ascorbic-acid and 5-iodoacetamidofluorescein

The biotin switch assay was developed to aid in the identification of S-nitrosylated proteins in different cell types. However, our work with microtubule proteins including tubulin and its associated proteins tau and microtubule-associated protein-2 shows that ascorbic acid is not a selective reductant of protein S-nitrosothiols as described in the biotin switch assay. Herein we show that ascorbic acid reduces protein disulfides in tubulin, tau, and microtubule-associated protein-2 that are formed by peroxynitrite anion. Reduction of microtubule-associated protein disulfides by ascorbic acid following peroxynitrite treatment restores microtubule polymerization kinetics to control levels. We also show that ascorbic acid reduces the disulfide dithiobis(2-nitrobenzoic acid), a reagent commonly used to detect protein thiols. Not only do we describe a new reactivity of ascorbic acid with microtubule proteins but we expose an important limitation when using the biotin switch assay to detect protein S-nitrosylation.

Topics: Animals; Ascorbic Acid; Biotin; Blotting, Western; Brain; Chelating Agents; Cystine; Dithionitrobenzoic Acid; Fluoresceins; Glutathione; Kinetics; Microtubule-Associated Proteins; Oxidation-Reduction; S-Nitrosoglutathione; Swine; Tubulin