The reductive cleavage of disulfide bonds and its application to problems of protein structure

The quantitative reduction of disulfide bonds in ribonuclease and lysozyme may be achieved at room temperature with thioglycolic acid in 8 M urea at pH 8.₅. Sulfhydryl groups of the resulting cysteine residues may be reacted with iodoacetic acid to yield the S-carboxymethyl derivatives of these residues. The S-carboxymethylcysteine content of reduced and alkylated proteins may be estimated by dinitrophenylation of acid hydrolysates followed by chromatographic isolation of DNP-SCMC. Under the conditions of reduction and alkylation employed, no significant modification, stable to the conditions of hydrolysis employed, of tyrosine, tryptophan, lysine, or histidine residues occurs. The method described is, therefore, of particular value as an initial step in the study of amino acid sequence in such tryptophan-rich proteins as lysozyme. The digestion of reduced and alkylated lysozyme and ribonuclease with trypsin proceeds rapidly and yields, in each case, a population of peptide fragments which is in good agreement with that to be expected from the lysine and arginine contents of these proteins.