), the degradation of the protoporphyrin IX and the oxidative disruption of the Met80-Fe(3+) bond. Noteworthy, the latter has been involved in the conversion of cyt c to a peroxidase. Finally, Met80 appears as the most sensitive residue towards hydroxyl but also superoxide free radicals mediated oxidation.     

Role of glycine-33 and methionine-35 in Alzheimer's amyloid beta-peptide 1-42-associated oxidative stress and neurotoxicity

Recent theoretical calculations predicted that Gly33 of one molecule of amyloid beta-peptide (1-42) (Abeta(1-42)) is attacked by a putative sulfur-based free radical of methionine residue 35 of an adjacent peptide. This would lead to a carbon-centered free radical on Gly33 that would immediately bind oxygen to form a peroxyl free radical. Such peroxyl free radicals could contribute to the reported Abeta(1-42)-induced lipid peroxidation, protein oxidation, and neurotoxicity, all of which are prevented by the chain-breaking antioxidant vitamin E. In the theoretical calculations, it was shown that no other amino acid, only Gly, could undergo such a reaction. To test this prediction we studied the effects of substitution of Gly33 of Abeta(1-42) on protein oxidation and neurotoxicity of hippocampal neurons and free radical formation in synaptosomes and in solution. Gly33 of Abeta(1-42) was substituted by Val (Abeta(1-42G33V)). The substituted peptide showed almost no neuronal toxicity compared to the native Abeta(1-42) as well as significantly lowered levels of oxidized proteins. In addition, synaptosomes subjected to Abeta(1-42G33V) showed considerably lower dichlorofluorescein-dependent fluorescence - a measure of reactive oxygen species (ROS) - in comparison to native Abeta(1-42) treatment. The ability of the peptides to generate ROS was also evaluated by electron paramagnetic resonance (EPR) spin trapping methods using the ultrapure spin trap N-tert-butyl-alpha-phenylnitrone (PBN). While Abeta(1-42) gave a strong mixture of four- and six-line PBN-derived spectra, the intensity of the EPR signal generated by Abeta(1-42G33V) was far less. Finally, the ability of the peptides to form fibrils was evaluated by electron microscopy. Abeta(1-42G33V) does not form fibrils nearly as well as Abeta(1-42) after 48 h of incubation. The results suggest that Gly33 may be a possible site of free radical propagation processes that are initiated on Met35 of Abeta(1-42) and that contribute to the peptide's toxicity in Alzheimer's disease brain.