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. 2018 Oct 30;8:16050. doi: 10.1038/s41598-018-34439-y

Figure 1.

Figure 1

The effect of GSH/GSSG on Lyzred aggregation, pKa of Lyzred cysteines and evidence for a transient Lyzred-GSSG complex. (a) Blue line: Lyzred (6 µM) incubated in 0.2 M urea (37 °C). Red line: Same Lyzred solution after immediate addition of GSH/GSSG (2 mM/0.4 mM). (b) Expanded kinetics of the experiment shown in (a). (c) Green line: Disappearance of Lyzred sulfhydryl groups after addition of 0.4 mM GSSG to a 1.25 µM Lyzred solution (10 µM –SH groups) at pH 7.4 and 0.2 M urea (25 °C). Blue line: Disappearance of the sulfhydryl groups of free cysteine (10 µM) incubated with 0.4 mM GSSG as in the experiment with Lyzred (pH 7.4, 25 °C). Red line: Theoretical sulfhydryl groups disappearance of an unperturbed protein cysteine (10 µM) during the reaction with 0.4 mM GSSG. (d) Expanded kinetics of the experiment shown in c. (e) Reduced GSH produced after 10 s incubation of Lyzred with 0.4 mM GSSG at pH 7.4. Titration of GSH using NBD-Cl and glutathione transferase P1-1 (GSTP1-1) (see Methods) (column 1). Same experiment in the absence of GSTP1-1 (column 2). Same experiment in which the produced GSH was titrated with DTNB after filtration of the mixture on Amicon Ultra 10 kDa cut-off filter to remove the protein (column 3). (f) Average pKa of the seven reactive cysteines in Lyzred as calculated using DTNB (pKa = 6.6) (blue line); average pKa of the four reactive cysteines using NBD-Cl (pKa = 7.1) (red line). Black line is the theoretical curve for unperturbed cysteine (pKa = 9.1). v0/Vmax are the initial velocities normalized to those at full deprotonation (see Methods). (g) Apparent first order kinetic constants for the reaction of the most hyper-reactive cysteine with variable GSSG concentrations at pH 7.4 and 25 °C. (h) Quenching of the intrinsic fluorescence at 340 nm (λex = 295 nm) of Lyzred (1.25 µM, pH 7.4) after addition of GSSG 0.4 mM (25 °C). See (c) for comparison. The very fast fluorescence perturbation after the addition of GSSG occurs within one second. (i) The dependence of the fast fluorescence perturbation (occurring within 1 s) on the GSSG concentration. The error bars represent the S.D. from three independent experiments.