A thermodynamic analysis of the binding of calcium and magnesium ions to parvalbumin.

Microcalorimetry and equilibrium dialysis were used to determine the thermodynamic functions delta H0, delta G0 and delta S0 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 Mg2+ with equilibrium constants of KCa = 2.7 X 10(9) M-1, and KMg = 9.5 X 10(4) 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+ is 2.8 X 10(7) M-1, and the Mg2+-Ca2+ exchange equilibrium constant equals 2.8 X 10(4) 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 delta H0 = -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 delta S0 are +55.2 J x mol-1 x K-1 for Ca2+ complex formation and +54.8 x mol-1 x K-1 for Mg2+ complex formation. Thus the respective metal binding processes are driven by both enthalpy and entropy conbinations, and are reminiscent of Ca2+ binding to troponin C. The reaction entropy observed during Mg2+-Ca2+ exchange (0.4 J x mol-1 x 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.