Several synthetic peptides, modelled from a Ca2+-binding loop of the EF-hand family of proteins, were prepared containing cysteine residues. The peptide, GDKNADGFICFEEL, was labelled covalently at the cysteine residue (loop position 9) with iodoacetamidosalicylic acid. This novel conjugate is a metal-binding loop containing a salicylic acid side chain that could not only chelate Tb3+ in conjunction with the other chelating groups in the sequence, but could also sensitize Tb3+ luminescence. The loop had a high Tb3+ affinity, with stoichiometric binding observed under experimental conditions. The luminescence from the Tb3+-peptide complex was more than 10-fold greater than the luminescence reported from a related peptide which contained Trp as the Tb3+ donor at loop position 7. This peptide has significant potential for use in lanthanide-based time-resolved luminescence immunoassays.
