FIGURE 6.
pKa determination of GrxS12 sulfhydryl groups. A, reaction of GrxS12 C87S with PDT-bimane was monitored at 343 nm at pH values ranging from 3.0 to 8.0. The increase at 343 nm results from the release of pyridyl-2-thione from PDT-bimane. Each curve was fit to the Michaelis-Menten equation. B, t½ for the reactions of PDT-bimane with GrxS12 C29S and C87S were plotted as a function of pH, and the results were fitted to a sigmoidal curve. From this plot, sulfhydryl pKa values of 2.84 ± 0.08 and 5.63 ± 0.17 were determined for Cys29 and Cys87, respectively.