Reevaluation of Copper(I) Affinity for Amyloid-ß Peptides by Competition with FerrozineuAn Unusual Copper(I) Indicator

The association constant of ferrozine (5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine-4,4''-disulfonic acid) with CuI to form the chromophoric [CuI(Fz)2]3- complex was determined by UV/Vis titration experiments in Hepes buffer (0.1?m, pH 7.4). An association constant close to 1012 M-2, which is significantly weaker than those of the well-known, water-soluble, CuI chelators bicinchoninic acid and 2,9-dimethyl-4,7-diphenyl-1,10-phenantroline disulfonic acid, was found. The [CuI(Fz)2]3- chromophore was used in UV/Vis competition experiments to determine CuI binding affinity for the amyloid-beta peptide involved in Alzheimers disease and for a series of pertinent mutants. An association constant of approximately 107 M-1 was found; this is much weaker than that reported for dithiothreitol and confirms that imidazoles are harder ligands than thiolates. Each His mutation (H6A, H13A, and H14A) impacts the peptide affinity for CuI. The native human amyloid-beta peptide was found to be a fourfold-stronger CuI ligand than the murine peptide, which differs by three point mutations (R5G, Y10F, and H13R) from the human one.