A specific kinetic assay for tripeptide aminopeptidase in serum

This is a method for measuring tripeptide aminopeptidase (EC 3.4.11.4) activity in serum. L- Leucylglycylglycine is used as substrate, and the reaction is followed by monitoring the absorbance increase at 340 nm when NAD+ is reduced to NADH in the presence of an excess of leucine dehydrogenase. This principle allows kinetic determination of the enzyme without interference by carboxypeptidases. Amastatin is added to the reaction mixture to prevent nonspecific hydrolysis of the substrate catalyzed by other aminopeptidases. As final reaction concentrations we recommend (per liter): 100 mmol of Tris buffer (pH 8.2), 4.0 mmol of L- leucylglycylglycine , 10 kU of leucine dehydrogenase, 3.8 mmol of NAD+, and 85 mumol of amastatin . The assay is suited to modern enzyme analyzers and has high precision.