L-Amino acid oxidase (LAO; 2 L-amino acid:O 2 oxidoreductase, EC 1.4.3.2) catalyzes the oxidative deamination of L-amino acids to produce the corresponding 2-oxo acids, hydrogen peroxide and ammonia. Many methods have been reported for the determination of LAO activity. They include measurement of oxygen consumption using a classic Warburg manometer (1, 2) or an oxygen-sensitive electrode (3-5) and measurements of hydrogen peroxide (6 -11), ammonia (12-15), and 2-oxo acid production (16,17). Such measurements of oxygen consumption require a well-controlled assay environment and are not suitable for rapidly processing numerous samples. Of the above methods, those detecting hydrogen peroxide production seem to be reasonably simple, sensitive, and reproducible. In these assays, LAO acts on its substrates to cogenerate H 2 O 2 , which is detected by the horseradish peroxidase (HRP)catalyzed oxidation of H 2 O 2 -sensitive probes, such as o-dianisidine (ODA) (6), guaiacol (7), aminoantipyrine (8), scopoletin (9), and 3,3ะ,5,5ะ-tetramethyl-benzidine (10).
Here we describe a spectrophotometric microplate assay based on the HRP-coupled reaction using o-phenylenediamine (OPD) as an H 2 O 2 probe, considering that in many laboratories absorption-detecting microplate readers are more available than fluorescencedetecting ones, although fluorometric measurement is more sensitive than spectrophotometric measurement.