A fluorescence-based assay for the reductase activity of protein disulfide isomerase.
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.
Tomazzolli
Rossella
R
CNR-ITC, Institute of Biophysics, Unit at Trento, Povo, Italy.
Serra
Mauro Dalla
MD
Bellisola
Giuseppe
G
Colombatti
Marco
M
Guella
Graziano
G
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
D005456:Fluorescent Dyes; D009842:Oligopeptides; C509808:glycyl-cystinyl-aspartic acid; D010088:Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase; D019704:Protein Disulfide-Isomerases; D016650:Fluorescein-5-isothiocyanate