Fig. 4: PDI- and GPx-like activity of synthetic compounds regulated by Ca2+ as an allosteric effector.
a General oxidative folding mechanisms. b Time course of recovered enzymatic activity of HEL during oxidative folding of reduced HEL using BAPDiSox (red) or DSTox (blue) as a catalyst in the presence (solid line) or absence (dashed line) of Ca2+. Reaction conditions: [reduced HEL]0 = 10 μM, [BAPDiSox]0 or [DSTox]0 = 20 μM in 100 mM Tris-HCl buffer solution containing no or 1 mM CaCl2 at 37 °C and pH 7.5 in the presence of 2 M urea under aerobic conditions. Data are shown as the means ± SEM (n = 3). c High-performance liquid chromatography (HPLC) chromatograms obtained from oxidative folding of reduced HEL using BAPDiSox in the absence (top) or presence (bottom) of Ca2+. The reaction conditions were the same as those in (b). Des intermediates, des[6-127], des[64–80], and des[76–94], represents key precursors of native HEL having three native S–S bonds but lacking one S–S linkage denoted in the brackets65,66,67. d Reductase-like function of compounds for reduction of H2O2 and protein S–S bonds coupled with NADPH oxidation. e Comparison of initial velocity for catalytic reduction of H2O2. Reaction conditions: [H2O2]0 = 0.25 mM, [GSH]0 = 4.0 mM, [NADPH]0 = 0.3 mM, [GR] = 4 unit mL−1, and [Cat.] = 0.1 mM at 25 °C and pH 7.2 in 100 mM Tris-HCl buffer solution containing 0.9 mM EDTA with or without 1.83 mM CaCl2. f Comparison of initial velocity for catalytic reduction of S–S bond in insulin. Reaction conditions: [insulin]0 = 60 μM, [GSH]0 = 3.7 mM, [NADPH]0 = 0.12 mM, [GR] = 4 unit mL−1, and [Cat.] = 30 μM at 25 °C and pH 7.2 in 100 mM Tris-HCl buffer solution containing 0.9 mM EDTA with or without 1.83 mM CaCl2.
