The role played by the metal ion in thermodynamics of azurin folding was addressed by studying the thermal denaturation of
the apo-form by differential scanning calorimetry (DSC), and by comparing the results with data concerning the holo protein.
The thermal unfolding experiments showed that at 25°C the presence of metal ion increases the thermodynamic stability of azurin
by 24 kJ mol−1. A comparison between the unfolding and the copper binding free energies allow us to assert that the unfolded polypeptide
chain binds copper and subsequently folds into native holo azurin, being this the thermodynamically most favourable process
in driving azurin folding.