Fluoresceinthiocarbamyl-insulin: a potential analytical tool for the assay of disulfide bond reduction |
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Authors: | AP Heuck RA Wolosiuk |
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Affiliation: | Instituto de Investigaciones Bioquímicas (Fundación Campomar, F.C.E.N.-U.B.A., IIBBA-CONICET), Buenos Aires, Argentina. |
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Abstract: | We describe the synthesis of fluorescent derivatives of bovine pancreas insulin and its use as substrates of disulfide bond reduction in a spectrofluorometric assay. Amino groups of insulin were chemically modified with fluorescein isothiocyanate and proteins bearing one, two and three fluorescent groups were purified by ion-exchange chromatography. Upon incubation with dithiothreitol, di- and tri-fluoresceinthiocarbamyl-insulin evinced the highest and the lowest enhancement of fluorescence emission, whereas the mono-substituted protein had intermediate enhancement. Using di-fluoresceinthiocarbamyl-insulin, the reliability of this novel feature for the estimation of disulfide bond cleavage was assessed by (i) the separation of two fluorescent bands using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) the linear response of the fluorescence signal within a range from 0.04 to 1 microM, and (iii) the correlation of the rate of fluorescence enhancement with concentrations of dithiothreitol ranging from 0.1 to 5 mM. Moreover, di-fluoresceinthiocarbamyl-insulin was a sensitive oxidant when the catalytic capacity of thioredoxin and protein disulfide isomerase was analyzed in the presence of dithiothreitol or glutathione, as reductants. On this basis, di-fluoresceinthiocarbamyl-insulin constitutes an analytical tool to test the capacity of biochemical preparations in the reduction of disulfide bonds. |
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