Figure 1.

Scheme of some PDI activity assays. (A) RNase with scrambled thiols can be used as initial substrate for PDI isomerase assay, while totally reduced RNAse is used for PDI-mediated oxidative refolding assay. RNase gain-of-activity is measured by hydrolysis of its substrates, namely RNA or cyclic CMP. (B) Peptides can be used for oxidase, reductase or isomerase PDI assays, based on energy transfer from a donor to an acceptor residue with changes in fluorescence intensity. (C) PDI reduction assay is usually performed with insulin as substrate, which precipitates upon reduction of its B chain thiols. Coupling insulin reduction with NADPH consumption (using thioredoxin or glutaredoxin reductase systems) provides more precise quantitative results. See text for details.