Self-association of Ca(2+)-binding peptides induced by lanthanide ions: a fluorescence study.

In this study a Ca(2+)-binding 14mer peptide was synthesized with the sequence GDKNADGCIEFEEL, allowing covalent attachment of sulfhydryl-reactive fluorescent molecules at position 7 of the 12-residue, metal-binding loop (underlined). This provided the opportunity to select donor molecules with suitable spectral characteristics for sensitized excitation of chelated terbium (Tb3+) or europium (Eu3+) ions. N-(1-Pyrene)-iodoacetamide and 7-diethylamino-3-((4'-iodoacetylamino)phenyl)-4-methylcoumarin were attached to the peptide and titrations carried out with terbium or europium stock solutions. It was possible to observe lanthanide ion binding to the loop in stoichiometric quantities, but maximal lanthanide luminescence was achieved with a large excess of lanthanide present, due to metal-induced peptide association. Obtaining maximal lanthanide luminescence is important in the development of systems for use in sensitive clinical diagnostic and time-resolved luminescence-based immunoassay applications.