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Figure 3
E82I PaDsbA1 exhibits wild-type protein activity and redox character. (a) WT and E82I PaDsbA1, in the presence of 2 mM oxidized glutathione, display a similar rate of oxidation of a fluorescently labelled peptide substrate. Reactions lacking glutathione, a protein catalyst or containing only buffer display negligible activity. The mean and SEM of four (WT) and three (E82I) independent biological replicates are plotted. For GSSG and buffer-only controls n = 1. (b) The disulfide reductase activity of WT and E82I PaDsbA1 was assessed using an insulin-reduction assay. WT (blue) and E82I (orange) PaDsbA1 catalyze the reduction of insulin in the presence of DTT to a similar extent. The mean and SEM of three independent biological replicates are plotted. (c) The fraction of oxidized and reduced E82I PaDsbA1 after equilibrium in redox buffers containing varying ratios of reduced glutathione:oxidized glutathione at pH 7.0 and 310 K was determined from the redox state-dependent fluorescence of PaDsbA1. These data were used to calculate Keq and subsequently the redox potential. Representative data of a single experiment of six performed are shown, plotted on a semi-log graph using a base 10 logarithmic scale for the x axis and a linear scale for the y axis. For final redox potential determination, mean and SEM were calculated (n = 6).

Journal logoBIOLOGICAL
ISSN: 1399-0047
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