Analytical Biochemistry 1996-05-01

Synthesis of peroxynitrite in a two-phase system using isoamyl nitrite and hydrogen peroxide.

R M Uppu, W A Pryor

Index: Anal. Biochem. 236(2) , 242-9, (1996)

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Abstract

A new method for the preparation of high concentrations of peroxynitrite (up to 1 M) is described. The synthesis uses a two-phase system and involves a displacement reaction by the hydroperoxide anion (in the aqueous phase) on isoamyl nitrite (in the organic phase). The product peroxynitrite remains in the aqueous phase, whereas isoamyl alcohol forms a new organic phase along with the unreacted isoamyl nitrite. The aqueous phase contains some 0.15 M isoamyl alcohol and the unreacted hydrogen peroxide, but no isoamyl nitrite. Removal of isoamyl alcohol or traces of isoamyl nitrite is accomplished by washing the aqueous phase with dichloromethane, chloroform, or hexane. A near total removal of hydrogen peroxide is then achieved by passing the solutions through a short column of manganese dioxide. The peroxynitrite in these postprocessed solutions has broad absorption spectrum with a maximum around 302 nm, follows a characteristic first-order decomposition at pH 7.2 and 25 degrees C (k = 0.34 +/- 0.1 s-1), and reacts with organic compounds to give either nitrated or one-electron transfer products. When stored frozen at -20 degrees C, these peroxynitrite solutions decompose at a rate of about 1.7 % per day and should be used within 2-4 weeks. For short-term storage of about 1 week or less, these solutions can be stored at refrigerator temperatures (approximately 5 degrees C) where peroxynitrite has a half-life of about 7 days.