PROBING THE INTERACTION OF THYROGLOBULIN WITH METAL-IONS BY TERBIUM(III) LUMINESCENCE SPECTROSCOPY

The binding of Ca2+ ions to bovine and human thyroglobulin (Tg) was de monstrated qualitatively by Ca-45 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 pr otein with Tb3+ ions. These have been used with several proteins as is omorphous replacement probes for Cn(2+) ions, as protein-bound Tb3+ io ns fluoresce, upon irradiation in the UV region, because of energy tra nsfer from tyrosyl and/or tryptophanyl residues. The fluorescence emis sion spectrum of Tg excited at 280 nm showed, upon addition of Tb3+ io ns, a peak at 546 nm and a marked decrease at 335 nm, indicating an ef ficient Forster energy transfer between bound Tb3+ ions and closely lo cated Tg intrinsic chromophores. Titration of Tg with Tb3+ ions, carri ed out by monitoring the emitted fluorescence at 546 nm, indicated the presence of 13.15 metal binding sites per Tg molecule. (C) 1998 Elsev ier Science Ireland Ltd. All rights reserved.