Studies of cyanolysis of the rhodanese-thionitrobenzoate complex

The reaction of sulfur-free rhodanese with 5-5'-dithio-bis (2-nitrobenzoic acid) produces a modified enzyme with 35% of residual activity. The resulting enzyme derivative has 1.6 moles of thionitrobenzoate/mole of enzyme bound to sulfhydryl groups of rhodanese. Determination of free sulfhydryl groups of this derivative shows that during the reaction with 5-5'-dithio-bis (2-nitrobenzoic acid) oxidative formation of an intramolecular disulfide bridge between two sulfhydryl groups of rhodanese occurs. Cyanolysis of the modified enzyme produces an enzyme-thiocyano derivative which partially beta-eliminates with release of thiocyanate. The cleavage of disulfide bonds present in the enzyme-thionitrobenzoate adduct using labeled cyanide shows an incorporation of radioactivity in the protein higher than would be expected. An electrostatic bond between cyanide and positively charged groups of the enzyme is suggested. Most of bound cyanide is released when samples are acidified for protein hydrolysis. In these conditions the thiocyanoalanine residue cyclizes to form 2-iminothiazolidine-4-carboxylic acid.