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

A sensitive and reproducible assay for the determination of chymotrypsin-li ke esterase activity is reported. This method is based on fluorimetric dete ction of a dansylated amino acid, 5-dimethylaminonaphthalene-1-sulfonyl-L-p henylalanine, enzymatically formed from the substrate 5-dimethylaminonaphth alene-1-sulfonyl-L-phenylalanine ethyl ester, after separation by high-perf ormance liquid chromatography using a C-18 reversed-phase column and isocra tic elution. This method is sensitive enough to measure 5-dimethylanminonap hthalene-1-sulfonyl-L-phenylalanine at concentrations as low as 40 pmol/ml yields highly reproducible results and requires less than 9.5 min per sampl e 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 muM and 0.241 p mol/mug 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 inhibit ed by N-tosyl-L-phenylalaline chloromethyl ketone. Among the mouse organs e xamined, the highest specific activity of the enzyme was found in lung. Thi s new method would be useful for clarification of the physiological role of this enzyme. (C) 2001 Elsevier Science B.V. All rights reserved.