Formation of enzyme—substrate disulfide linkage during catalysis by protein disulfide isomerase

During the regeneration of native ribonuclease A (RNase) from the disulfide scrambled molecule by protein disulfide isomerase (PDI), the substrate forms a covalent intermediate with the enzyme through disulfide linkage(s). This has been shown by the appearance of a band at the molecular weight position expected in SDS-PAGE at the same time as the increase in RNase activity. The new band decreased when the regeneration of RNase activity approached completion and disappeared by treatment of the reaction mixture with excess dithiothreitol.