The three disulfide bonds of pepsinogen and pepsin are reduced by β-mercaptoethanol in the absence of urea with a concomitant loss of the potential enzymic activity. The potential pepsin activity of the zymogen and the proteolytic activity of the enzyme can be restored if only two of the three disulfide linkages are reduced initially. Both the initial concentration of the reducing agent and of the protein are critical in the reoxidation process. However, the enzyme is less susceptible to reduction by mercaptoethanol than the zymogen.
Reoxidized pepsinogen was similar to the native zymogen in its kinetic parameters, ultraviolet absorption, optical rotatory dispersion, and circular dichroism. Reoxidized pepsin, however, differed from the native enzyme in its catalytic and optical properties, suggesting impairment in the refolding of this protein.