Investigation of cation-binding properties of cardiac troponin C peptides by circular-dichroism spectroscopy

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Abstract
Bovine cardiac troponin C was cleaved at residues cysteine-35 and cysteine-84. Three peptides, N-terminal (residues 1-34), central (residues 35-83) and C-terminal (residues 84-161), of cardiac troponin C were obtained in a homogeneous state. Saturation of troponin C or its C-terminal peptide with Ca2+ or Mg2+ is accompanied by an increase in the ellipticity at 222 nm in the c.d. [circular dichroism] spectrum. The half-maximal changes in the ellipticity of troponin C were observed at 32 nM-Ca2+ or 56 .mu.M-Mg2+. The corresponding values for the C-terminal peptide are 7.1 nM for Ca2+ and 4.5 .mu.M for Mg2+. The ellipticity of the central peptide (residues 35-83) containing the 2nd cation-binding site was decreased on saturation with Ca2+. The half-maximal changes in the ellipticity occur at 80 .mu.M-Ca2+. Study of the c.d. spectra suggests that the .alpha.-helices flanking the 2nd cation-binding site of cardiac troponin C exist independently of Ca2+. Saturation of the 3rd and 4th sites with these cations is associated with a considerable increase in the .alpha.-helix content, probably due to the formation of an .alpha.-helix flanking the 3rd site on the N-terminus.