Luminescence and circular-dichroism analysis of terbium binding by pig intestinal calcium-binding protein (relative mass = 9000)

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Abstract
The structure and conformations of pig intestinal Ca-binding protein (CaBP) were studied by terbium luminescence enhancement and circular dichroism. The 2 cation-binding sites bind Tb3+ sequentially; the affinity of the 1st site is > 107 M-1 and the affinity of the 2nd site is .apprx. 105 M-1. Filling of the 1st site enhances the fluorescence of the single tyrosine residue, whereas Tb3+ in the 2nd site quenches the fluorescence. Excitation spectra of the Tb3+-bound forms of CaBP show that considerable energy transfer takes place from phenylalanine residues to the bound Tb3+, although some transfer from tyrosine is also detected. The sequence in which the sites are filled was deduced from these results and the published 3-dimensional structure of the cow intestinal CaBP. Tb3+ bound .apprx. 20 .ANG. (1 .ANG. = 0.1 nm) from the tyrosine induced a large increase in the optical activity of this residue. A potentially important conformational change apparently is induced in CaBP by cation binding.