Open AccessMeasurement of Cysteine Dioxygenase Activity and Protein Abundance
Author(s) -
Stipanuk Martha H.,
Dominy Jr. John E.,
Ueki Iori,
Hirschberger Lawrence L.
Publication year - 2008
Publication title -
current protocols in toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.449
H-Index - 23
eISSN - 1934-9262
pISSN - 1934-9254
DOI - 10.1002/0471140856.tx0615s38
Subject(s) - dioxygenase , cysteine , chemistry , hydroxylamine , enzyme , substrate (aquarium) , thiol , cofactor , catalysis , biochemistry , stereochemistry , biology , ecology
Cysteine dioxygenase is an iron (Fe 2+ )–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.