Refinement and critical evaluation of a fluorometric method for measuring leucine aminopeptidase activity in serum or tissues.

The fluorometric method for measuring leucine aminopeptidase (EC 3.4.11.1) activity in biological systems, with L-leucyl--naphthylamide as the substrate [C/in. Chem. 16, 412 (1970)] was critically evaluated and refined. In this enzyme assay system, a few precautions are necessary in determining /3-naphthylamine liberated from the substrate by the enzyme action. In water, tris(hydroxymethyl)aminomethane buffer, and ethanol, /3-naphthylamine, excited at 280 nm, fluoresces at 400 nm with a greater intensity than when an excitation wavelength of 340 nm is used. However, in the presence of protein or substrate the fluorescence intensity with an excitation wavelength of 280 nm is markedly diminished, and is less intense than that with 340 nm. Therefore, an excitation wavelength of 340 nm is preferred to 280 nm, to obtain increased sensitivity. Bilirubin and hemoglobin also slightly diminish the fluorescence intensity.