Lanthanide ion luminescence probes. Characterization of metal ion binding sites and intermetal energy transfer distance measurements in calcium-binding proteins. 2. Thermolysin.

Eu(III) laser excitation spectroscopy of the 7F0 leads to 5D0 transition reveals spectral features characteristic of the occupation of Eu(III) ions in the S(1), S(3), and S(4) Ca-(II)-binding sites of thermolysin. Various hybrid Eu(III) thermolysin species were prepared in which the Eu(III) ion resides in the S(1) or S(3) and S(4) or all three Eu(III)-binding sites of the metalloprotein. The Eu(III) ion in the S(1) site coordinates to one H2O molecule while the other two sites coordinate to about three to four H2O molecules. Thermolysin hybrids in which the donor Ln(III) ion occupies S(1) and acceptor Ln(III) ions occupy sites S(3) and S(4) were prepared. Nonradiative energy transfer between Eu(III) and Tb(III) acting as luminescent donors and various other Ln(III) ions serving as acceptors was observed by monitoring the excited-state lifetimes of the donor ions using a pulsed dye laser apparatus. Virtually all energy transfer occurs between sites S(1) and S(4) while less than 1% of the donor's energy is transferred from S(1) to S(3). With the assumption of a Förster-type dipole--dipole mechanism, the experimental inter-binding-site distance estimates between sites S(1) and S(4) are in good agreement with the distance (11.7 A) obtained by X-ray crystallography. R0 values (critical distances for 50% energy transfer) in H2O solution range from 9.3 A for the Tb-(III)--Ho(III) donor--acceptor pair down to 5.3 A for the Eu-(III)--Ho(III) pair.