Simplified cysteine dioxygenase activity assay allows simultaneous quantitation of both substrate and product.
A high-performance liquid chromatography (HPLC) method for enzyme activity assays using a hydrophilic interaction liquid chromatography (HILIC) column in combination with an evaporative light scattering detector was developed. The method was used to measure the activity of the non-heme mono-iron enzyme cysteine dioxygenase. The substrate cysteine and the product cysteine sulfinic acid are very weak chromophores, making direct ultraviolet (UV) detection without derivatization rather insensitive; moreover, derivatization of cysteine is often not efficient. Using the system described, underivatized substrate and product in samples from cysteine dioxygenase activity assays could be separated and analyzed. Furthermore, it was possible to quantify cysteic acid, the noncatalytic oxidation product of cysteine sulfinic acid. Acetone was used both to stop the enzymatic reaction by protein precipitation and as an organic mobile phase, making sample preparation very easy and the assay highly reproducible.
Siakkou
Eleni
E
Department of Chemistry, University of Otago, Dunedin 9054, New Zealand.
Wilbanks
Sigurd M
SM
Jameson
Guy N L
GN
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003544:Cysteic Acid / Q000032:analysis; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis*; D050558:Cysteine Dioxygenase / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays; D008027:Light; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012542:Scattering, Radiation
D011994:Recombinant Proteins; D003544:Cysteic Acid; D050558:Cysteine Dioxygenase; D003545:Cysteine; C013461:cysteine sulfinic acid