Figure 6.

Effect of pH, time, and temperature on cleavage of fusion protein with CoCl₂ and CuCl₂. MBP-H10-AVI was incubated with 10 molar equivalents of CoCl₂ and MBP-H6-AVI with an equimolar amount of CuCl₂ at standard reaction conditions. (A) Effect of pH on cleavage. The fusion proteins were incubated at pH values varying between 3 and 10 for 60 min at room temperature. The pH values of the cleavage reactions are shown above the lanes. The concentration of ascorbate and H₂O₂ were 4.6 mM and 0.35 mM, respectively. The incubations with copper were performed in 50 mM Na-acetate at pH 5 in the presence of 171 mM NaCl, and the incubations with cobalt were performed in water. (B) Effect of time on cleavage of fusion proteins under standard conditions for different times. The incubation times in minutes are shown above the lanes. (C) Effect of temperature on cleavage. The fusion proteins were incubated at different temperatures for 20 min (CuCl₂) or 60 min (CoCl₂), with 4.6 mM ascorbate and 0.35 mM hydrogen peroxide. The incubation temperatures in °C are shown above the lanes.