title: A chromogenic assay of substrate depletion by thiol dioxygenases.
abstract: A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the nonheme mono-iron enzyme cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed standard kinetic parameters to be determined for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitates a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addition, the relative specificity of Ellman's reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.
authors.affiliation: Department of Chemistry, University of Otago, Dunedin 9054, New Zealand.
authors.forename: Matthias
authors.initials: M
authors.lastname: Fellner
authors.affiliation: Department of Chemistry, University of Otago, Dunedin 9054, New Zealand.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Doughty
authors.affiliation: Department of Chemistry, University of Otago, Dunedin 9054, New Zealand. Electronic address: gjameson@chemistry.otago.ac.nz.
authors.forename: Guy N L
authors.initials: GN
authors.lastname: Jameson
authors.affiliation: Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand. Electronic address: sigurd.wilbanks@otago.ac.nz.
authors.forename: Sigurd M
authors.initials: SM
authors.lastname: Wilbanks
mesh_terms: D000818:Animals; D002863:Chromogenic Compounds / Q000737:chemistry*; D003545:Cysteine / Q000378:metabolism; D050558:Cysteine Dioxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry*; D020536:Enzyme Activators / Q000494:pharmacology; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity
chemical_list: D002863:Chromogenic Compounds; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D004228:Dithionitrobenzoic Acid; D050558:Cysteine Dioxygenase; D003545:Cysteine
keywords: Cysteine dioxygenase; Ellman’s reagent; Enzyme assay; Thiols