This research focusses on oxidative modification of biopolymers, especially of proteins. The reaction is typically initiated by the binding of a metal, such as iron or copper, to a cation binding site on the targeted protein. Oxygen reacts at that site to generate an activated species, which then oxidizes amino acid residues at the binding site. This covalent modification has been implicated in important physiologic and pathologic processes. These include the aging processes, atherosclerosis, arthritis, carcinogenesis, gene regulation, hypertension, intracellular protein turnover, oxygen toxicity, and reperfusion injury after ischemia. Determination of the actual roles of oxidative modification in these processes requires the application of specific assays for modified proteins, identification of the structural and functional changes induced by modification, and understanding of factors which modulate the rate and specificity of oxidative modification in vivo. These are the current aims of the project. We continued to utilize and refine methodologies for assessing oxidative modifications, including an automated HPLC technique capable of analyzing complex samples. This method was applied to human plasma samples to assess the effect of vitamin E supplementation, to mouse plasma during aging, to rat tissues during hyperoxic stress, to cultured human lymphocytes exposed to oxidative stresses, and to extracts of flies to study inherited differences in lifespan. We also developed a sensitive and rapid technique to locate covalent modifications in proteins. Rather than purifying individual peptides following proteolytic cleavage, the entire peptide collection was simultaneously sequenced. The method was successful in identifying (1) oxo-histidine as the oxidation product of histidine in glutamine synthetase; (2) the site of glutathiolation of carbonic anhydrase; and (3) the sites of methionine sulfoxide formation in proteins exposed to oxidizing conditions.