A novel superoxide dismutase-based trap for peroxynitrite used to detect entry of peroxynitrite into erythrocyte ghosts

Peroxynitrite (ONOO^-) is a relatively stable oxidant produced by activated macrophages and neutrophils. To detect peroxynitrite, a novel human superoxide dismutase (SOD) trap was developed by substituting a tyrosine near the copper in the active site. The copper can catalyze nitration of this tyrosine by peroxynitrite. The nitrated tyrosine can serve as a reporter for peroxynitrite by measuring the extent of nitration with Western blots developed with a nitrotyrosine antibody. The new SOD mutant differs from bovine SOD whose sole tyrosine is far removed from the active site. Nitration of bovine SOD was second-order with respect to SOD concentration, whereas nitration of the new mutant SODs followed first-order kinetics with respect to peroxynitrite. The tyrosine SODs were used to assess whether peroxynitrite crosses erythrocyte membranes through the band 3 anion exchange protein. Tyrosine-containing SOD entrapped within normal human erythrocyte ghosts became nitrated in proportion to peroxynitrite concentration. The band 3 anion exchange protein inhibitors, phenyl isothiocyanate (PITC) and 4,4'- diisothiocyanatostilbene-2,2'-disulfonate (DIDS), inhibited up to 90% of the nitration. The erythrocyte membrane proteins, spectrin, band 3 anion exchange protein, and proteins 4.1 and 4.2, were also nitrated. Nitration of erythrocyte membrane proteins was also inhibited by PITC and DIDS. These data suggest that the band 3 anion exchange protein is the major route for the entry of peroxynitrite into erythrocytes. The ability of peroxynitrite to cross cell membranes can contribute to its toxicity by allowing access to intracellular target molecules.