A colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters.
Urate oxidase (E.C.1.7.3.3; uricase, urate oxygen oxidoreductase) is an enzyme of the purine breakdown pathway that catalyzes the oxidation of uric acid in the presence of oxygen to allantoin and hydrogen peroxide. A 96-well plate assay measurement of urate oxidase activity based on hydrogen peroxide quantitation was developed. The 96-well plate method included two steps: an incubation step for the urate oxidase reaction followed by a step in which the urate oxidase activity is stopped in the presence of 8-azaxanthine, a competitive inhibitor. Hydrogen peroxide is quantified during the second step by a horseradish peroxidase-dependent system. Under the defined conditions, uric acid, known as a radical scavenger, did not interfere with hydrogen peroxide quantification. The general advantages of such a colorimetric assay performed in microtiter plates, compared to other methods and in particular the classical UV method performed with cuvettes, are easy handling of large amounts of samples at the same time, the possibility of automation, and the need for less material. The method has been applied to the determination of the kinetic parameters of rasburicase, a recombinant therapeutic enzyme.
Fraisse
Laurent
L
Biochemistry Unit, Sanofi-Synthelabo Recherche, Innopole, Voie No. 1, BP 137, 31676 Labège Cedex, France. laurent.fraisse@sanofi-synthelabo.com
Bonnet
Marie Claude
MC
de Farcy
Jacques Philippe
JP
Agut
Christophe
C
Dersigny
Didier
D
Bayol
Alain
A
D000481:Allantoin / Q000138:chemical synthesis; D002138:Calibration; D003124:Colorimetry / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis*; D007700:Kinetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012016:Reference Values; D012044:Regression Analysis; D015203:Reproducibility of Results; D014503:Urate Oxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D014527:Uric Acid / Q000737:chemistry; D014970:Xanthines / Q000494:pharmacology
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D014970:Xanthines; C469709:rasburicase; C018221:8-azaxanthine; D014527:Uric Acid; D000481:Allantoin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D014503:Urate Oxidase