On the mechanism of energy transfer to Tb3+ ions in proteins. A time-resolved luminescence study of the Tb-elastase complex.

Intraprotein energy transfer to terbium ions is widely used for probing distances of calcium sites in proteins. In this work we have performed a time-resolved study of the sensitized luminescence in elastase using a pulsed laser excitation at 265 nm. Terbium-sensitized luminescence was found to build-up within about 150 microseconds, which indicates that the protein transfers energy at a rate several orders of magnitude slower than expected for a singlet state donor. From the rise time of the signal and from its variation with the oxygen concentration, it can be deduced that 80% of the transfer originates from the first triplet excited state of one unique aromatic residue. From the comparison of protein fluorescence and sensitized terbium luminescence excitation spectra the sensitizer was identified as a tryptophan, presumably Trp141, which is situated only 0.7-0.9 nm away from the Tb site. The results are at variance with the usual assumption that energy is transferred from the first excited singlet state of aromatic residues according to a long-range dipole-dipole interaction and are more consistent with a short-distance exchange mechanism.