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J. Biochem, 1984, Vol. 95, No. 6 1767-1774
© 1984 Japanese Biochemical Society

research-article

Stoichiometry and Location of Terbium and Calcium Bindings to Porcine Intestinal Calcium-Binding Protein

Kenzo CHIBA, Takao OHYASHIKI and Tetsuro MOHRI

Department of Physiological Chemistry, School of Pharmacy, Hokuriku University Kanagawa-machi, Kanazawa, Ishikawa 920-11

Quantitative analyses were carried out on Tb3+ binding to porcine intestinal cal cium-binding protein (CaBP). Tb3+ (emission at 547 nm) and intrinsic tyrosine (emission at 303 nm) fluorescences upon excitation at 260 nm increase almost in parallel with increasing Tb3+ concentration up to a molar ratio of 2 against the protein in the CaBP solution. The pH dependence profile of Tb3+ fluorescence of the Tb3+-CaBP complex suggests for Ca2+ that some free carboxylate groups are involved in the binding, as also suggested for Ca2+ binding. The results of fluorometric titration of Tb3+ and intrinsic tyrosine fluorescences of the CaBP complex with Tb3+ or Ca2+ led us to conclude that Tb3+ and Ca2+ have two common binding sites for each CaBP molecule. An equilibrium dialysis experiment showed that the dissociation constants of the two Tb3+-binding sites are 0.29 and 3.51 µM. Tb3+ strongly inhibits 45Ca binding to one of the two Ca2+ sites in the CaBP.

All of these and previous results indicate that each Tb2+ ion can bind to either of two high-affinity Ca2+-binding sites in porcine intestinal CaBP with an affinity different from that for Ca2+ ion. We discuss the localization of the Ca2+ and Tb3+-binding sites in the CaBP.


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