Di-fluoresceinthiocarbamyl-insulin: a fluorescent substrate for the assay of protein disulfide oxidoreductase activity

Analytical Biochemistry
A P Heuck, R A Wolosiuk

Abstract

We have developed a novel method for the continuous assay of protein disulfide oxidoreductase activity using as substrate bovine pancreas insulin in which both N-terminal amino groups are chemically modified with fluorescein isothiocyanate. The reduction of intercatenary disulfide bonds of di-fluoresceinthiocarbamyl-insulin with dithiothreitol initially lowers but subsequently enhances the emission intensity. In this biphasic kinetics, the rate of increase is sensitive enough for the estimation of Escherichia coli thioredoxin concentrations from 5 nM (0.06 microgram/ml) to 500 nM (6 micrograms/ml). Neither changes of pH over a range of 6.2 to 8.4 nor neutral salts (K+, Mg2+, and Ca2+) at concentrations lower than 100 mM affect this simple reaction system. Moreover, the fluorometric method is functional for measuring the reductive capacity of Brassica napus protein disulfide isomerase. Hence, a highly reproducible and accurate one-state assay for protein disulfide oxidoreductase activity not only greatly improves the sensitivity compared to the commonly used turbidimetric assay but also represents a reliable alternative to assays based on accessory enzymes or radiolabeled substrates.

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Citations

Jul 19, 2005·Biochemical and Biophysical Research Communications·Gábor NardaiPéter Csermely
Jan 26, 2006·Analytical Biochemistry·Rossella TomazzolliGraziano Guella
Dec 3, 2014·Biochimica Et Biophysica Acta·Diego S VazquezJavier Santos
Feb 18, 2014·Chemico-biological Interactions·Caterina GrilloLaura Cervoni
Nov 30, 2016·Scientific Reports·Elisa GaucciSilvia Chichiarelli
Aug 1, 2008·The Journal of Antibiotics·Elisa GaucciCarlo Turano
Jul 3, 1999·The Journal of Biological Chemistry·J ChenC C Wang
Jan 6, 2016·The Journal of Biological Chemistry·Harshavardhan KencheAnna Blumental-Perry
Jan 10, 2002·The Journal of Biological Chemistry·Mijung KwonDavid M Waisman
Jan 9, 2001·Archives of Biochemistry and Biophysics·G NardaiP Csermely
Sep 27, 2000·Biochimica Et Biophysica Acta·X X SunC C Wang
Apr 11, 2001·Biochimica Et Biophysica Acta·P D Duek, R A Wolosiuk
Jan 13, 2004·Analytical Biochemistry·Daniel J ClineJoel P Schneider

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