The hydrogen peroxide/copper ion system, but not other metal-catalyzed oxidation systems, produces protein-bound dityrosine.

Abstract

Dityrosine formation leads to the cross-linking of proteins intra- or intermolecularly. The formation of dityrosine in lens proteins oxidized by metal-catalyzed oxidation (MCO) systems was estimated by chemical and immunochemical methods. Among the four MCO systems examined (H(2)O(2)/Cu, H(2)O(2)/Fe-ethylenediaminetetraacetic acid (Fe-EDTA), ascorbate/Cu, ascorbate/Fe-EDTA), the treatment with H(2)O(2)/Cu preferentially caused dityrosine formation in the lens proteins. The success of oxidative protein modification with all the MCO systems was confirmed by carbonyl formation estimated using 2,4-dinitrophenylhydrazine. The loss of tyrosine by the MCO systems was partly due to the formation of protein-bound 3,4-dihydroxyphenylalanine. The formation of dityrosine specific to H(2)O(2)/Cu was confirmed by using poly-(Glu, Ala, Tyr) and N-acetyl-tyrosine as a substrate. The dissolved oxygen concentration in the H(2)O(2)/Cu system hardly affected the amount of dityrosine formation, suggesting that dityrosine generation by the H(2)O(2)/Cu system is independent of oxygen concentration. Moreover, the combination of copper ion with H(2)O(2) is the most effective system for dityrosine formation among various metal ions examined. The addition of reducing agents, glutathione or ascorbic acid, into the H(2)O(2)/Cu system suppressed the generation of the dityrosine moiety, suggesting effective quench of tyrosyl radicals by the reducing agents.