Figure 5. MsrA repairs oxidized methionines in cyt c and de-aggregates oxidized cyt c. A: Colloidal blue staining of a Tricine-SDS-PAGE gel following CNBr cleavage of oxidized cyt c (5 µg). Lane 1 untreated cyt c, lane 2 - 0.4 mM HOCl (2:1 molar ratio), lane 3 - 0.8 mM HOCl (4:1 molar ratio) and lane 4 - 1.2 mM HOCl (6:1 molar ratio). Lanes 5-7 are Cyt c (with the same ratios HOCl) repaired by 1.9 µM MsrA and 15 mM DTT. B: SDS-PAGE gel analysis of cyt c oxidized (5 µg) with high doses of HOCl using colloidal blue staining. Lanes 1 – 3, high doses of HOCl (5, 7.5 and 10 mM HOCl). Lane 4 contains the cyt c standard. C: Colloidal blue staining of a Tricine-SDS-PAGE gel of cyt c (5 µg) following CNBr cleavage. Lane 1 – Untreated cyt c cleaved with CNBr. Lane 2 – Oxidized cyt c (0.2 mM cyt c treated with 0.8 mM HOCl, 4:1 molar ratio) cleaved with CNBr. Lane 3 – Oxidized cyt c (291 µM) treated with MsrA (1.9 µM) and DTT (15 mM) for 2 h at 37 ºC and cleaved with CNBr.