Protein thiol modification by peroxynitrite anion and nitric oxide donors.
Oxidation and modification of protein cysteines can have profound effects on protein structure and function. Using tubulin and microtubule-associated proteins (MAP) tau and MAP2 as examples, this chapter summarizes methods employed to characterize total cysteine modification using thiol-specific reagent 5-iodoacetamido-fluorescein labeling. Western blot analysis of peroxynitrite-damaged tubulin under nonreducing conditions reveals the formation of higher molecular weight dimers and tetramers. Disulfides in microtubule proteins are substrates for both the thioredoxin reductase system and the glutaredoxin/glutathione reductase system. The yield of disulfides formed by peroxynitrite anion is quantitated by monitoring the oxidation of NADPH, a cofactor required by the thioredoxin reductase system. Treatment of proteins with S-nitrosothiols, including S-nitrosoglutathione and S-nitroso-N-acetyl penicillamine, can yield either disulfides or protein S-nitrosation. In the case of tubulin, both types of cysteine modification were detected.
Landino
Lisa M
LM
Department of Chemistry, The College of William and Mary, Williamsburg, Virginia, USA.
D000818:Animals; D000838:Anions / Q000378:metabolism*; D006801:Humans; D020030:Nitric Oxide Donors / Q000737:chemistry* / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects* / Q000502:physiology*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013113:Spin Labels; D019097:Spin Trapping; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D000838:Anions; D020030:Nitric Oxide Donors; D011506:Proteins; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D030421:Peroxynitrous Acid