Figure 2.

Spurious inhibitors and detergents as PDI reductase assay interferents in biological samples. (A) PDI overexpression (~3-fold increase vs. endogenous PDI) increased, while PDI silencing (by ca.70%) did not change insulin reduction in endothelial cell homogenates. Freshly prepared homogenates (150 μg) obtained by mechanical lysis (in the absence of detergents) were incubated with insulin (1 mg/mL) and PDI (1.5 μM) in the presence of DTT (1.5 mM) and insulin turbidity was assessed at 540nm. pPDI = purified PDI, mock = cells treated with transfection reagent, wtPDI = cells transfected with wild type PDI, siRNA = cells transfected with siRNA against PDI. Cells were lysed 24h after transfection procedure. Note the higher lag time for insulin reduction when exogenous PDI is incubated together with homogenates (mock), as compared with exogenous PDI alone (pPDI), suggesting that biological samples might have endogenous unknown PDI inhibitors. (B) Detergents commonly used in buffers in different experimental protocols can inhibit or increase insulin reduction mediated by PDI in vitro, depending on their concentration. PDI (1.5 μM) was incubated with insulin at the same experimental conditions of (A). Detergents (nonionic Triton X-100, anionic sodium dodecyl sulfate or anionic sodium deoxycholate) were added at concentrations 100-fold (150 μM) to 0.1-fold (0.15 μM) vs. PDI concentration. Lag time was calculated as time for absorbance reaching 0.1 unit.