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J. Biol. Chem., Vol. 279, Issue 50, 51836-51843, December 10, 2004

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Production of the Carbonate Radical Anion during Xanthine Oxidase Turnover in the Presence of Bicarbonate^*

Marcelo G. Bonini, Sayuri Miyamoto, Paolo Di Mascio, and Ohara Augusto

From the Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26077, São Paulo CEP 05513-970, Brazil

Xanthine oxidase is generally recognized as a key enzyme in purine catabolism, but its structural complexity, low substrate specificity, and specialized tissue distribution suggest other functions that remain to be fully identified. The potential of xanthine oxidase to generate superoxide radical anion, hydrogen peroxide, and peroxynitrite has been extensively explored in pathophysiological contexts. Here we demonstrate that xanthine oxidase turnover at physiological pH produces a strong one-electron oxidant, the carbonate radical anion. The radical was shown to be produced from acetaldehyde oxidation by xanthine oxidase in the presence of catalase and bicarbonate on the basis of several lines of evidence such as oxidation of both dihydrorhodamine 123 and 5,5-dimethyl-1-pyrroline-N-oxide and chemiluminescence and isotope labeling/mass spectrometry studies. In the case of xanthine oxidase acting upon xanthine and hypoxanthine as substrates, carbonate radical anion production was also evidenced by the oxidation of 5,5-dimethyl-1-pyrroline-N-oxide and of dihydrorhodamine 123 in the presence of uricase. The results indicated that Fenton chemistry occurring in the bulk solution is not necessary for carbonate radical anion production. Under the conditions employed, the radical was likely to be produced at the enzyme active site by reduction of a peroxymonocarbonate intermediate whose formation and reduction is facilitated by the many xanthine oxidase redox centers. In addition to indicating that the carbonate radical anion may be an important mediator of the pathophysiological effects of xanthine oxidase, the results emphasize the potential of the bicarbonate-carbon dioxide pair as a source of biological oxidants.

Received for publication, June 22, 2004 , and in revised form, September 20, 2004.

^* This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Financiadora de Estudos e Projetos, and the John Simon Guggenheim Memorial Foundation (P. D. Mascio Fellowship). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of an FAPESP fellowship.

To whom correspondence should be addressed. Tel.: 55-11-30913873; Fax: 55-11-30912186 or 55-11-38155579; E-mail: oaugusto{at}iq.usp.br.

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