Fig. 5

Determination of the native molecular mass by gel filtration. A Elution of native AtsR, reduced AtsR:C123SC187S, and oxidized AtsR from size exclusion column. Native AtsR and reduced AtsR:C123SC187SAtsR (20 µM AtsR was incubated with 50 mM DTT) were indicated by clear circle and clear square, respectively. AtsR previously incubated with H₂O₂ resulted in a mixture of native (dimer; clear circle) and oxidized (dimer; arrow) species. B Elution of native AtsR, oxidized AtsR, and reduced AtsR:C123SC187S from size exclusion column indicated by clear circle, arrow, and clear square, respectively. For calibration, a premixed protein molecular mass marker containing the following proteins was used: ribonuclease A (13,700 Da), carbonic anhydrase (29,000 Da), ovalbumin (44,000 Da), conalbumin (75,000 Da), and alcohol dehydrogenase (150,000 Da) (GE Healthcare, Piscataway, NJ). V_o was determined with blue dextran (2000 kDa). C Redox response of His₆-AtsR, His₆-AtsR:C123S, His₆-AtsR:C187S, and His₆-AtsR:C123SC187S detected by nonreducing SDS-PAGE. Proteins were incubated with or without H₂O₂ and CHP for 30 min, respectively, and then, when indicated, added with DTT and incubated for another 30 min. All samples were separated by 15% nonreducing SDS-PAGE and stained with Coomassie Brilliant Blue. M represented protein molecular mass marker. “−” expressed purified protein. D Quantification of free thiol levels in His₆-AtsR, His₆-AtsR:C123S, His₆-AtsR:C178S, and His₆-AtsR:C123SC178S. DTT−, H₂O₂−, and CHP-treated protein (20 µM) were mixed 5 mM with DTNB in 50 mM Tris-HCl buffer (pH 8.0), respectively, and the absorbance was monitored at 412 nm against a 2 mM DTNB solution as reference. These data were means of the values obtained from three independent assays. E Spectrophotometric analysis of NBD-labeled AtsR mutants. Proteins treated with DTT or without H₂O₂ were modified with NBD-Cl. The resulting proteins were analyzed spectrophotometrically at 200 to 600 nm