FIG. 8.

Optimization of helicase reaction conditions for the full-length NS3 protein. The activity was quantified by determining the percentage of unwinding on 1 nM dsRNA substrate by 40 nM enzyme, as described in Materials and Methods. (A) MOPS (pH 6.5) or HEPES (pH 6.6, 7, 7.5, and 8) (each at 25 mM) compared in reactions performed at 0 mM NaCl, 3 mM MgCl₂, 3 mM ATP. (B) Determinations of optimal pH were performed in 25 mM MOPS (pH 6.5, 7, 7.5, and 8), as in panel A. (C) Mg²⁺ and Mn²⁺ titration was performed in 25 mM MOPS (pH 7) at 0 mM NaCl and 3 mM ATP. (D) Na⁺ titration was performed in 25 mM MOPS (pH 7)–3 mM MgCl₂–3 mM ATP.