SPECTROPHOTOMETRIC ASSAY OF AMINOPEPTIDASE WITH THERMOSTABLE ALANINE DEHYDROGENASE FROM BACILLUS-STEAROTHERMOPHILUS

A spectrophotometric assay for aminopeptidase in biological fluids has been developed using a thermostable alanine dehydrogenase (AlaDH, EC 1.4.1.1) from Bacillus stearothermophilus, L-Alanine produced by the aminopeptidase with L-alanine amide or L-leucyl-L-alanine as the substrate, is oxidatively deaminated to pyruvate in the presence of NAD(+) by the action of AlaDH. Aminopeptidase activity was continuously monitored by measuring the absorbance at 340 nm corresponding to NADH production. The measured aminopeptidase activity was found to be linear up to 700-800 units/liter at 37 degrees C. The reagents were stable in solution for at least 4 weeks at 10 degrees C. This method was applicable to the assay of serum aminopeptidase. The assays had a high degree of precision even at low aminopeptidase activity and correlated well with the conventional assay methods.