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

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• 作者：Rossella Tomazzolli ; Mauro Dalla Serra ; Giuseppe Bellisola ; Marco Colombatti and Graziano Guella
• 关键词：Thioredoxin reductase ; Protein disulfide isomerase ; Fluorescence self-quenching ; FITC derivatives ; NMR characterization
• 刊名：Analytical Biochemistry
• 出版年：2006
• 出版时间：1 March 2006
• 年：2006
• 卷：350
• 期：1
• 页码：105-112
• 全文大小：446 K

文摘

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-NH₂ 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.