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.
Department of Chemistry, The College of William and Mary, PO Box 8795, Williamsburg, VA 23187-8795, USA.
Hillary M
HM
Clark
Department of Chemistry, The College of William and Mary, PO Box 8795, Williamsburg, VA 23187-8795, USA.
Tara D
TD
Hagedorn
Department of Chemistry, The College of William and Mary, PO Box 8795, Williamsburg, VA 23187-8795, USA. Electronic address: lmland@wm.edu.
Lisa M
LM
Landino
D000818:Animals; D002021:Buffers; D003488:Cyanogen Bromide / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D005990:Glycerol / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects*; D020836:Protein Structure, Quaternary; D013552:Swine; D013861:Thiocyanates / Q000494:pharmacology*; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*
D002021:Buffers; D004220:Disulfides; D016877:Oxidants; D013861:Thiocyanates; D014404:Tubulin; C517667:hypothiocyanous acid; D003545:Cysteine; D003488:Cyanogen Bromide; D005990:Glycerol
Cysteine oxidation; Disulfide; Hypochlorous acid; Hypothiocyanous acid; Tubulin