Determination of the Ca²⁺ and Mg²⁺ affinity constants of troponin C from eel skeletal muscle and positioning of the single tryptophan in the primary structure

The complete amino acid sequence of troponin C (ETnC) from the white muscle of the European eel has been determined by Edman degradation procedures. Its single tryptophan residue is situated in helix H at amino acid position 152 of the aligned sequence; the tryptophan is the first residue on the C-terminal side of Ca²⁺ binding loop IV. The increase of tryptophan fluorescence emission intensity occurring upon titration of ETnC with Ca²⁺ has been used to determine the affinity constants of ETnC for Ca²⁺. The calculated affinity of ETnC for Ca²⁺ results in a K_(Ca) of 1.3 10⁷ M^-1, typical of the Ca²⁺-Mg²⁺ sites of the second domain of fast skeletal muscle TnCs. Moreover, a direct competition between Ca²⁺ and Mg²⁺ was also observed. The calculated affinity of ETnC for Mg²⁺ is K_(Mg)=1.2 10³ M^-1. In order to probe the affinity constants of the Ca²⁺ binding sites of the regulatory domain, ETnC was labelled with dansylaziridine (Danz). The Danz fluorescent signal was used to estimate the affinity constants of ETnC-Danz for Ca²⁺ and also for Mg²⁺ (assuming a competitive behaviour between these two metal ions). The calculated affinity constants are K_(Ca)=9.4 10⁵ M^-1 and K_(Mg)=2.0 10² M^-1, respectively. These values are typical of the Ca²⁺-specific sites of the regulatory domain of fast skeletal muscle TnCs.