Figure 7.

Effect of concentration of ascorbate and H₂O₂ in the linker cleavage. (A, B) The cleavage of MBP-H8-AVI with a molar equivalent of CuCl₂ and various amounts of ascorbate and hydrogen peroxide is shown. (A) Lane 1, only protein; lane 2, hydrogen peroxide at 0.7 mM. In lanes 3–8 the hydrogen peroxide concentration was kept constant at 0.7 mM and ascorbate concentration was increased as follows: 1.14 mM (lane 3), 2.3 mM (lane 4), 4.6 mM (lane 5), 9.1 mM (lane 6), 18.3 mM (lane 7), and 41.4 mM (lane 8). (B) Lane 1, only protein; lane 2, ascorbate at 9 mM. In lanes 3–8 the ascorbate concentration was kept at 9 mM and the hydrogen peroxide concentration was increased as follows: 0.07 mM (lane 3), 0.17 mM (lane 4), 0.7 mM (lane 5), 1.4 mM (lane 6), 2.7 mM (lane 7), and 5.5 mM (lane 8). (C, D) The cleavage of MBP-H10-AVI with 10 molar equivalents of CoCl₂ is shown. (C) Lane 1, only protein; lane 2, hydrogen peroxide at 0.4 mM. In lanes 3–5, the hydrogen peroxide concentration was kept constant at 0.4 mM and ascorbate concentration was increased as follows: 35 μM (lane 3), 70 μM (lane 4), and 0.14 mM (lane 5). (D) Lane 1, only protein; lane 2, ascorbate at 4.6 mM. In lanes 3–8 the ascorbate concentration was kept at 4.6 mM and the hydrogen peroxide concentration was increased as follows: 0.09 mM (lane 3), 0.17 mM (lane 4), and 0.35 mM (lane 5).