D-Amino oxidase is a peroxisomal enzyme that catalyzes the oxidative deami-nation of D-amino acids to give the corresponding a-keto acids, ammonia, and hydrogen peroxide. In this assay, a-ketovaleric acid from D-valine was quantitated.
The quinoxalinol derivatives of a-ketovaleric acid and ketovaleric acid (internal standard) were separated on a LiChrospher 100 RP-8 column (4 mm X 250 mm). The mobile phase was a 60:40 ratio of 0.35 M ammonium acetate and acetonitrile. The eluate was monitored by fluorescence with the excitation and emission wavelengths set at 340 and 420 nm, respectively.
The reaction mixture contained in 1.0 mL: 20 /xmol of D-Val, 10 nmol of FAD, 10 /xg of catalase, and 2 nmol of ketovaleric acid in 0.1 M pyrophosphate buffer (pH 8.5). The enzymatic reaction was started by adding 10 to 100 fjiL of beef kidney homogenate. At different times, 200 /xL aliquots were withdrawn and added to 100 fiL of ice-cold 6 M HC1. After centrifugation, a
200 fxL aliquot of o-phenylenediamine solution (0.44 M in 2 M HC1 containing 5 /¿L/mL 2-mercaptoethanol) was added. The resulting mixture was flushed with nitrogen and then heated for 15 minutes at 80°C in a sand bath. After cooling, 0.3 mL of 6 M sodium acetate was added and 20 fiL aliquots were injected into the HPLC. The assay was linear for up to 20 min and with up to 10 of a commercial preparation of D-amino acid oxidase.
Sources of enzyme were partially purified type II D-amino acid oxidase from porcine kidney, and the supernate from a beef kidney homogenate prepared in 2.5 volumes of 0.1 M pyrophosphate buffer (pH 8.5) containing 10 ij,M FAD. The supernate obtained by centrifugation was used.
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