The interaction of 2,6-dichloroindophenol and protein sulfhydryl groups

The sulfhydryl proteins, d-aminoacid oxidase (EC 1.4.3.3) (porcine kidney), bovine-liver glutamate dehydrogenase (EC 1.4.1.3) and ovalbumin, all in a denatured state, and in addition, thiolated gelatin are all capable of reacting with 2,6-dichloroindophenol in a stoichiometry approaching 1 mole of indophenol per protein sulfhydryl residue. The product of this type of reaction is a protein-reduced indophenol adduct which can be oxidized by suitable electron acceptors including oxygen to a protein dye derivative. The indophenol component is bound tightly to the protein and resists removal by exhaustive dialysis. Pretreatment of the initial protein with p-chloromercuriphenylsulfonate completely inhibits the reduction and binding of the indophenol. These observations are discussed in terms of a thiol-substitution reaction to a ring of the indophenol dye, similar to the production of an S-glutathionyldichloroindophenol described in an earlier investigation in this laboratory¹.