Recombinant Metal-Free Apo-SOD1 Protein Production

All experiments were performed using recombinant, metal-free apo-SOD1 protein expressed and purified from BL21-Gold(DE3) PLysS E. coli cells (Strategene, Inc., Cedar Creek, TX, USA), as previously described ^{24 (link)}. The two free cysteines, Cys6 and Cys111, were mutated to Ala and Ser, respectively, to avoid intermolecular disulfide scrambling; the glycine-to-alanine substitution (G93A) was introduced into this pseudo WT background^{18 (link)}. L-Histidine, L-methionine, 30% hydrogen peroxide, leucine enkephalin acetate hydrate, catalase, trifluoroethanol (TFE), urea, dithiothreitol (DTT), iodoacetamide (IAM), ammonium acetate, formic acid (FA), trifluoroacetic acid (TFA), HPLC-grade solvents, and phosphate buffer saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO). Sequencing-grade trypsin was purchased from Promega (Madison, WI).