| Assay Method Information | |
| | In Vitro Enzyme Assay |
| Description: | The proteolytic cleavage of N-acyl aminocoumarins by cathepsins was conducted in Dynatech Microfluor fluorescence 96-well microtiter plates, and readings were taken on a Molecular Devices Spectra Max Gemini XS instrument. The excitation wavelength was 355 nm and the emission wavelength was 450 nm for peptidyl-AMC substrates. Generation of AMC was monitored over 5 min. The dissociation constants (Ki) were determined from the IC50 values taken from plots of Vi/Vo versus inhibitor concentration, where Vo is the velocity in the absence of the inhibitor and Vi is the velocity with the inhibitor. The IC50 values were converted to Ki by the equation Ki = IC50 - Et/2, where Et is the enzyme concentration. |
| Affinity data for this assay | |
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