3.7. β-galactosidase Refolding Assay

Investigation of the ability of the T. brucei Hsp70s to refold chemically denatured β-galactosidase alone or in combination with Tbj2 was carried out as previously described [37]. The catalytic hydrolysis activity of the refolded β-galactosidase using 2-Nitrophenyl β-D-galactopyranoside (ONPG) as a chromogenic substrate [41] was measured in relation to native β-galactosidase and used as a means to determine the refoldase activity of Hsp70. After denaturation of β-galactosidase (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 30 °C in denaturation buffer (25 mM HEPES, pH 7.5, 5 mM MgCl₂, 50 mM KCl, 5 mM β-mercaptoethanol, and 6 M guanidine-HCl), denatured β-galactosidase to a final concentration of 3.4 nM was diluted 1:125-fold into refolding buffer (25 mM HEPES pH 7.5, 5 mM MgCl₂, 50 mM KCl, 2 mM ATP, and 10 mM DTT) supplemented with the T. brucei Hsp70s alone or in the presence of Tbj2 at indicated concentrations, and incubated for 2 h at 37 °C. Reactions containing 1.6 µM BSA (no molecular chaperones) with native and denatured β-galactosidase were conducted to serve as positive and negative controls, respectively. The negative control consisted of BSA (1.6 µM) in the presence of denatured β-galactosidase enzyme to determine the kinetics of spontaneous refolding of the denatured β -galactosidase enzyme. The activity of β-galactosidase was measured at various time points by mixing 10 µL of each refolding reaction with 10 µL of ONPG, followed by incubation at 37 °C for 15 min. Assays were terminated by the addition of 0.5 M sodium carbonate. Absorbance was recorded at 420 nm for each reaction. The percentage refolding activity was calculated relative to the activity of native β-galactosidase. The assay was conducted in triplicate on three independently purified batches of protein.