A Conformation Change of the Porcine Intestinal Calcium Binding Protein on Binding of Ca²⁺

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The intrinsic tyrosine fluorescence of the porcine intestinal calcium binding protein (CaBP, 7μM) was quenched by the addition of ∼170μM ethyleneglycol-bis (2-amino ethylether)-N, N, N', N'-tetraacetic acid (EGTA), returning progressively to its original level with increasing concentration of subsequently added Ca³⁺ up to 117μM, in a concentrationdependent manner. In the presence of an excess of EGTA, the intrinsic fluorescence of the CaBP was further quenched by 1M or less of guanidine-HCl, While it was enhanced by 2-4M guanidine. In the presence of an excess of Ca²⁺, the fluorescence intensity increased monotonically with increasing concentration of guanidine (∼4м). Quenching of the intrinsic fluorescence of the CaBP by alkaline pH's (above 8) was moderated by addition of EGTA compared to that measured in the presence of Ca²⁺. KCl (∼100mм) showed a quenching effect on the fluorescence in the presence of 83μм EGTA, an enhancing effect in the presence of 1 mм EGTA, and no effect in the presence of Ca²⁺ at a concentration sufficient to saturate the CaBP. These experimental results suggest that Ca²⁺ binding to the CaBP induces microenvironmental and also significant conformational changes in the tyrosine-containing region of the protein.