A continuous spectrophotometric assay for ureidoglycolase activity with lactate dehydrogenase or glyoxylate reductase as coupling enzyme.
A spectrophotometric assay for ureidoglycolase activity (both ureidoglycolate lyase and hydrolase), based on the reduction of glyoxylate to glycolate catalyzed by glyoxylate reductase or lactate dehydrogenase with the stoichiometric and continuous NADH oxidation, is described. The assay has been optimized for the amount of coupling enzyme, reagent concentrations, buffers, and the nonenzymatic degradation of ureidoglycolate. Under optimal assay conditions, ureidoglycolase activity can be followed with either lactate dehydrogenase or glyoxylate reductase as coupling enzyme and reaction can be started by addition of substrate or enzyme extract. Once the reaction was started, NADH oxidation was linear with time after a lag phase of 1-2 min. This linear NADH oxidation was directly proportional to enzyme concentration in the assay mixture until changes in absorbance of 0.12 per minute. This method is easy and reliable for the accurate determination of ureidoglycolase activity in crude and purified extracts from Chlamydomonas reinhardtii cells and no notable interferences have been detected. Since lactate dehydrogenase is much cheaper and has a lower Km for its substrate than glyoxylate reductase and can be used as supplied, its use as the coupling enzyme of choice is recommended.
Pineda
M
M
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Córdoba, Spain.
Piedras
P
P
Cárdenas
J
J
D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D019760:Amidine-Lyases; D000818:Animals; D002384:Catalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D006016:Glycolates / Q000737:chemistry*; D006038:Glyoxylates / Q000737:chemistry*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D016014:Linear Models; D008190:Lyases / Q000032:analysis* / Q000737:chemistry; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D006016:Glycolates; D006038:Glyoxylates; C031149:glycolic acid; D000429:Alcohol Oxidoreductases; C020830:glyoxylate reductase; D007770:L-Lactate Dehydrogenase; D008190:Lyases; D019760:Amidine-Lyases; C033757:ureidoglycollate lyase; C031150:glyoxylic acid