Physicochemical properties of calcium-binding protein isolated from rat liver cytosol: Ca2+-induced conformational changes.

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Abstract
The physicochemical properties of a calcium-binding protein (CaBP) isolated from rat liver cytosol was investigated. Isoelectric focusing in a polyacrylamide gel plate using the Broad pI Calibration Kit showed that the isoelectric point is 5.20. The ultraviolet (UV) absorption spectrum of CaBP showed a maximum at 278 nm. The conformational changes induced by Ca2+-binding to CaBP were examined by measuring the UV difference, fluorescence emission, and circular dichroism (CD) spectra. These spectra were altered by titration of CaBP with 1.0 mM Ca2+. The alterations could be attributed to an increased exposure of tyrosine and tryptophan residues to a more aqueous environment, resulting in an increased hydrophobicity of CaBP. From the CD spectrum, the apparent .alpha.-helical content of CaBP in Ca2+-free buffer was estimated to be 34%. This value was decreased by 1.0 mM Ca2+ addition. The results suggest that Ca2+-binding induces conformational changes of CaBP, and that the protein contains distinct and specific ligand-binding sites for Ca2+.