Temperature dependence of calcium and magnesium induced caseinate precipitation in H2O and D2O

Casein is a well-known and highly studied protein present in milk found in the form of large colloidal particles called casein micelles. Here we assessed the influence of two solvents, H₂O and D₂O, on the precipitation temperatures of sodium caseinate induced by calcium and magnesium ions. The intrinsic fluorescence of casein was correlated with the suspended and precipitated protein fractions in order to extrapolate the precipitation temperature. The precipitation temperature values were determined for several calcium and magnesium concentrations in H₂O, demonstrating the strong differences in the association behaviour induced by the two divalent cations. The precipitation temperatures of caseinate, in the presence of calcium induced by heating the samples in D₂O, were significantly lower compared with the one performed in H₂O. On the contrary, the precipitation temperatures of casein in the presence of magnesium were very close to those obtained in the samples tested in H₂O. Thus, calcium interaction with the protein depends on temperature and solvents, two parameters that modify the accessibility of the binding site of the cation. Conversely, the interaction of the divalent magnesium ion with casein was shown to depend only upon the temperature. Remarkably, this evidence indicates that the two ions have different binding sites on the protein, suggesting that the D₂O solvent plays an important role in the detection of the different binding sites for calcium and magnesium. Additionally, the results obtained with the simultaneous presence of both cations suggest the existence of a cooperative mechanism between the two ions, in which the presence of calcium makes more sites available for the binding of magnesium.