Dieosin disulfide reductase assay

Dieosin glutathione disulfide, Di-Eo-GSSG, was synthesized by the reaction of eosin isothiocyanate with GSSG. Di-E-GSSG had low fluorescence which increased upon reduction of its disulfide bond⁵¹. Reductase activity of purified enzymes was monitored in 96-well fluorescence microtiter plates. Recombinant ERp5, ERp72, PDI and ERp57 were assayed at the concentrations indicated in the absence or presence of anti-PDI antibody directed at an epitope in the b′ domain (H-160; Santa Cruz) or at an N-terminal epitope in PDI (PH4B; Aviva) (3 μM). Di-Eo-GSSG (150 nM) was added to enzyme in the presence of 5 μM DTT and the increase in fluorescence due to release of eosin-glutathione for ERp5, ERp72, ER57 and PDI was determined by excitation at 520 nm and emission at 545 nm in a Synergy 4 plate reader,Winooski VT). The reduction of 150 nM di-E-GSSG by 5 μM DTT alone served as a negative control.