Fluoresceinthiocarbamyl-insulin: a potential analytical tool for the assay of disulfide bond reduction

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.