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

Andrew J Macfadyen, Christopher Reiter, Yingxin Zhuang, Joseph S. Beckman

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)223-229
Number of pages7
JournalChemical Research in Toxicology
Volume12
Issue number3
DOIs
StatePublished - Mar 1 1999

Fingerprint

Peroxynitrous Acid
Erythrocyte Membrane
Superoxide Dismutase
Nitration
Chloride-Bicarbonate Antiporters
Erythrocyte Anion Exchange Protein 1
Tyrosine
Anions
Copper
Catalytic Domain
Membrane Proteins
Antiporters
Spectrin
Macrophages
Cell membranes
Oxidants
Toxicity
Neutrophils
Erythrocytes
Western Blotting

ASJC Scopus subject areas

  • Toxicology

Cite this

A novel superoxide dismutase-based trap for peroxynitrite used to detect entry of peroxynitrite into erythrocyte ghosts. / Macfadyen, Andrew J; Reiter, Christopher; Zhuang, Yingxin; Beckman, Joseph S.

In: Chemical Research in Toxicology, Vol. 12, No. 3, 01.03.1999, p. 223-229.

Research output: Contribution to journalArticle

Macfadyen, Andrew J ; Reiter, Christopher ; Zhuang, Yingxin ; Beckman, Joseph S. / A novel superoxide dismutase-based trap for peroxynitrite used to detect entry of peroxynitrite into erythrocyte ghosts. In: Chemical Research in Toxicology. 1999 ; Vol. 12, No. 3. pp. 223-229.
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