Cu(II)-catalyzed oxidation of β-amyloid peptide targets His13 and His14 over 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 muM betaAP with 15-150 muM Cu(II) and (physiologically relevant) 720 muM 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 His(13) and His(14), which are converted to 2-oxo-His, whereas the other two metal-binding residues, His(6) and Tyr(10), remain intact. Longer oxidation times then also target His(6). Even in betaAP1-40 the oxidation of His(13) and His(14) precedes the oxidation of Met(35). Especially, the insensitivity of Tyr(10) is noteworthy and may be explained by electron withdrawal from the Tyr side chain through complexation of Cu(II). The insensitivity of His(6) to initial oxidation may be rationalized by a proposed bridging of two Cu(II)-betaAP congeners, lowering the electron density on His(6), comparable to similar results on a Cu(II)and Zn(II)-bridging His(61) residue of bovine Cu,Zn superoxide dismutase.