Cu,Zn-superoxide dismutase-driven free radical modifications: copper- and carbonate radical anion-initiated protein radical chemistry

摘要

pThe understanding of the mechanism, oxidant(s) involved and how and what protein radicals are produced during the reaction of wild-type SOD1 (Cu,Zn-superoxide dismutase) with Hsub2/subOsub2/sub and their fate is incomplete, but a better understanding of the role of this reaction is needed. We have used immuno-spin trapping and MS analysis to study the protein oxidations driven by human (h) and bovine (b) SOD1 when reacting with Hsub2/subOsub2/sub using HSA (human serum albumin) and mBH (mouse brain homogenate) as target models. In order to gain mechanistic information about this reaction, we considered both copper- and COsub3/subsup??/sup (carbonate radical anion)-initiated protein oxidation. We chose experimental conditions that clearly separated SOD1-driven oxidation via COsub3/subsup??/sup from that initiated by copper released from the SOD1 active site. In the absence of (bi)carbonate, site-specific radical-mediated fragmentation is produced by SOD1 active-site copper. In the presence of (bi)carbonate and DTPA (diethylenetriaminepenta-acetic acid) (to suppress copper chemistry), COsub3/subsup??/sup produced distinct radical sites in both SOD1 and HSA, which caused protein aggregation without causing protein fragmentation. The COsub3/subsup??/sup produced by the reaction of hSOD1 with Hsub2/subOsub2/sub also produced distinctive DMPO (5,5-dimethylpyrroline-iN/i-oxide) nitrone adduct-positive protein bands in the mBH. Finally, we propose a biochemical mechanism to explain COsub3/subsup??/sup production from COsub2/sub, enhanced protein radical formation and protection by (bi)carbonate against Hsub2/subOsub2/sub-induced fragmentation of the SOD1 active site. Our present study is important for establishing experimental conditions for studying the molecular mechanism and targets of oxidation during the reverse reaction of SOD1 with Hsub2/subOsub2/sub; these results are the first step in analysing the critical targets of SOD1-driven oxidation during pathological processes such as neuroinflammation./p