Lisa M. Landino

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.

Hypothiocyanous acid oxidation of tubulin cysteines inhibits microtubule polymerization.

Thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. In addition, protein thiol redox reactions are increasingly identified as a mechanism to regulate protein structure and function. We assessed the effect of hypothiocyanous acid on the cytoskeletal protein tubulin. Total cysteine oxidation by hypothiocyanous and hypochlorous acids was monitored by labeling tubulin with 5-iodoacetamidofluorescein and by detecting higher molecular weight inter-chain tubulin disulfides by Western blot under nonreducing conditions. Hypothiocyanous acid induced nearly stoichiometric oxidation of tubulin cysteines (1.9 mol cysteine/mol oxidant) and no methionine oxidation was observed. Because disulfide reducing agents restored all the polymerization activity that was lost due to oxidant treatment, we conclude that cysteine oxidation of tubulin inhibits microtubule polymerization. Hypothiocyanous acid oxidation of tubulin cysteines was markedly decreased in the presence of 4% glycerol, a component of the tubulin purification buffer. Due to its instability and buffer- and pH-dependent reactivity, hypothiocyanous acid studies require careful consideration of reaction conditions.

Fluorescein-labeled glutathione to study protein S-glutathionylation

Numerous studies of S-glutathionylation of cysteine thiols indicate that this protein modification plays a key role in redox regulation of proteins. To facilitate the study of protein S-glutathionylation, we developed a synthesis and purification to produce milligram quantities of fluorescein-labeled glutathione. The amino terminus of the glutathione tripeptide reacted with fluorescein isothiocyanate readily in ammonium bicarbonate. Purification by solid phase extraction on C8 and C18 columns separated excess reactants from desired products. Both oxidized and reduced fluorescein-labeled glutathione reacted with a variety of thiol-containing proteins to yield fluorescent proteins.