A Conformation Change of the Porcine Intestinal Calcium Binding Protein on Binding of Ca^<2+>

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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 Ca^<3+> 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 Ca^<2+>, 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 Ca^<2+>. 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 Ca^<2+> at a concentration sufficient to saturate the CaBP. These experimental results suggest that Ca^<2+> binding to the CaBP induces microenvironmental and also significant conformational changes in the tyrosine-containing region of the protein.
1983-03-25