Figure 3.

Effect of oxygen on disulfide bond formation in CrtJ in vivo and in vitro. (A) In vivo analysis of CrtJ disulfide bond formation after exposure to oxygen. Lanes 1 and 6 depict the SDS/PAGE migration of CrtJ from anaerobically grown cells that were treated with AMS. Lanes 2 and 3 are of anaerobically grown cells that were exposed to 1.0 mM H₂O₂ for 10 and 30 min, respectively, before treatment with AMS. Lanes 4 and 5 are of anaerobically grown cells that were exposed to O₂ for 10 or 30 min, respectively, before treatment with AMS. (B) In vitro analysis of CrtJ disulfide bond formation after exposure to oxygen. Lanes 3, 4, and 5 show effect of exposure of CrtJ to oxygen for 1, 5, and 10 min before treatment with AMS, respectively. Lanes 6–8 show the effect of a 5 min treatment of CrtJ to 0.01, 0.1, and 1.0 mM H₂O₂ before treatment with AMS, respectively. Lane 1 is a control that shows the migration of CrtJ that has not been exposed to AMS, whereas lane 2 is a control that shows the migration of reduced CrtJ that has been exposed to AMS. For optimal separation between oxidized and reduced CrtJ, the isolated protein used for this analysis contained a Cys → Ala mutation at position 22, which is a residue that does not undergo disulfide bond formation.