FIGURE 2.

SNO-induced changes in the thiol-disulfide and oligomerization state of Prx1. A, reduced Prx1 was incubated with CysNO (250 μm) for different times at 37 °C, and its redox state was then assessed by non-reducing SDS-PAGE. The Prx1 monomer is indicated by an asterisk, the dimer is indicated by double asterisk, and the high molecular weight species are indicated by triple asterisks. B, reduced Prx1, WT, or Cys to Ser mutants were incubated with or without CysNO (250 μm) for 60 min at 37 °C. The samples were then analyzed by non-reducing SDS-PAGE. C, shown are gel filtration chromatograms of reduced, nitrosylated, and oxidized Prx1. Reduced Prx1 was either left untreated or exposed to CysNO (1 mm, 60 min) or to H₂O₂ (100 μm, 60 min). Protein samples (0.66 mg) were fractionated using Superdex 200 size exclusion chromatography as described under “Experimental Procedures.” D, reduced Prx1 (either WT or C52S) were left untreated or exposed to CysNO (250 μm, 60 min). Protein samples (0.66 mg) were analyzed as in C. The elution profile of standard proteins was as follows: ferritin, 10.7 ml; aldolase, 12.8 ml; conalbumin, 14.5 ml; ovalbumin, 15 ml; ribonuclease, 18 ml.