. Amyloid beta-Cu2+ complexes in both monomeric and fibrillar forms do not generate H2O2 catalytically but quench hydroxyl radicals. Biochemistry. 2008 Nov 4;47(44):11653-64. PubMed.

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  1. This paper sets out to try and confirm the observation originally made by Ashley Bush and colleagues (Huang et al., 1999a) and expanded upon in their further papers (e.g., Huang et al., 1999b; Opazo et al., 2002) that the Aβ peptide can generate H2O2 directly from molecular oxygen through an interaction with redox-active metal ions. This is highly relevant to the debate on the mechanism of toxicity of Aβ and its oligomers.

    In their initial experiments, Nadal and co-workers incubated 10 μM Aβ40 or Aβ42 at 37oC for 60 minutes in the presence or absence of an equimolar concentration of CuCl2 and measured the amount of H2O2 generated using an assay system based on o dianisidine (ODAD) and peroxidase. In some instances, ascorbate was also included as a reducing agent to enhance the amount of H2O2 generated in the presence of the peptide. These experimental conditions are similar to those reported by Ashley Bush’s group, although the H2O2 detection method and the reducing agent are different. Bush (and others) have proposed that in the absence of ascorbate, or other reducing agent, the peptide itself could possibly provide the electrons required for H2O2 formation when bound to an appropriate redox-active metal ion.

    Nadal reports that in the absence of ascorbate or copper ions, Aβ (in monomeric or fibrillar form) does not generate detectable levels of H2O2. However, turbidity in the ODAD assay system limited the accurate measurement of H2O2 levels to those greater than 5 μM. This could be a serious problem, because the incubation of 10 μM Aβ under these experimental conditions would be expected to generate levels of H2O2 that are much lower than this relatively poor detection limit. Our group at Lancaster University have confirmed the ability of Aβ to generate H2O2 and have also extended this observation to some other amyloidogenic proteins and peptides (reviewed in Allsop et al., 2008). The method used to detect this source of H2O2 in our initial experiments was based on its conversion into hydroxyl radicals, which were then trapped and detected by ESR spectroscopy. The generation of H2O2 by Aβ was later confirmed by an alternative method using Amplex red dye (Masad et al., 2007), the limit of detection of which is better than 0.1 μM. These experiments with Aβ are dependent on the presence of the low levels of redox-active metal ions that are unavoidably present as trace contaminants in the buffer reagents, or are associated with the peptide samples, and are equivalent to Nadal’s experiments without added copper ions or ascorbate. In our experiments, the incubation of 100 μM Aβ40 or Aβ42 produced H2O2 levels of no more than 1 μM. Thus, the 10 μM peptide concentration used by Nadal would be expected to generate levels of H2O2 that are well below the detection limit of the ODAD method. Another possible problem is the fact that the one-hour incubation time employed by Nadal may not correspond to the time when maximum H2O2 concentrations are achieved.

    Nadal et al. also report undetectable levels of H2O2 when Aβ (10 μM) was incubated with equimolar concentrations of copper ions. However, our experience with various amyloidogenic peptides is that when additional redox-active metal ions are required to generate detectable levels of H2O2, their optimal concentration is significantly lower than that of the peptide (see, for example, Turnbull et al., 2003). In fact, high levels of the metal ion (i.e., equimolar with the peptide) in a redox-active complex (in which some of the metal ions will be in the correct oxidation state) would be expected to destroy any H2O2 virtually as soon as it is formed, and so would be counterproductive.

    The only conditions resulting in a high concentration of H2O2 (in the range 15-20 μM) in Nadal’s experiments were when Aβ, copper ions, and ascorbate were incubated together. Notably, high levels of H2O2 were also observed when just copper ions and ascorbate were incubated alone (i.e., with no peptide present). We have obtained similar results to Nadal in this respect (unpublished data). Thus, we agree with Nadal that the amount of H2O2 generated by Aβ is not always increased beyond that of a non-peptide control in the presence of an additional reducing agent.

    In Nadal’s paper, the next set of experiments was carried out under similar conditions before, but this time looking for the production of hydroxyl radicals using coumarin-3-carboxylic acid (3CCA). The authors did not observe any such radicals formed from H2O2 after one-hour incubation of the Aβ peptides in the presence of Cu(II) (both at 10 μM), but their experimental conditions are such that H2O2 levels could be too low for any measurable concentration (of H2O2) to be formed. Consequently, any hydroxyl radicals formed from peptide-generated H2O2 would not be expected to be easily observed. In the presence of ascorbate, however, the well-known reaction between ascorbate and Cu(II) gives rise to significant levels of H2O2 (observed) and the authors (correctly) note that hydroxyl radicals generated from this latter peroxide can be trapped by 3 CCA. We generally agree with the authors’ results here in that, in the presence of the peptide, many of the hydroxyl radicals formed will attack the peptide and are, therefore, removed from the system before 3-CCA trapping can take place. For this reason, decreased levels of trapped hydroxyl radicals would be expected. If this conclusion is correct, then oxidation of the peptide would also be expected and, indeed, this is observed by the authors in their experiments, using NMR spectroscopy.

    Nadal also demonstrates in this paper that both Aβ40 and Aβ42 have an intrinsic ability to reduce Cu(II) to Cu(I), an observation which supports previously published research.

    In conclusion, the experimental conditions employed by the authors in this paper mitigate against H2O2 production, and this is compounded by the poor sensitivity of the ODAD detection method. Their choice of title, “Amyloid β-Cu2+ Complexes in both Monomeric and Fibrillar Forms Do Not Generate H2O2 Catalytically but Quench Hydroxyl Radicals,” is, therefore, unfortunate and misleading with respect to the overall situation concerning H2O2 generation and is only true under certain experimental conditions, such as those employed by these authors.

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