2.10. Ellman Assay

The quantitation of free sulfhydryl groups was performed by Ellman reagent using molar extinction coefficient for the leaving group (NTB) of 13700 M⁻¹cm⁻¹ at 412 nm. Protein was diluted in the denaturation buffer (50 mM Na₂HPO₄–NaH₂PO₄ pH 7.4, 8 M urea, 1% SDS, 1 mM EDTA) to a final concentration of 50–100 μM, then an excess of Ellman reagent was added, and absorbance at 412 nm was read over 30 min period. The maximum signal was reference-subtracted, divided by protein concentration, and averaged over three replicates. To measure the number of disulfide bonds experimentally, we used an assay with 2-nitro-5-thiosulfobenzoate (NTSB) [30]. The denatured protein was reduced by an excess of sodium sulfite, and total number of cysteines was measured by reaction with NTSB, which has the same leaving group as Ellman reagent. The NTSB was synthesized from Ellman reagent by reaction with sodium sulfite and O₂, as described in the original paper [30]. The protein solution was mixed with freshly prepared denaturing buffer (0.1 M glycine–NaOH pH 9.4, 6 M guanidine thiocyanate, 0.2 M Na₂SO₃, 5 mM EDTA) and NTSB, afterwards the analysis was performed as described for Ellman assay.