Diffusion of peroxynitrite across erythrocyte membranes

Diffusion of peroxynitrite across erythrocyte membranes

^†Department of Physical Biochemistry, Facultad de Ciencias, and ^‡Department of Biochemistry, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

Edited by Irwin Fridovich, Duke University Medical Center, Durham, NC, and approved January 26, 1998 (received for review October 28, 1997)

Abstract

Peroxynitrite anion (ONOO⁻) 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 k ₂ = (10.4 ± 0.3) × 10³ M⁻¹⋅s⁻¹ 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 (pK_a = 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.

Footnotes

↵ § To whom reprint requests should be addressed at: Depto. de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. Gral. Flores 2125, 11800 Montevideo, Uruguay. e-mail: rradi{at}fmed.edu.uy.
This paper was submitted directly (Track II) to the Proceedings Office.
Abbreviations: ONOO⁻, peroxynitrite anion; ONOOH, peroxynitrous acid; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; ^·NO, nitric oxide; O_2⨪, superoxide anion; RBC, red blood cells; BCECF, 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein; NEM, N-ethylmaleimide; dtpa, diethylenetriaminepentaacetic acid.
↵ ¶ The International Union of Pure and Applied Chemistry recommended names for peroxynitrite anion (ONOO⁻), peroxynitrous acid (ONOOH), and nitric oxide (^·NO) are oxoperoxonitrate(1−), hydrogen oxoperoxonitrate, and nitrogen monoxide, respectively. The term peroxynitrite is used to refer to the sum of ONOO⁻ and ONOOH.
↵ ‖ The concentration of hemoglobin used in intact erythrocyte suspension experiments will be expressed as the concentration of oxyhemoglobin (concentration of heme, μM) released after lysis, which is achieved by pelleting the cells and resuspending them in the same volume of distilled water.