Fluorometric microassays for the determination of cathepsin L and cathepsin S activities in tissue extracts

We established a continuous semi-microassay, and for large-scale studies bo th a stopped and a continuous microtiter plate assay for the fluorometric d etermination of cathepsin L and cathepsin S activities in body fluids, tiss ues or cell extracts in the presence of cathepsin B. For the detection of e nzymatic activities we used the synthetic substrate Z-Phe-Arg-AMC, and for discrimination between cathepsin L, S and cathepsin B the specific inhibito r CA-074 for blocking interfering cathepsin B activities was applied. Furth ermore, we took advantage of the stability of cathepsin S at pH 7.5 for fur ther differentiation between cathepsin L and cathepsin S activities. The ki netic assays were characterized in terms of imprecision, analytical sensiti vity, accuracy and substrate concentration. The within-run coefficients of variation were found to be 4.9%-7.2% for the continuous semi-microassay, 10 .3%-11.7% for the stopped, and 4.5%-11.8% for the continuous microtiter pla te assay. The between days coefficients of variation for the continuous sem i-microassay were 8.1%-8.9%, while for the stopped and continuous microtite r plate assays the coefficients were 11.2%-13.5% and 5.8%-12.2%, respective ly. Compared to the continuous semi-microassay, the stopped and the continu ous microtiter plate assays showed 3-fold and 11-fold higher sensitivity, r espectively. Comparison between the continuous enzyme activity assays at su bstrate concentrations of 40 mu M and 200 mu M demonstrated a significant c orrelation of r = 0.97 and r = 0.99, respectively.