Assay Method Information

Assay Name:  Inhibition Assay
Description:  The inhibition of full-length hepatitis C NS3 protease enzyme was measured essentially as described in Poliakov, 2002 Prot Expression & Purification 25 363 371. Briefly, the hydrolysis of a depsipeptide substrate, Ac-DED(Edans)EEAbuψ[COO]ASK(Dabcyl)-NH2 (AnaSpec, San Jos , USA), was measured spectrofluorometrically in the presence of a peptide cofactor, KKGSVVIVGRIVLSGK (Åke Engstr m, Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden) (Landro, 1997 Biochem 36 9340-9348). The enzyme (1 nM) was incubated in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 7.5, 10 mM dithiothreitol, 40% glycerol, 0.1% n-octyl-D-glucoside, with 25 μM NS4A cofactor and inhibitor at 30° C. for 10 min, whereupon the reaction was initiated by addition of 0.5 μM substrate. Inhibitors were dissolved in DMSO, sonicated for 30 sec and vortexed. The solutions were stored at −20° C. between measurements.
Affinity data for this assay
 

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