Figure 6. SH content and aggregates of TOP treated with 10 mM (100% reduced cysteines) and 1 mM (40% reduced cysteines) TCEP.

A) WT) wild type TOP, A) + 10 mM TCEP, B) + 10 mM TCEP and 5 µM H₂O₂, C) + 10 mM TCEP and 0.5 µM Mb, D) + 10 mM TCEP and 0.5 µM Mb and 5 µM H₂O₂, E) + 10 mM TCEP and 0.5 µM HRP, F) + 10 mM TCEP and 0.5 µM HRP and 5 µM H₂O₂, A') + 1 mM TCEP, B') + 1 mM TCEP and 5 µM H₂O₂, C') + 1 mM TCEP and 0.5 µM Mb, D') + 1 mM TCEP and 0.5 µM Mb and 5 µM H₂O₂, E') + 1 mM TCEP and 0.5 µM HRP, F') + 1 mM TCEP and 0.5 µM HRP and 5 µM H₂O₂. B) SDS-PAGE of TOP at the following conditions: lanes 1 to 7 correspond, respectively, to molecular weight standard, 100% reduced TOP, + H₂O₂, + Mb, + H₂O₂, and Mb, + HRP, + HRP and H₂O₂. C) SDS-PAGE of 40%-reduced TOP at the following conditions: lanes 1 to 7 correspond, respectively, to molecular weight standard, 40% reduced TOP, + H₂O₂, + Mb, + H₂O₂, and Mb, + HRP, + HRP and H₂O₂. D) This panel shows the large amount of aggregates in aged TOP. The reaction mixtures were incubated for 2 hours in buffer Tris 20 mM Chelex-100 ®, pH 7.4, at 37° and contained 5 µM TOP. The SDS-PAGE of 100% reduced TOP was done with 10 μM TOP.