Diffusion of peroxynitrite across erythrocyte membranes (nitric oxideysuperoxidey4,4*-diisothiocyanatostilbene-2,2*-disulfonic acidyanion transportyhemoglobin)

Peroxynitrite anion (ONOO2) is a reactive species of increasingly recognized biological relevance that contributes to oxidative tissue damage. At present, however, there is limited knowledge about the mechanisms of peroxynitrite diffusion through biological compartments. In this work we have studied the diffusion of peroxynitrite across erythrocyte membranes. In solution, peroxynitrite rapidly reacts with oxyhemoglobin to yield methemoglobin, with k2 5 (10.4 6 0.3) 3 103 M21zs21 at pH 7.4 and 25°C. Addition of peroxynitrite to intact erythrocytes caused oxidation of intracellular oxyhemoglobin to methemoglobin. Oxidation yields in red blood cells at pH 7.0 were approximately 40% of those obtained in solution, which results mostly from competition of other cytosolic components for peroxynitrite. Indeed, rather small differences were observed between oxidation yields in lysates compared with intact erythrocytes, in particular at acidic and neutral pH values, indicating that membrane was not precluding peroxynitrite diffusion. Incubation of erythrocytes at pH 7.0 with 4,4*-diisothiocyanatostilbene-2,2*disulfonic acid (DIDS), a specific inhibitor of anion exchange, resulted in up to 50% inhibition of oxyhemoglobin oxidation by peroxynitrite. More protection by DIDS was achieved at alkaline pH, while no effect was observed at pH 5.5, where 95% of peroxynitrite is in the acidic form, ONOOH (pKa 5 6.8). In addition, peroxynitrite caused nitration of intracellular hemoglobin, in a process that was enhanced in thiol-depleted erythrocytes. Our results indicate that peroxynitrite is able to cross the erythrocyte membrane by two different mechanisms: in the anionic form through the DIDS-inhibitable anion channel, and in the protonated form by passive diffusion. Peroxynitrite anion (ONOO2), the product of the reaction between superoxide anion (O2 .) and nitric oxide (zNO) and its conjugated acid, peroxynitrous acid (ONOOH) (pKa 5 6.8; refs. 1 and 2), are potent oxidants known to be formed in vivo (3–5).¶ At physiological pH, 80% of peroxynitrite is present in the anionic form, but once protonated it rapidly rearranges to nitrate (kobs 5 0.9 s21 at 37°C and pH 7.4; ref. 2). The biological half-life of peroxynitrite is low (,0.1 s) because of the protoncatalyzed isomerization to nitrate but mainly because of reactions with target molecules. zNO 1 O2 . 3ONOO^ONOOH^ONOOH*3NO3 The chemistry of peroxynitrite is complex and strongly pH dependent (6, 7). Peroxynitrite can directly oxidize a variety of biomolecules (1, 8–14) by oneor two-electron oxidations or rearrange after protonation to a highly oxidizing species (ONOOH*, E0 5 12.1 V) with a reactivity close to that of hydroxyl radical. In addition, peroxynitrite is capable of nitrating aromatics (15, 16) in a process that can be enhanced by metal centers and CO2 (17). The reaction between peroxynitrite and CO2 leads to the formation of nitrosoperoxocarbonate (ONO2CO2 ), a reactive intermediate that represents one of the major routes of peroxynitrite consumption in biological systems