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

Anal Biochem. 2006 Mar 1;350(1):105-12. doi: 10.1016/j.ab.2005.11.037. Epub 2005 Dec 20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescein-5-isothiocyanate / chemistry
  • Fluorescence
  • Fluorescent Dyes / chemistry
  • Nuclear Magnetic Resonance, Biomolecular
  • Oligopeptides / chemistry*
  • Oxidation-Reduction
  • Oxidoreductases / analysis
  • Protein Disulfide-Isomerases / metabolism*
  • Spectrometry, Mass, Electrospray Ionization
  • Thioredoxin-Disulfide Reductase / metabolism

Substances

  • Fluorescent Dyes
  • Oligopeptides
  • glycyl-cystinyl-aspartic acid
  • Oxidoreductases
  • Thioredoxin-Disulfide Reductase
  • Protein Disulfide-Isomerases
  • Fluorescein-5-isothiocyanate