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J Biol Chem, Vol. 274, Issue 40, 28233-28239, October 1, 1999

Bicarbonate Enhances the Peroxidase Activity of Cu,Zn-Superoxide Dismutase
ROLE OF CARBONATE ANION RADICAL

From the Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

We examined the effect of bicarbonate on the peroxidase activity of copper-zinc superoxide dismutase (SOD1), using the nitrite anion as a peroxidase probe. Oxidation of nitrite by the enzyme-bound oxidant results in the formation of the nitrogen dioxide radical, which was measured by monitoring 5-nitro--tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H₂O₂-mediated nitration of -tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H₂O₂-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H₂O₂-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H₂O₂, suggest that SOD1/H₂O₂ oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H₂O₂ by bicarbonate is attributed to the intermediary role of the diffusible oxidant, the carbonate radical anion. We conclude that, contrary to a previous report (Sankarapandi, S., and Zweier, J. L. (1999) J. Biol. Chem. 274, 1226-1232), bicarbonate is not required for peroxidase activity mediated by SOD1 and H₂O₂. However, bicarbonate enhanced the peroxidase activity of SOD1 via formation of a putative carbonate radical anion. Biological implications of the carbonate radical anion in free radical biology are discussed.

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