The binding of Ca2+ ions to bovine and human thyroglobulin (Tg) was demonstrated qualitatively by 45Ca overlay on polyvinylidene difluoride (PVDF) membranes. A quantitative analysis of the interaction of metal ions with bovine Tg was conducted by fluorimetric titration of the protein with Tb3+ ions. These have been used with several proteins as isomorphous replacement probes for Ca2+ ions, as protein-bound Tb3+ ions fluoresce, upon irradiation in the UV region, because of energy transfer from tyrosyl and/or tryptophanyl residues. The fluorescence emission spectrum of Tg excited at 280 nm showed, upon addition of Tb3+ ions, a peak at 546 nm and a marked decrease at 335 nm, indicating an efficient Förster energy transfer between bound Tb3+ ions and closely located Tg intrinsic chromophores. Titration of Tg with Tb3+ ions, carried out by monitoring the emitted fluorescence at 546 nm, indicated the presence of 13.15 metal binding sites per Tg molecule.
Probing the interaction of thyroglobulin with metal ions by Terbium(III) luminescence spectroscopy
GENTILE, Fabrizio;
1998-01-01
Abstract
The binding of Ca2+ ions to bovine and human thyroglobulin (Tg) was demonstrated qualitatively by 45Ca overlay on polyvinylidene difluoride (PVDF) membranes. A quantitative analysis of the interaction of metal ions with bovine Tg was conducted by fluorimetric titration of the protein with Tb3+ ions. These have been used with several proteins as isomorphous replacement probes for Ca2+ ions, as protein-bound Tb3+ ions fluoresce, upon irradiation in the UV region, because of energy transfer from tyrosyl and/or tryptophanyl residues. The fluorescence emission spectrum of Tg excited at 280 nm showed, upon addition of Tb3+ ions, a peak at 546 nm and a marked decrease at 335 nm, indicating an efficient Förster energy transfer between bound Tb3+ ions and closely located Tg intrinsic chromophores. Titration of Tg with Tb3+ ions, carried out by monitoring the emitted fluorescence at 546 nm, indicated the presence of 13.15 metal binding sites per Tg molecule.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
