This research focusses on metal-catalyzed oxidative modification of biopolymers, especially of proteins. The reaction is enabled 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 oxidation leads to an apparently irreversible, covalent modification of proteins which 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 this project. Emphasis was placed in the last year on the development of methodology for the detection of oxidative modification of proteins. Two methods were successfully established. The first is a sensitive assay for both HPLC and immunochemical detection of oxidatively modified proteins. The immunochemical method allows detection of picomole quantities of oxidized proteins, making it sensitive enough for application to human fluids or biopsy specimens. It was used to determine the relative sensitivity of plasma proteins to oxidative attack. It revealed a particular sensitivity of fibrinogen to attack, a finding of potential significance in inflammatory conditions. The second method facilitates identification of the specific residues which are oxidized in sensitive proteins. The control and oxidized proteins are cleaved by chemical or enzymatic means while bound to a resin. The resulting collection of peptides is then sequenced on the resin. Comparison of the cycle yields allows identification of the altered residues. The method was applied to oxidatively modified glutamine synthetase to establish that oxidation occurs at the two metal binding sites, and that several residues are attacked at each site.
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