Mass spectral evidence for carbonate-anion-radical-induced posttranslational modification of tryptophan to kynurenine in human Cu, Zn superoxide dismutase

Free Radic Biol Med. 2004 Dec 15;37(12):2018-26. doi: 10.1016/j.freeradbiomed.2004.08.026.

Abstract

Previously, we showed that oxidation of tryptophan-32 (Trp-32) residue was crucial for H(2)O(2)/bicarbonate (HCO(3)(-))-dependent covalent aggregation of human Cu,Zn SOD1 (hSOD1). The carbonate anion radical (CO(3)(-))-induced oxidation of Trp-32 to kynurenine-type oxidation products was proposed to cause the aggregation of hSOD1. Here we used the matrix-assisted laser desorption ionization-time of flight mass spectroscopy, high-performance liquid chromatography-electrospray ionization mass spectroscopy, and liquid chromatography mass spectroscopy methods to characterize products. Results show that a peptide region (31-36) of hSOD1 containing the Trp-32 residue (VWGSIK) is oxidatively modified to the N-formylkynurenine (NFK)- and kynurenine (Kyn)-containing peptides (V(NFK)GSIK) and (V(Kyn)GSIK) during HCO(-)-dependent peroxidase activity of hSOD1. Also, UV photolysis of a cobalt complex that generates authentic CO(3)(-) radical induced a similar product profile from hSOD1. Similar products were obtained using a synthetic peptide with the same amino acid sequence (i.e., VWGSIK). We propose a mechanism involving a tryptophanyl radical for CO(3)(-)-induced oxidation of Trp-32 residue (VWGSIK) in hSOD1 to V(NFK)GSIK and V(Kyn)GSIK.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Anions / chemistry
  • Anions / pharmacology
  • Carbonates / chemistry
  • Carbonates / pharmacology*
  • Chromatography, High Pressure Liquid
  • Copper / metabolism
  • Electron Spin Resonance Spectroscopy
  • Free Radicals / chemistry
  • Free Radicals / pharmacology*
  • Humans
  • Kynurenine / chemistry
  • Kynurenine / metabolism*
  • Models, Molecular
  • Oxidation-Reduction / drug effects
  • Protein Processing, Post-Translational / drug effects*
  • Protein Structure, Tertiary
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Superoxide Dismutase / chemistry*
  • Superoxide Dismutase / metabolism*
  • Tryptophan / chemistry
  • Tryptophan / metabolism*
  • Zinc / metabolism

Substances

  • Anions
  • Carbonates
  • Free Radicals
  • Kynurenine
  • Copper
  • Tryptophan
  • Superoxide Dismutase
  • Zinc