Amperometric titration of disulfide and sulfhydryl in proteins in 8 M urea.

Of the numerous methods which have been used in the estimation of disulfide bonds and sulfhydryl groups in proteins and amino acids (l), the amperometric, argentimetric titration procedure, with appropriate modifications, is especially well suited for this purpose. This paper describes a modification and extension of known methods whereby these groups may be titrated in 8 M urea. The total number of available groups is theoretically accessible in 8 M urea and, conversely, such groups may be inaccessible in an aqueous medium although the protein may have been subjected to the denaturing effect of 8 M urea initially. The protein molecule is “unfolded” by the urea, thereby exposing the disulfide bonds to the action of SOS” with the subsequent release of 1 mole of sulfhydryl for rach mole of disulfide. The kinetics of the equilibrium reaction of SOS” on disulfide has been studied by Stricks and Kolthoff (2), and by Cecil and McPhee (3-5). The results obtained with seven different proteins, the majority of which are of known disulfide composition, are reproducible, indicate rapid quantitative cleavage of all disulfide linkages, and show good linear current-volume (titration) curves with sharp, long lasting end points.