A CONTINUOUS SPECTROPHOTOMETRIC ASSAY FOR UREIDOGLYCOLASE ACTIVITY WITH LACTATE-DEHYDROGENASE OR GLYOXYLATE REDUCTASE AS COUPLING ENZYME

A spectrophotometric assay for ureidoglycolase activity (both ureidogl ycolate lyase and hydrolase), based on the reduction of glyoxylate to glycolate catalyzed by glyoxylate reductase or lactate dehydrogenase w ith the stoichiometric and continuous NADH oxidation, is described. Th e assay has been optimized for the amount of coupling enzyme, reagent concentrations, buffers, and the nonenzymatic degradation of ureidogly colate. Under optimal assay conditions, ureidoglycolase activity can b e followed with either lactate dehydrogenase or glyoxylate reductase a s 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 oxidat ion was directly proportional to enzyme concentration in the assay mix ture until changes in absorbance of 0.12 per minute. This method is ea sy and reliable for the accurate determination of ureidoglycolase acti vity in crude and purified extracts from Chlamydomonas reinhardtii cel ls and no notable interferences have been detected. Since lactate dehy drogenase is much cheaper and has a lower K-m for its substrate than g lyoxylate reductase and can be used as supplied, its use as the coupli ng enzyme of choice is recommended. (C) 1994 Academic Press, Inc.