Gdmhcl

CD analyses were performed as described by Kelly et al. (79 (link)). Briefly, the spectra of each hemocyanin were measured in the far-UV region (200–260 nm) in a model J-1500 JASCO spectropolarimeter (JASCO, Japan) of the Facultad de Ciencias Químicas y Farmacéuticas from the Universidad de Chile (Santiago, Chile). For all experiments, hemocyanin samples were analyzed at a concentration of 0.25 mg/ml in 0.1-cm trays with an optical passage at 25 °C. For the analysis of reduced hemocyanins, proteins were incubated with 2% β-mercaptoethanol. To generate unfolding curves, hemocyanins were incubated with increasing concentrations of GdmHCl (0–6.7 m) (Sigma) for 24 h. For the refolding curves, hemocyanins were incubated for 24 h with 6.7 m GdmHCl and then diluted in PBS to obtain different concentrations of GdmHCl (0.5–6.7 m). For all samples, spectra were taken between 200 and 260 nm, and similarly, the dichroism signal was measured at 222 nm for 1 min.

Sodium chloride, trizma, glycine, SDS, EDTA, ANS, Gdm-HCl, PMSF, and imidazole were obtained from Sigma Chemical Co. Nickel–nitrilotriacetate (Ni–NTA) agarose–based resin was brought from Qiagen. Mono-Q ion-exchange and superdex-75 (10/300 GL) gel filtration columns were purchased from GE Healthcare. LB agar, LB broth, glycerol, ammonium persulphate, IPTG, and kanamycin were purchased from Himedia Laboratories. Syringe filters (0.22 and 0.02 μm cutoff) were purchased from Millipore Corporation. All chemicals and reagents used were of analytical grade.