Microcalorimetry and equilibrium dialysis were used to determine the thermodynamic functions ΔHΔ, ΔG° and δS° guiding the interaction of Ca²⁺ and Mg²⁺ with purified carp muscle isoparvalbumin of pI 4.25. The equilibrium dialysis studies indicate equal affinities of the two metal sites of parvalbumin for either Ca²⁺ or Mg ²⁺ with equilibrium constants of K_Ca= 2.7 × 10⁹ M⁻¹, and K_Mg= 9.5 × 10⁴ M⁻¹. Binding of the two metal ions is fully competitive with no indication for cooperative effects. The apparent Ca²⁺ affinity constant K'_Ca in the presence of 1 mM Mg²⁺ 2.8 × 10⁷M⁻¹, and the Mg²⁺−Ca²⁺ exchange equilibrium constant equals 2.8 × 10⁴ M⁻¹.

Microcalorimetry shows that parvalbumin exhibits negative reaction enthalpies of –37.2 kJ/mol metal site for Ca²⁺-binding and –25.1 kJ/mol site for Mg²⁺-Ca²⁺ exchange, yielding ΔH°=–12.1 kJ/mol site for Mg²⁺ complex formation. Enthalpy changes are linearly dependent upon the amount of metal bound to the protein, thus corroborating the equal affinities of the two sites. Reaction entropies ΔS° are + 55.2 J · mol⁻¹− K⁻¹ for Ca²⁺ complex formation and + 54.8 J · mol-⁻¹·K⁻¹ for Mg²⁺ complex formation. Thus the respective metal binding processes are driven by both enthalpy and entropy contributions, and are reminiscent of Ca²⁺ binding to troponin C. The reaction entropy observed during Mg²⁺−Ca²⁺ exchange (0.4 J. mol⁻¹ - K⁻¹) is negligible in spite of the markedly different hydration entropies for Ca²⁺ and Mg²⁺ This indicates that the two metal complexes of parvalbumin do not have the same conformation entropy. Since no variation in the intrinsic protein fluorescence was observed upon metal exchange, the conformation differences must be restricted to the immediate environment of the metal binding sites.