Assays of Thiol Isomerase Activity

Thiol isomerases are oxidoreductases that mediate disulfide bond formation in nascent proteins of the endoplasmic reticulum to ensure their structural integrity. In addition to its role in protein folding, thiol isomerases can modify allosteric disulfide bonds in both intracellular and extracellular proteins, thereby controlling protein function. The process of disulfide bond formation and cleavage is strictly regulated and responsive to redox conditions. Understanding disulfide bond regulation under different redox environments is critical to understanding physiological and pathological processes related to disulfide bond chemistry. Here, we describe protocols for the measurement of disulfide bond modulation by thiol isomerases. These assays include reductase, oxidase, denitrosylase, and sulfenylation assays. These methods can be applied to study recombinant thiol isomerases and thiol isomerases in cellular settings.