FIGURE 1.

Assay of SoxB activity. The components present in each reaction are shown in the top panel. The assay buffer was 10 mm Hepes-NaOH, pH 7.5. The reactions were incubated for 3 h at the indicated temperature. Where DTT is specified, it was added to the sample at the end of the incubation. Reduced SoxYZ has a free active site cysteine. In the S-thiosulfonate adduct the cysteine is covalently linked to thiosulfate. An analysis of the reaction products by SDS-PAGE is shown in the middle panel. Reactions 2–13 were placed at 100 °C for 2 min prior to loading, whereas reaction 1 was kept at 22 °C. The identities of the observed species are indicated to the left of the gel. A summary of the electrospray ionization mass spectrometry results for the same samples is shown in the bottom panel. The original mass data are shown in supplemental Fig. S2. Complementary analysis of the reaction products by size exclusion chromatography is given in supplemental Fig. S1.