Formation and Loss of Nitrated Proteins in Peroxynitrite-Treated Rat Skin in Vivo

Abstract

Peroxynitrite is a reactive cytotoxic species, capable of nitrating tyrosine residues to form 3-nitrotyrosine. Little is known about the formation and loss of nitrated proteins in vivo. We have measured nitrated proteins, by enzyme-linked immunosorbent assay, in rat skin after exposure to peroxynitrite. Peroxynitrite (100–200 nmol site⁻¹) was injected into the skin of anesthetized rats. At the highest dose 78.6 ± 9.5 pmol mg⁻¹ protein of nitrated BSA equivalents were measured at 4 h and a significant increase was observed for 24 h after administration in skin samples. The loss of nitrated proteins from skin appeared biphasic with an initial (t_1/2 = 2 h) and slower loss (t_1/2 = 22 h). A major nitrated protein was identified as albumin by Western blot analysis. The data demonstrate that a single exposure to peroxynitrite can lead to the presence of nitrated proteins in skin for at least 24 h. The sustained presence of nitrated proteins may influence the inflammatory process in skin disease.