Workflow for Accurate Measurement of PDI Reductase Kinetics Using a Fluorescent Disulfide Substrate.

Protein disulfide isomerase (PDI) is essential for oxidative protein folding, proteostasis, and redox regulation and has become a promising target for thromboinflammation, cancer, and neurodegenerative disorders. Fluorogenic disulfide substrates like Bodipy FL L-cystine (BDSS) are essential for mechanistic studies but have low affinity for PDI, necessitating high micromolar concentrations to measure Michaelis-Menten kinetics. These conditions can lead to optical artifacts, requiring development of correction methods to enable rigorous characterization of PDI inhibitors and allosteric modulators. Here, we present a simple workflow for correcting fluorescence quenching during BDSS reduction assays and provide an example of its utility with two allosteric modulators.