Quantitative analysis of calcium binding to porcine intestinal calcium-binding protein.
An equilibrium dialysis study has revealed that porcine intestinal calcium-binding protein (CaBP) has two binding sites for Ca2+ whose dissociation constants (Kd) are the same, 0.56 microM. 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 Kd value is 0.51 microM. 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 alpha-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 accompanying Ca2+ binding in the vicinity of single tyrosine and phenylalanine residues of the protein molecule.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D007421:Intestine, Small / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013552:Swine
D002135:Calcium-Binding Proteins; D002118:Calcium