کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1176735 961873 2006 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A fluorescence-based assay for the reductase activity of protein disulfide isomerase
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
A fluorescence-based assay for the reductase activity of protein disulfide isomerase
چکیده انگلیسی

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 350, Issue 1, 1 March 2006, Pages 105–112
نویسندگان
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