High-performance liquid chromatographic-fluorimetric assay for cathepsin A(lysosomal protective protein) activity

A rapid and sensitive assay for the determination of cathepsin A activity i s reported. This method is based on fluorimetric detection of a dansylated peptide, 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe enzymatically formed f rom the substrate 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe-L-Leu, after separation by high-performance liquid chromatography using a C-18 reversed- phase column and isocratic elution. This method is sensitive enough to meas ure 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe at concentrations as low as 300 fmol, yields highly reproducible results and requires less than 7.0 mi n per sample for separation and quantitation, The optimum pH for cathepsin A activity was 4.5-5.0. The K-m and V-max values were respectively 14.9 mu M and 27.91 pmol/mu g/h with the use of enzyme extract obtained from mouse kidney. Cathepsin A activity was strongly inhibited by Ag+, Hg2+, diisoprop ylfluorophosphate and p-chloromercuriphenylsulphonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in kidney. The sensitivity and selectivity of this method will aid in effo rts to examine the physiological role of this peptidase. (C) 1999 Elsevier Science B.V. All rights reserved.