Aj. Macfadyen et al., A novel superoxide dismutase-based trap for peroxynitrite used to detect entry of peroxynitrite into erythrocyte ghosts, CHEM RES T, 12(3), 1999, pp. 223-229
Peroxynitrite (ONOO-) is a relatively stable oxidant produced by activated
macrophages and neutrophils. To detect peroxynitrite, a novel human superox
ide dismutase (SOD) trap was developed by substituting a tyrosine near the
copper in the active site. The copper can catalyze nitration of this tyrosi
ne by peroxynitrite. The nitrated tyrosine can serve as a reporter for pero
xynitrite 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 bovin
e SOD was second-order with respect to SOD concentration, whereas nitration
of the new mutant SODs followed first-order kinetics with respect to perox
ynitrite. The tyrosine SODs were used to assess whether peroxynitrite cross
es erythrocyte membranes through the band 3 anion exchange protein. Tyrosin
e-containing SOD entrapped within normal human erythrocyte ghosts became ni
trated in proportion to peroxynitrite concentration. The band 3 anion excha
nge protein inhibitors, phenyl isothiocyanate (PITC) and 4,4'-diisothiocyan
atostilbene-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 mem
brane 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 pero
xynitrite into erythrocytes. The ability of peroxynitrite to cross cell mem
branes can contribute to its toxicity by allowing access to intracellular t
arget molecules.