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. 2017 Jun 20;292(32):13154–13167. doi: 10.1074/jbc.M117.794586

Figure 4.

Figure 4.

Thiol–disulfide exchange reactions between CcmG, CcmH, and apocyt c1. A, schematic representation of the DTNB-based thiol–disulfide exchange assay. The thiolate of a fully reduced single Cys mutant derivative of CcmG (e.g. CcmGCys-78) initiates a nucleophilic attack on the mixed disulfide of purified CcmH–TNB adduct (e.g. CcmHCys-45–TNB), releasing TNB2− ions. B, TNB2− ions release kinetics during the reaction between CcmGCys-78 and CcmHCys-45–TNB. As an illustrative example, the data that are obtained using 1 μm CcmHCys-45–TNB and different concentrations of reduced CcmGCys-78 (1–30 μm) in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA buffer, are shown. During these reactions, the increase in A412 nm due to the release of the TNB2− ions was monitored in function of time. Under pseudo-first order kinetics conditions, the time-dependent absorbance changes follow single exponential curves. In each case, the initial rates were converted to kobs using 1 μm protein–TNB adduct (e.g. CcmHCys-45-TNB) and the absorption coefficient at 412 nm of TNB2− or the logarithmic linear plot to determine the rate. Reduced and IOA-alkylated CcmGCys-78, which is incapable of resolving the CcmHCys-45-TNB mixed disulfide, was used as a control. Similar assays were repeated with all selected Cys pairs between CcmG, CcmH, and apocyt c1, as appropriate. C, bimolecular rate constants for the thiol–disulfide exchange reactions. kobs values obtained above were plotted in function of the concentration of reduced protein (e.g. CcmGCys-78). Selected Cys pairs with high k values for TNB2− ion release are shown. CcmGCys-78 × CcmHCys-45-TNB (□), CcmGCys-75 × apocyt c1Cys-34-TNB (○), apocyt c1Cys-34 × CcmHCys-45-TNB (×), and CcmHCys-45× apocyt c1Cys-34-TNB (▿) are shown. In each case, the data points are average of at least two assays, and the linear curve is the best fit to the data points. The slope of this line represents the bimolecular rate constant (k) of the thiol–disulfide exchange reactions between the indicated Cys residues. Cumulative data obtained with all tested Cys pairs between CcmG, CcmH, and apocyt c1 are presented in Table 2.