Reaction of protein disulfide groups with Ellman's reagent: A case study of the number of sulfhydryl and disulfide groups in Aspergillus oryzae α-amylase, papain, and lysozyme

Abstract

Ellman's reagent, 5,5′-dithiobis[2-nitrobenzoic acid] (DTNB and abbreviated here as Ø-S-S-Ø), is a standard reagent for the determination of reactive sulfhydryl groups by absorbance measurement at 412 nm. The color produced is due to the formation of a thio anion, 3-carboxylato-4-nitro-thiophenolate (Ø-S −). In the determination of the number of sulfhydryl groups of porcine pancreatic α-amylase, we observed that the protein contained more Ø-S-S-groups than could be accounted for from the original sulfhydryl analysis (reaction 1). We consequently postulated that disulfide groups were reacting in addition to sulfhydryl groups (reactions 2 and 3). The reaction was postulated to release one Ø-S − for every free sulfhydryl group, and the protein was postulated to be derivatized so that one -S-S-Ø group is formed for every free sulfhydryl group and two -S-S-Ø groups are formed for every disulfide group. This hypothesis was tested by using three diverse proteins ( A. oryzae α-amylase, papain, and lysozyme) whose numbers of sulfhydryl and disulfide groups are known. Ø-S − was released from the derivatized protein by incubation at pH 10.5 (reaction 4). Determination of the amount of Ø-S −/mole of protein before and after pH 10.5 permitted determination of the number of sulfhydryl and disulfide groups in the protein. The results of the study of the three proteins confirmed the hypothesis and established methods for the fast and accurate determination of the number of sulfhydryl and disulfide groups in proteins. 1. (1) Protein-S − + Ø-S-S-Ø → Protein-S-S-Ø + Ø-S − 2. (2) Protein-S-S-Protein + Ø-S − → Protein-S-S-Ø + Protein-S − 3. (3) Protein-S − + Ø-S-S-Ø → Protein-S-S-Ø + Ø-S − 4. (4) 3 Protein-S-S-Ø + DTT or pH 10.5 → 3 Protein-S − + 3 Ø-S −