A comprehensive study on the generation of reactive oxygen species in Cu-Aβ-catalyzed redox processes

In the amyloid plaques, a signature of AD, abnormally high Cu2+ concentrations are found bound to A beta. Most of previous studies reported that Cu-A beta could contribute to oxidative stress, as H2O2 and center dot OH are catalytically generated by Cu-A beta with the assistance of biological reductant, with only one recent report stated that free O-2(center dot-) is also generated in the Cu-A beta catalyzed processes, where an indirect technique was applied. To comprehensively investigate the free radicals produced during this Cu-A beta-mediated process with a biological reductant, DNA-cleavage assay, an indirect method, and two direct methods including electron paramagnetic resonance (EPR) spectroscopy and transient absorption spectroscopy (TAS), both having qualitative and quantitative power, were employed in this work. All the experimental results obtained from the three methods demonstrated that Cu-A beta in the biological reducing environment was not only able to catalyze the production of H2O2 and center dot OH, but also to generate free O-2(center dot-). The results further indicated that O-2(center dot-) was the precursor of H2O2 and center dot OH. It is also important to note that the results obtained from EPR spectroscopy and TAS provided direct evidence for the presence of O-2(center dot-) and center dot OH. By virtue of the direct techniques, we also found that the longest peptide fragments of A beta(16), A beta(40), and A beta(42) produced the least radicals with a lowest rate. More interestingly, the fibrillar forms of A beta generated less O-2(center dot-) and center dot OH compared with oligomeric and monomeric forms.