UNIT 6.15 Measurement of Cysteine Dioxygenase Activity and Protein Abundance
Published Online: 1 NOV 2008
Copyright © 2008 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Toxicology
How to Cite
Stipanuk, M. H., Dominy Jr., J. E., Ueki, I. and Hirschberger, L. L. 2008. Measurement of Cysteine Dioxygenase Activity and Protein Abundance. Current Protocols in Toxicology. 38:6.15:6.15.1–6.15.25.
- Published Online: 1 NOV 2008
- Published Print: NOV 2008
Cysteine dioxygenase is an iron (Fe2+)–dependent thiol dioxygenase that uses molecular oxygen to oxidize the sulfhydryl group of cysteine to generate 3-sulfinoalanine (commonly called cysteinesulfinic acid). Cysteine dioxygenase activity is routinely assayed by measuring cysteinesulfinate formation from substrate l-cysteine at pH 6.1 in the presence of ferrous ions to saturate the enzyme with metal cofactor, a copper chelator to diminish substrate oxidation, and hydroxylamine to inhibit pyridoxal 5′-phosphate-dependent degradation of product. The amount of cysteine dioxygenase may be measured by immunoblotting. Upon SDS-PAGE, cysteine dioxygenase can be separated into two major bands, with the upper band representing the 23-kDa protein and the lower band representing the mature enzyme that has undergone formation of an internal thioether cross-link in the active site. Formation of this cross-link is dependent upon the catalytic turnover of substrate and produces an enzyme with a higher catalytic efficiency and catalytic half-life. Curr. Protoc. Toxicol. 38:6.15.1-6.15.25. © 2008 by John Wiley & Sons, Inc.
- cysteine dioxygenase;
- cysteinesulfinic acid;