title: Methods of measuring protein disulfide isomerase activity: a critical overview.
abstract: Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.
authors.affiliation: Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine São Paulo, Brazil.
authors.forename: Monica M
authors.initials: MM
authors.lastname: Watanabe
authors.affiliation: Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine São Paulo, Brazil.
authors.forename: Francisco R M
authors.initials: FR
authors.lastname: Laurindo
authors.affiliation: Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine São Paulo, Brazil.
authors.forename: Denise C
authors.initials: DC
authors.lastname: Fernandes
keywords: chaperone; isomerization; oxidation; protein disulfide isomerase; redox signaling; reduction; thiols