Cu(II)-Catalyzed Oxidation of β-Amyloid Peptide Targets His13and His14over His6:  Detection of 2-Oxo-histidine by HPLC-MS/MS

The interaction of beta-amyloid peptide (betaAP) with Cu(II) leads to the formation of reactive oxygen species, neurotoxicity, and the chemical modification of the peptide. To study product formation and the potential selectivity of oxidation, we have exposed specific betaAP congeners, betaAP1-16, betaAP1-28, and betaAP1-40, to ascorbate/Cu(II)-induced metal-catalyzed oxidation and electrospray ionization-time-of-flight (ESI-TOF) MS/MS analysis. Incubation of 30 microM betaAP with 15-150 microM Cu(II) and (physiologically relevant) 720 microM ascorbate in 20 mM phosphate buffer, pH 7.4, leads to significant oxidation of the peptides within remarkably short reaction times of as low as 6 min. Initial oxidation targets are His13 and His14, which are converted to 2-oxo-His, whereas the other two metal-binding residues, His6 and Tyr10, remain intact. Longer oxidation times then also target His6. Even in betaAP1-40 the oxidation of His13 and His14 precedes the oxidation of Met35. Especially, the insensitivity of Tyr10 is noteworthy and may be explained by electron withdrawal from the Tyr side chain through complexation of Cu(II). The insensitivity of His6 to initial oxidation may be rationalized by a proposed bridging of two Cu(II)-betaAP congeners, lowering the electron density on His6, comparable to similar results on a Cu(II)- and Zn(II)-bridging His61 residue of bovine Cu,Zn superoxide dismutase.