Cu(II)-Catalyzed Oxidation of beta -Amyloid Peptide Targets His super(13) and His super(14) over His super(6): Detection of 2-Oxo-histidine by HPLC-MS/MS

The interaction of beta -amyloid peptide ( beta AP) 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 beta AP congeners, beta AP1-16, beta AP1-28, and beta AP1-40, to ascorbate/Cu(II)-induced metal-catalyzed oxidation and electrospray ionization-time-of-flight (ESI-TOF) MS/MS analysis. Incubation of 30 mu M beta AP with 15-150 mu M Cu(II) and (physiologically relevant) 720 mu M 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 super(13) and His super(14), which are converted to 2-oxo-His, whereas the other two metal-binding residues, His super(6) and Tyr super(10), remain intact. Longer oxidation times then also target His super(6). Even in beta AP1-40 the oxidation of His super(13) and His super(14) precedes the oxidation of Met super(35). Especially, the insensitivity of Tyr super(10) is noteworthy and may be explained by electron withdrawal from the Tyr side chain through complexation of Cu(II). The insensitivity of His super(6) to initial oxidation may be rationalized by a proposed bridging of two Cu(II)- beta AP congeners, lowering the electron density of His super(6), comparable to similar results on a Cu(II)- and Zn(II)-bridging His super(61) residue of bovine Cu,Zn superoxide dismutase.