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A fluorescence-based assay for the reductase activity of protein disulfide isomerase.

Authors
  • Tomazzolli, Rossella
  • Serra, Mauro Dalla
  • Bellisola, Giuseppe
  • Colombatti, Marco
  • Guella, Graziano
Type
Published Article
Journal
Analytical biochemistry
Publication Date
Mar 01, 2006
Volume
350
Issue
1
Pages
105–112
Identifiers
PMID: 16434015
Source
Medline
License
Unknown

Abstract

We report on a new spectrofluorimetric assay for the measurement of reductase activity of proteins belonging to the superfamily of thioredoxins such as protein disulfide isomerase (PDI). The assay relies on the preparation of a fluorescence-quenched substrate easily accessible in two steps through functional group transformations of the peptide Gly-Cys-Asp. In the first step fluorescein isothiocyanate is linked to the Gly-NH(2) terminus and in the second step the Cys-SH groups are converted into a disulfide bond. Both intermediate and final substrate have been fully characterized by mass spectrometric and nuclear magnetic resonance measurements. Dimethyl sulfoxide is here reported to be a mild oxidizing agent allowing us to obtain in good overall yield the assay substrate in a single synthetic step. A reliable estimation of PDI reductase activity is obtained via the detection of a strong fluorescence enhancement after enzymatic reduction. Moreover, our assay provides further support for the key role played by thioredoxin reductase in enabling disulfide reductase activity of PDI.

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