Ascorbic acid reduction of microtubule protein disulfides and its relevance to protein S-nitrosylation assays |
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Authors: | Landino Lisa M Koumas Maria T Mason Courtney E Alston Jane A |
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Affiliation: | Department of Chemistry, The College of William and Mary, P.O. Box 8795, Williamsburg, VA 23187-8795, USA. lmland@wm.edu |
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Abstract: | 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. |
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Keywords: | Peroxynitrite Ascorbic acid Tubulin Microtubule-associated proteins Tau MAP2 Cysteine oxidation Disulfide bonds S-nitrosylation Biotin switch |
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