Reactions of Peroxynitrite and Nitrite with Organic Molecules and Hemoglobin

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De-jia Li, Run-wei Yan, Hua Luo, and Guo-lin Zou^*

College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, PR China; fax: +86-27-87669560; E-mail: dejia_li@hotmail.com

^* To whom correspondence should be addressed.

Received September 9, 2004; Revision received November 15, 2004

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In this work, the reactions of nitrite (NO₂^-) and peroxynitrite (ONOO^-) with organic molecules as well as with hemoglobin (Hb) were examined and the potential interference with the detection of hydrogen peroxide and Hb was investigated. ONOO^- at low concentrations (35-140 µM) induced a concentration-dependent oxidation of o-phenylenediamine and guaiacol, and this process can be improved by the addition of Hb in a concentration-dependent manner. This enhancing effect of Hb was possibly due to the formation of such highly reactive species as ferrylHb during the reaction of ONOO^- and Hb. NO₂^- also oxidized the aromatic amine o-phenylenediamine, but its efficiency was much lower than that of ONOO^-. A 300-fold excess of NO₂^- over hydrogen peroxide inhibited the oxidation of Pyrogallol Red mediated by hydrogen peroxide and Hb, which was due in part to the reaction of NO₂^- with Hb ferryl species compound I and compound II and the phenoxyl radical. These data suggest that ONOO^- and NO₂^- can interfere with the detection of hydrogen peroxide. The overestimation or underestimation of the hydrogen peroxide detected is dependent upon the organic molecule utilized for detection and the relative rate of NO₂^-, superoxide, and ONOO^- generation.

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KEY WORDS: peroxynitrite, nitrite, hemoglobin, o-phenylenediamine

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