FLUORESCEINTHIOCARBAMYL-INSULIN - A POTENTIAL ANALYTICAL TOOL FOR THEASSAY OF DISULFIDE BOND REDUCTION

We describe the synthesis of fluorescent derivatives of bovine pancrea s insulin and its use as substrates of disulfide bond reduction in a s pectrofluorometric assay. Amino groups of insulin were chemically modi fied 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-fluoresceinthiocarba myl-insulin evinced the highest and the lowest enhancement of fluoresc ence emission, whereas the mono-substituted protein had intermediate e nhancement. Using di-fluoresceinthiocarbamyl-insulin, the reliability of this novel feature for the estimation of disulfide bond cleavage wa s assessed by (i) the separation of two fluorescent bands using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) the linear r esponse of the fluorescence signal within a range from 0.04 to 1 mu M, and (iii) the correlation of the rate of fluorescence enhancement wit h concentrations of dithiothreitol ranging from 0.1 to 5 mM; Moreover, di-fluoresceinthiocarbamyl-insulin was a sensitive oxidant when the c atalytic capacity of thioredoxin and protein disulfide isomerase was a nalyzed in the presence of dithiothreitol or glutathione, as reductant s. On this basis, di-fluoresceinthiocarbamyl-insulin constitutes an an alytical tool to test the capacity of biochemical preparations in the reduction of disulfide bonds. (C) 1997 Elsevier Science Ireland Ltd.