Ascorbic acid reduction of microtubule protein disulfides and its relevance to protein S-nitrosylation assays.
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.
Landino
Lisa M
LM
Department of Chemistry, The College of William and Mary, P.O. Box 8795, Williamsburg, VA 23187-8795, USA. lmland@wm.edu
Koumas
Maria T
MT
Mason
Courtney E
CE
Alston
Jane A
JA
D000818:Animals; D001205:Ascorbic Acid / Q000502:physiology*; D001710:Biotin; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002614:Chelating Agents / Q000737:chemistry; D003553:Cystine / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D005452:Fluoresceins; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D026422:S-Nitrosoglutathione; D013552:Swine; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism
D002614:Chelating Agents; D005452:Fluoresceins; D008869:Microtubule-Associated Proteins; D014404:Tubulin; D003553:Cystine; D026422:S-Nitrosoglutathione; C015296:5-iodoacetamidofluorescein; D001710:Biotin; D004228:Dithionitrobenzoic Acid; D005978:Glutathione; D001205:Ascorbic Acid