Digestion of troponin C with trypsin in the presence and absence of Ca2+. Identification of cleavage points

The rate of tryptic digestion of troponin C has been shown to be dependent on Ca²⁺ (Drabikowski et al., Biochim. Biophys. Acta 490, 216–224). We have characterized the tryptic peptides produced both in the presence and absence of Ca²⁺ using amino acid composition and end-group analyses. In the presence of Ca²⁺ trypsin cleaves TnC at Arg-8, Lys-84 and Lys-88, leading to the formation of two large peptides, one containing the two low-affinity sites (TR₁C), the other, the two high-affinity Ca²⁺-binding sites (TR₂C). In the absence of Ca²⁺ (1 mM EDTA), digestion proceeds much more rapidly and takes place first at Arg-100, followed by Arg-104, Arg-120, Lys-153, Arg-8 and others. The data suggest that the points of cleavage are determined by the Ca²⁺-dependent conformational states of TnC, particularly in the C-terminal half of the protein where the cation is known to induce secondary structure.