Quantitative Analysis of Calcium Binding to Porcine Intestinal Calcium-Binding Protein

Abstract

An equilibrium dialysis study has revealed that porcine intestinal calcium-binding protein (CaBP) has two binding sites for Ca²⁺ whose dissociation constants (K_d) are the same, 0.56 μM. The intrinsic fluorescence spectrum of the CaBP shows a peak (at 303 nm) in tyrosine band. Ca²⁺ binding to the CaBP induces a monophasic increase in the intensity of intrinsic fluorescence without any shift to either excitation or emission maximum. The change in the fluorescence intensity induced by Ca²⁺ binding is complete at a bound Ca²⁺/CaBP molar ratio of about 2, and the apparent K_d value is 0.51 μM. The same bound Ca²⁺/CaBP molar ratio has been obtained from the maximal changes in UV absorption and CD spectrum of the CaBP upon addition of Ca²⁺. A CD study has shown an about 5% increase in α-helix content in the CaBP at the maximal binding of Ca²⁺. All these results indicate that the porcine intestinal CaBP has two high-affinity binding sites with equal affinity for Ca²⁺ and suggest that it undergoes a quantitative conformation change accompany-ing Ca²⁺ binding in the vicinity of single tyrosine and phenylalanine residues of the protein molecule.