Determination of calcium-binding sites in rat brain calbindin D(28K) by electrospray ionization mass spectrometry

Calbindin D(28K), a member of the troponin-C superfamily of calcium- binding proteins, contains six putative EF-hand domains. Calcium-binding studies of the protein by different groups of investigators have yielded discordant results with respect to the stoichiometry of calcium-binding. It has been suggested that the protein binds anywhere from 3-6 mol of calcium/mol of protein. We used negative ion electrospray ionization mass spectrometry in order to definitively determine the exact calcium-binding stoichiometry of calbindin D(28K) and two mutant forms of the protein, one lacking EF-hand 2 (Δ2) and the other lacking EF-hands 2 and 6 (Δ2,6). The full-length protein bound 4 mol of calcium/mol of protein, while both of the deletion mutants bound 3 mol of calcium. Since terbium has been used extensively as a probe for the determination of the calcium-binding stoichiometries of calcium-binding proteins, we also examined the binding of terbium to the three proteins under the same conditions. Full-length calbindin D(28K) bound 4 mol of terbium/mol of protein, while calbindin Δ2 and Δ2, 6 each bound 3 mol. These results clearly show that calbindin D(28K) binds 4 mol of calcium/mol of protein and that terbium-binding stoichiometry is similar to that of calcium.