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Novel Fluorescence-Based Microplate Assay for Screening Inhibitors of Thioredoxin and Protein Disulfi de IsomeraseDownload
April 21, 2010
Authors: Eric Chan, Dee Shen, Jack Coleman, Lijun Dai, Praveen Pande, Andrew Whiteley and Wayne F. Patton; Enzo Life Sciences, Farmingdale, NY
Thioredoxin (Trx) is a small globular protein, present in the cytosol, nucleus and mitochondria, which catalyzes reduction of protein disulfi de bonds, while protein disulfi de isomerase (PDI) is primarily located in the endoplasmic reticulum where it is also capable of catalyzing disulfi de exchange, leading to the rearrangement of disulfi de bonds within proteins. Trx possesses a highly active site made up of two neighboring cysteine residues in a conserved active site motif, CGPC, while the redox active sites in PDI are slightly different, being CGHC. The motif difference leads to an increase in PDI’s redox potential, which in turn dramatically increases its catalytic activity for disulfi de formation. The enzymatic activities of Trx and PDI were measured using a homogenous fl uorescence-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 fl uorogenic dye. Relative to analogous turbidometric assays, the fl uorescence-based assay provides a superior assay signal window, improved lower detection limit, and higher Z’-score (>0.8). Intra-plate and inter-plate CVs using the assay are typically 3-4%. Concentration-response plots were employed to determine the effects of bacitracin on PDI and Trx activity. These experiments were performed at constant enzyme and substrate concentrations while systematically varying bacitracin concentration. Compared with PDI, Trx was determined to be relatively insensitive to bacitracin. The described assay has been specifi cally developed for use with a fl uorescence microplate reader and can potentially be applied to the identifi cation of inhibitors that selectively act upon PDI or Trx.