Work

Measurement of Cysteine Dioxygenase Activity and Protein Abundance

Year: 2008

Type: article

Abstract: 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.

Source: Current Protocols in Toxicology

DOI: 10.1002/0471140856.tx0615s38

Authors: Martha H. Stipanuk, John E. Dominy Jr., Iori Ueki, Lawrence L. Hirschberger

Institution: Cornell University

Language: English

Topic: Redox biology and oxidative stress

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funder: National Institute of Diabetes and Digestive and Kidney Diseases

Award: Nutritional Regulation of Cysteine Dioxygenase