title: A fluorescence-based assay for the reductase activity of protein disulfide isomerase.
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.
authors.affiliation: CNR-ITC, Institute of Biophysics, Unit at Trento, Povo, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Tomazzolli
authors.forename: Mauro Dalla
authors.initials: MD
authors.lastname: Serra
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Bellisola
authors.forename: Marco
authors.initials: M
authors.lastname: Colombatti
authors.forename: Graziano
authors.initials: G
authors.lastname: Guella
mesh_terms: D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000032:analysis; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D009842:Oligopeptides; C509808:glycyl-cystinyl-aspartic acid; D010088:Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase; D019704:Protein Disulfide-Isomerases; D016650:Fluorescein-5-isothiocyanate