Novel Fluorescence-Based Microplate Assay for Screening Inhibitors of Thioredoxin and Protein Disulfide Isomerase

Thioredoxin (Trx) is a small globular protein, present in the cytosol, nucleus and mitochondria, which catalyzes reduction of protein disulfide bonds, while protein disulfide isomerase (PDI) is primarily located in the endoplasmic reticulum where it is also capable of catalyzing disulfide exchange, leading to the rearrangement of disulfide bonds within proteins. In this poster, the enzymatic activities of Trx and PDI were measured using a homogenous fluorescence-based assay wherein the reduction of insulin in the presence of dithiothreitol leads to the formation of insulin aggregates which subsequently bind avidly to a novel red-emitting fluorogenic dye.