Elsevier

Methods in Enzymology

Volume 269, 1996, Pages 201-209
Methods in Enzymology

[19] Antibodies that recognize nitrotyrosine

https://doi.org/10.1016/S0076-6879(96)69022-3Get rights and content

Publisher Summary

This chapter discusses the antibodies that recognize nitrotyrosine. Peroxynitrite is a potent oxidant that reacts with a variety of biological molecules, oxidizing thiols, initiating lipid peroxidation, inactivating ion channels, and damaging DNA. Tyrosine nitration can inactivate enzymes and receptors that depend on tyrosine residues for their activity. Nitration prevents phosphorylation of tyrosine residues important for signal transduction. Nitration of phenolics by peroxynitrite occurs spontaneously however is also catalyzed by low molecular mass transition metals and by superoxide dismutase and other metalloproteins. Nitrotyrosine in proteins occurs spontaneously when peroxynitrite is added to purified solutions of protein. The existence of nitrotyrosine is important indirect evidence that peroxynitrite is involved in pathophysiological processes. Nitration of tyrosine residues in purified nonheme proteins is relatively easy to detect by visible spectroscopy owing to the characteristic yellow color. Nitrotyrosine can be identified by gas chromatography, mass spectroscopy, and high-performance liquid chromatography (HPLC). Peroxynitrite is prepared by quenching the reaction of acidified nitrite and hydrogen peroxide with an excess of sodium hydroxide.

References (11)

  • H. Ohshima et al.

    Food Chem. Toxicol.

    (1990)
  • H. Ischiropoulos et al.

    Arch. Biochem. Biophys.

    (1992)
  • B.L. Martin et al.

    J. Biol. Chem.

    (1990)
  • J.S. Beckman et al.

    Arch. Biochem. Biophys.

    (1992)
  • J.S. Beckman et al.

    Methods Enzymol.

    (1994)
There are more references available in the full text version of this article.

Cited by (185)

  • Detection and quantification of nitric oxide-derived oxidants in biological systems

    2019, Journal of Biological Chemistry
    Citation Excerpt :

    Poly- and monoclonal antibodies have been raised and are now commercially available. Immunohistochemical methods, immunoblotting techniques, and ELISAs have been developed and applied to the study of biological samples (184, 231–234). The typical readout of antibody-based methods is the difference in immunoreactivity in samples versus controls.

  • Peroxynitrite supports a metabolic reprogramming in merlin-deficient Schwann cells and promotes cell survival

    2019, Journal of Biological Chemistry
    Citation Excerpt :

    VDAC (1:1,000, Cell Signaling Technology, catalog number 4866, RRID:AB_2272627); Hsp60 (1:1,000, Cell Signaling Technology, catalog number 12165, RRID:AB_2636980); complex IV (1;1,000, Cell Signaling Technology, catalog number 4850, RRID:AB_2085424); complex I (1:5,000, ThermoFisher Scientific, catalog number 459100, RRID:AB_2532223), cytochrome c (1:1,000, Cell Signaling Technology, catalog number 4280S, RRID:AB_10695410); pyruvate dehydrogenase (1:1,000, Cell Signaling Technology, catalog number 3205S, RRID:AB_2162926); SDHA (1:1,000, Cell Signaling Technology, catalog number 11998); Hsp90 (1:2,000, Santa Cruz Biotechnology, catalog number SC-7947); β-actin (1:30,000, Cell Signaling Technology, catalog number 3700, RRID:AB_2242334); α-tubulin (1:30,000, Cell Signaling Technology, catalog number 3873, RRID:AB_1904178); merlin (1:2,000, Cell Signaling Technology, catalog number 12888, RRID:AB_2650551); MnSOD (1:1,000, Millipore Sigma, catalog number 06-984); nNOS (Santa Cruz Biotechnology, catalog number sc-648, RRID:AB_630935); iNOS (Santa Cruz Biotechnology, catalog number sc-650, RRID:AB_631831); and eNOS (1:1,000, BD Biosciences, catalog number 610296, RRID:AB_397690). The mouse mAb against nitrated Hsp90 (1:2,000) was developed and characterized in-house (28), as well as the rabbit polyclonal antibody against nitrotyrosine (1:2,000) (70). Human Schwann cells mitochondrial content was assayed by measuring MitoTracker Green FM fluorescence in live cells according to the manufacturer's instructions (200 nm, Molecular Probes/ThermoFisher Scientific, catalog number M7514) using a Synergy H1 multi-mode reader (BioTek, Winooski, VT).

View all citing articles on Scopus
View full text