Lanthanide ion luminescence probes. Characterization of metal ion binding sites and intermetal energy transfer distance measurements in calcium-binding proteins. 1. Parvalbumin

Abstract

Eu(III) laser excitation spectroscopy of the 7F0 .fwdarw. 5D0 transition reveals spectral features characteristic of the occupation of the CD and EF Ca(II)-binding sites of carp parvalbumin by Eu(III) ions. In addition, at pH 6.5, a signal attributable to binding at a 3rd site is observed. This feature is abolished upon lowering the pH to 3.8. At pH 6.5, the appearance of this feature correlates with the decrease in luminescence intensity during titrations of parvalbumin with Eu(III) or Tb(III) after more than .apprx. 1.8 equivalent of either of these ions has been added. Eu(III) ions in the primary sites coordinate 0-2 H2O molecules while the 3rd site involves about 3 coordinated H2O molecules. Parvalbumins in which mixed pairs of lanthanide ions, Ln(III), occupy the CD and EF sites 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. With the assumption of a Forster-type dipole-dipole mechanism, inter-binding site distance estimates were made and are in reasonable agreement with the distance (11.8 .ANG.) obtained by X-ray crystallography, especially when Eu(III) is the donor. RO values (critical distances for 50% energy transfer) in H2O solution range from 9.2 .ANG. for the Tb(III)-Ho(III) donor-acceptor pair down to 5.7 .ANG. for the Eu(III)-Ho(III) pair.