ANALYSIS OF NATIVE HUMAN PLASMA-PROTEINS AND HEMOGLOBIN FOR THE PRESENCE OF BITYROSINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Generation of reactive oxygen species in vivo results in oxidative-dam age to cellular components, including proteins. Due to the relatively long half-lives of several blood proteins the cumulative formation of oxidatively damaged proteins might serve as a biomarker for reactive o xygen species formation. The most prominent sources of reactive oxygen species in vivo are site-specific metal ion-catalyzed reactions of th e Fenton and Haber-Weiss types and the H2O2/peroxidase system. In vitr o oxidation of L-tyrosine using a peroxidase or Cu++/H2O2 system gives rise to the formation of a highly fluorescent substance, bityrosine. High-performance liquid chromatography (HPLC) analysis of acid hydroly zed serum albumin after oxidation with peroxidase/H2O2 or with Cu++/H2 O2 showed that bityrosine had been formed whereas oxidation of this pr otein with Fe(III)/ascorbate did not result in the formation of bityro sine. Bityrosine could not be detected in human plasma proteins or hae moglobin with the detection limit of one pmol per mg protein.