title: Fluoresceinthiocarbamyl-insulin: a potential analytical tool for the assay of disulfide bond reduction.
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.
authors.affiliation: Instituto de Investigaciones Bioquímicas (Fundación Campomar, F.C.E.N.-U.B.A., IIBBA-CONICET), Buenos Aires, Argentina.
authors.forename: A P
authors.initials: AP
authors.lastname: Heuck
authors.forename: R A
authors.initials: RA
authors.lastname: Wolosiuk
mesh_terms: D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D007328:Insulin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction; D012967:Sodium Dodecyl Sulfate
chemical_list: D004220:Disulfides; D007328:Insulin; C028647:insulin, fluoresceinthiocarbamyl(B29)-; D012967:Sodium Dodecyl Sulfate; D004229:Dithiothreitol