Nitrated and Oxidized Products of a Single Tryptophan Residue in Human Cu,Zn-Superoxide Dismutase Treated with Either Peroxynitrite-Carbon Dioxide or Myeloperoxidase-Hydrogen Peroxide-Nitrite

Abstract

We reported previously that a single tryptophan residue, Trp32, in human Cu,Zn-superoxide dismutase is specifically modified by peroxynitrite-CO2 [Yamakura et al. (2001) Biochim. Biophys. Acta 1548, 38-46]. In this study, we modified Cu,Zn-superoxide dismutase by using a combination of myeloperoxidase, hydrogen peroxide, and nitrite. The modified enzyme showed no loss of copper and zinc, and 15% less enzymatic activity. Trp32 was the only significant amino acid lost. After trypsin digestion of the modified SOD with peroxynitrite-CO2 and the myeloperoxidase system, six newly appearing peptides containing tryptophan derivatives were observed on microLC-ESI-Q-TOF mass analyses and HPLC with a photodiode-array detector. The derivatives of the tryptophan residue exhibiting mass increases of 4, 16 (2 peaks), 32, 45 (major), and 45 Da (minor) were identified as kynurenine, oxindole-3-alanine and its derivatives, dihydroxytryptophan, 6-nitrotryptophan and 5-nitrotryptophan, respectively. We further identified 6-nitrotryptophan from the 1H-NMR spectrum for the pronase-digested product and calculated the yield of 6-nitrotryptophan as being about 30% for each of the modification methods. The tryptophan residue in the modified human Cu,Zn-superoxide dismutase gave the same spectra for the products including 6-nitrotryptophan as the major nitrated product with the two different modification systems.

Disulfide Bond As a Switch for Copper-Zinc Superoxide Dismutase Activity in Asthma

“…Consistent with these reports, in our study, a peptide-containing modified histidine (2-oxo histidine at H120) was found, but at very low abundance in asthma and control samples, indicating that nonspecific oxidation or fragmentation was not a primary cause of activity loss. Structurally, human CuZnSOD does not have a tyrosine residue, a potential target of nitrative modification; rather, it has a single tryptophan residue (W32), the modification of which has been described to cause up to 15% loss of activity (48). In our study, several peptides with oxidative modification of tryptophan were identified (Table 2); however, again the amount was similar among control and asthmatic samples, indicating that nonspecific oxidation was not the mechanism of CuZnSOD inactivation.…”

Peroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin

“…Calibration curves were generated using authentic commercial standards or authentic samples of 6-nitroTrp, which was synthesized as described previously [29]. The identity and purity of this material was confirmed by 1 H NMR spectroscopy and comparison with literature data [30]. In order to examine the potential involvement of free Cys residues in the observed reactions, studies were carried out using Cys-specific fluorescent probes.…”

Reactive nitrogen species in cellular signaling

“…69 Hsp90 has multiple cellular functions and is one of the most common proteins in the cell, making up 1-2% of the total protein profile in the cytoplasm. This chaperone has a very broad range of functions: protein folding, stabilization, translocation, activity modulation, and degradation.…”