Figure 4. Specificity of diarylcarbonates.

A. Papain was preincubated with inhibitor (100 μM) for 30 min prior to addition of Z-Arg-AMC (300 μM). B. Ficin was preincubated with inhibitor (100 μM) for 30 min prior to addition of Z-Arg-AMC (300 μM). C. His₆-tagged catalytic domain of SENP6 (aa 628-1112) (200 nM) was treated with inhibitor for 30 min at room temperature followed by addition of SUMOylated His₆-tagged catalytic domain of RanGap1 containing the SUMO modification site (aa 418-588) (3 μM) for 10 min at 37 °C after which time lysates were quenched with Laemeli buffer and resolved by SDS-PAGE then blotted and probed with anti-His₆ antibodies. D, E. Serine proteases catalyze the hydrolysis of C17 (squares) and C22 (circles). D. Diarylcarbonates were incubated in the presence and absence of chymotrypsin (80 nM). E. Velocity versus substrate concentration plot for trypsin catalyzed hydrolysis. F. Velocity versus substrate concentration plot for chymotrypsin catalyzed hydrolysis. The fits to the Michaelis-Menten equation are shown.