Calcium Binding to Troponin C. II. A Ca2+ Ion Titration Study with a Ca2+ Ion Sensitive Electrode

Abstract

The effects of pH, Mg²⁺, and ionic strength on Ca²⁺ binding to rabbit skeletal troponin C were studied by using a Ca²⁺ sensitive electrode. Troponin C has two high affinity and two low affinity sites and the Ca²⁺ affinity of both sites was increased by increasing pH in a pH range from pH 5.6 to 10.4. The affinity was decreased by increasing ionic strength. The change of the Ca²⁺ affinity can be explained by the electrostatic interaction between Ca²⁺ and the protein. At alkaline pH, the four Ca²⁺ binding sites bind Ca²⁺ with the same affinity and the distinction between the high and the low affinity sites vanished. This result shows that the difference of the Ca²⁺ affinity is owing to differences of the secondary or the tertiary structure of the Ca²⁺ binding sites, not owing to a difference of the primary structures of the Ca²⁺ binding sites. The two high affinity sites bound two Ca²⁺ ions cooperatively in neutral pH. The cooperativity was diminished at both acidic and alkaline pH. Mg²⁺ ion decreased the affinity of the low affinity sites.