Rapid and irreversible reduction of protein disulfide bonds

This report describes the development of a method that enables a rapid (less than 20 s), quantitative, and irreversible reduction and inactivation of disulfide-containing proteins at room temperature (20 to 25 °C). The formula comprises the ingredients of optimized concentrations of denaturant, reductant, and hydroxide ion. The novelty of this formula is the application of a potent hydroxide ion in the concoction. The component of hydroxide ion serves two major functions. (1) It accelerates the cleavage of disulfide bonds mediated by the reducing agent and denaturant, leading to an instant and quantitative reduction of disulfide proteins. (2) It triggers a rapid covalent destruction of sulfhydryl groups and disulfide bonds via the mechanism of base-catalyzed β-elimination, thus leading to the irreversible and permanent abolition of disulfide bonds. The usefulness of this formula has been demonstrated here with the effective and rapid reduction of numerous highly stable disulfide-containing proteins, including cardiotoxin and prion aggregates.