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

Figure 4.

Figure 4

The effect of urea on the hyper-reactivity of cysteines in Lyzred and the inhibition of the aggregation of the hemi-reduced lysozyme by GSH/GSSG. (a) Inhibition of the hyper-reactivity of cysteines in Lyzred toward DTNB by variable urea concentrations (1.25 µM Lyzred was reacted with 20 mM DTNB at pH 5.0). (b) Red line: Hemi-reduction of native Lyz (6 µM) incubated with 60 µM DTT in 0.01 M borate buffer pH 8.5 without urea. No aggregation occurs during the reduction at this pH. Blue line: Aggregation of the hemi-reduced Lyz when the pH was lowered to 7.4 with phosphate buffer 0.1 M. Light blue line: Inhibition of aggregation by addition of GSH/GSSG (2 mM/0.4 mM). (c) Expanded kinetics of inhibition of the protein aggregation reported in b after addition of GSH/GSSG (2 mM/0.4 mM). (d) Disappearance of three hyper-reactive sulfhydryls in the hemi-reduced Lyz (1.25 µM) after reaction with 0.4 mM GSSG at pH 7.4. Note that the first sulfhydryl reacts within 7 seconds. Errors are reported as S.D. from three independent experiments in panels (a–d). (e) Scheme of the protection mechanism of Lyzred to avoid the protein aggregation. Note that the natural Cys76-Cys94 is the last bridge to be formed11,14,24,25.