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Copper, zinc‐superoxide dismutase catalyzes peroxynitrite‐mediated protein nitration in mouse tissue homogenates

We have previously demonstrated that knockout of copper,zinc‐superoxide dismutase (SOD1) blocked acetaminophen‐induced hepatic protein nitration in mice. The objective of the present study was to determine whether SOD1 was directly involved in catalyzing peroxynitrite (PN)‐mediated protein nitration. Bovine serum albumin (BSA) and liver, kidney and brain homogenates from the wild‐type (WT) and SOD knockout (SOD1‐/‐) mice (2‐month old, Cu and Zn adequate) were prepared in 50 mM potassium phosphate buffer, pH 7.8, 0.1% Triton X‐100, and 1.34 mM diethylenetriaminopenta‐acetic acid. For each reaction, 150 μg of sample proteins was incubated with 0.8 mM PN for 5 min at 37°C in the presence or absence of purified SOD1 protein (2.5 U/μl) twice. Protein nitration was analyzed by Western blot using monoclonal anti‐nitrotyrosine antibody. The PN treatment caused less (P < 0.05) protein nitration in the liver and kidney homogenates of the SOD1‐/‐ mice than those of the WT. In contrast, more protein nitration was formed in the PN‐treated brain homogenate of the SOD1‐/‐ mice than that of the WT. The addition of SOD1 protein enhanced the PN‐mediated nitrotyrosine formation in all tissue homogenates. Nitration of BSA was catalyzed by PN and the reaction was enhanced by the addition of SOD1 protein (P < 0.05). The SOD1 protein added in the BSA mixture was also nitrated by the PN treatment with a reduced activity (P < 0.05). In conclusion, knockout of SOD1 attenuated PN‐mediated protein nitration in liver and kidney homogenates, while the reaction was promoted by the addition of SOD1 protein in the mixture. [NIH DK53108 to XGL]