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 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.
Centro di Endocrinologia e Oncologia Sperimentale del Consiglio Nazionale delle Ricerche and Dipartimento di Biologia e Patologia Cellulare e Molecolare, University Federico II, Naples, Italy.
F
F
Gentile
E
E
Crescenzi
C
C
Pellegrini
M
M
Tecce
G
G
Palumbo
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013725:Terbium; D013954:Thyroglobulin / Q000302:isolation & purification / Q000378:metabolism*; D017186:Titrimetry / Q000379:methods
D008024:Ligands; D013725:Terbium; D013954:Thyroglobulin; D002118:Calcium