Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein.
Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.
Hofmann
T
T
Department of Biochemistry, University of Toronto, Ontario, Canada.
Eng
S
S
Lilja
H
H
Drakenberg
T
T
Vogel
H J
HJ
Forsén
S
S
D000818:Animals; D001665:Binding Sites; D064026:Calbindins; D002135:Calcium-Binding Proteins / Q000032:analysis*; D002942:Circular Dichroism; D005063:Europium; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000032:analysis*; D007834:Lasers; D064030:S100 Calcium Binding Protein G / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D013552:Swine
D064026:Calbindins; D002135:Calcium-Binding Proteins; D064030:S100 Calcium Binding Protein G; D005063:Europium