Fig. 4. RRSP does not require a metal cofactor.

(A, B) SDS-PAGE gels showing cleavage of 10 μM rKRAS that had been buffer-exchanged to remove MgCl₂ by 10 μM RRSP that had been pre-incubated with increasing amounts of EDTA (A) or 1,10-phenathroline (B) for 30 min. (C) Cleavage of 10 μM MgCl₂-free rKRAS by 10 μM thermolysin (T) that had been pre-incubated with EDTA (A) or 1,10-phenathroline (B) for 30 min. SDS-PAGE gels are representatives of three independent biological replicates. (D-E) Half maximal concentration (EC₅₀) curves for cleavage of 10 μM MgCl₂-free rKRAS by increasing concentrations of untreated (Unt) rRRSP compared to rRRSP that had been pre-treated with 5 mM EDTA (D) or 1,10-phenanthroline treated (E). At the highest concentration of rRRSP, KRAS:rRRSP was 1:1 as in panels A and B. (F) EC₅₀ curves for cleavage of 10 μM MgCl₂-free rKRAS by rRRSP in the presence of 5 mM MgCl₂ or ZnCl₂ or by rRRSP pre-treated with 1,10-phenanthroline. All curves are derived from SDS-PAGE analysis (N=3 independent experiments) reported as the means ± SD. EC₅₀ values calculated from non-linear curve fit ± the standard error.