Di-fluoresceinthiocarbamyl-insulin: A Fluorescent Substrate for the Assay of Protein Disulfide Oxidoreductase Activity

• Published in: Analytical biochemistry 1997-05, Vol.248 (1), p.94-101
• Main Authors: Heuck, Alejandro P., Wolosiuk, Ricardo A.
• Format: Article
• Language: English
• Subjects: Feasibility Studies; Fluorometry; Insulin - analogs & derivatives; Insulin - chemistry; Isomerases - analysis; Nephelometry and Turbidimetry; Protein Disulfide Reductase (Glutathione) - analysis; Protein Disulfide-Isomerases; Reproducibility of Results; Spectrometry, Fluorescence; Thioredoxins - analysis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1997.2123

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 ofEscherichia colithioredoxin concentrations from 5 nm(0.06 μg/ml) to 500 nm(6 μg/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 mmaffect this simple reaction system. Moreover, the fluorometric method is functional for measuring the reductive capacity ofBrassica napusprotein disulfide isomerase. Hence, a highly reproducible and accurate one-stage 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.