• Human amyloid β peptide;
  • Copper;
  • Affinity;
  • Stoichiometry;
  • Alzheimer's disease;
  • Binding affinity method;
  • Chelators

Abstract: Cu and Zn have been shown to accumulate in the brains of Alzheimer's disease patients. We have previously reported that Cu2+ and Zn2+ bind amyloid β (Aβ), explaining their enrichment in plaque pathology. Here we detail the stoichiometries and binding affinities of multiple cooperative Cu2+-binding sites on synthetic Aβ1-40 and Aβ1-42. We have developed a ligand displacement technique (competitive metal capture analysis) that uses metal-chelator complexes to evaluate metal ion binding to Aβ, a notoriously self-aggregating peptide. This analysis indicated that there is a very-high-affinity Cu2+-binding site on Aβ1-42 (log Kapp = 17.2) that mediates peptide precipitation and that the tendency of this peptide to self-aggregate in aqueous solutions is due to the presence of trace Cu2+ contamination (customarily ∼0.1 μM). In contrast, Aβ1-40 has much lower affinity for Cu2+ at this site (estimated log Kapp = 10.3), explaining why this peptide is less self-aggregating. The greater Cu2+-binding affinity of Aβ1-42 compared with Aβ1-40 is associated with significantly diminished negative cooperativity. The role of trace metal contamination in inducing Aβ precipitation was confirmed by the demonstration that Aβ peptide (10 μM) remained soluble for 5 days only in the presence of high-affinity Cu2+-selective chelators.