Cyanide-Binding and Oxy-Form Generation of BvPgb1.2

To prepare the cyanide form of BvPgb1.2, the purified protein was dialyzed in 10 mM potassium ferricyanide and 1 mM potassium cyanide dissolved in 50 mM MOPS for 8 h in 0.5 L solution; this process was performed twice. The cyanide-protein solution was passed through a PD10 column (Cytvia Life Science) to remove excess cyanide and the protein was concentrated using 10 kDa Vivaspin^® 20 mL ultrafiltration units (Vivascience). The oxy form of BvPgb1.2 was generated by passing the purified Hb through a PD10 column (Cytvia Life Science) in order to equilibrate with the oxygenated buffer (50 mM MOPS).