High-performance liquid chromatographic–fluorimetric assay of chymotrypsin-like esterase activity

A sensitive and reproducible assay for the determination of chymotrypsin-like esterase activity is reported. This method is based on fluorimetric detection of a dansylated amino acid, 5-dimethylaminonaphthalene-1-sulfonyl-l-phenylalanine, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-l-phenylalanine ethyl ester, after separation by high-performance liquid chromatography using a C₁₈ reversed-phase column and isocratic elution. This method is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-l-phenylalanine at concentrations as low as 40 pmol/ml, yields highly reproducible results and requires less than 9.5 min per sample for quantitation. The optimum pH for chymotrypsin-like esterase activity was 7.7–8.3. The K_m and V_max values were, respectively 25 μM and 0.241 pmol/μg protein/h with the use of enzyme extract obtained from mouse kidney. The approximate molecular mass of this enzyme was estimated to be 67 000 by gel filtration. Chymotrypsin-like esterase activity was strongly inhibited by N-tosyl-l-phenylalaline chloromethyl ketone. Among the mouse organs examined, the highest specific activity of the enzyme was found in lung. This new method would be useful for clarification of the physiological role of this enzyme.