| Assay Method Information | |
| | Cathepsn S Inhibition Assay |
| Description: | Enzyme assays were run using fluorescence resonance energy transfer-based substrates, (Aedens)EKARVLAEAA(Dabcyl)K-amide and cathepsin S cleaves between amino acids Leu-6 and Ala-7. The fluorescence of the aedens group is quenched by the dabcyl moiety in the intact peptide. Upon cleavage by cathepsin S, the quenching is released and the fluorescence of the aedens group can be measured. The increase in fluorescence was measured on a Cytofluor II (Applied Biosystems, Foster City, CA) with an excitation filter of 360/40 nm and an emission filter of 460/40 nm. A reading was made every minute for 20 to 60 min, and the slope obtained from a linear regression of this time course was used as the reaction rate. The reaction rates were determined for different inhibitor concentrations, and the percentage of inhibition was determined by comparison with the reaction rate in the absence of inhibitor. IC50 values were determined by four parameter fits of plots of the percentage of inhibition versus inhibitor concentration using the program GraFit (Erithacus Software Ltd.). |
| Affinity data for this assay | |
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