On the Molecular Mechanism of the Calcium-Induced Gelation of Pectate. Different Steps in the Binding of Calcium Ions by Pectate

Pectic acid/sodium pectate is one of the most widespread hydrocolloid used in the food industry. It is able to form strong ionotropic gels by the addition of ions, in particular, calcium ions. The initial steps of binding Ca²⁺ ions to a sample of sodium pectate with a composition close to 90% of ideal Na⁺-poly(galacturonate) were investigated by means of circular dichroism (CD), microcalorimetry, dilatometry, viscosity, and membrane osmometry, as a function of increasing R_j, R_j being the ratio of the molar concentrations of Ca²⁺ and pectate repeating units. Data were collected in aqueous NaClO₄ at 25 °C. The key instrument of interpretation has been the counterion condensation theory (CCT) of linear polyelectrolytes, modified to include the presence of both specific affinity of the divalent counterion for the polysaccharide ("territorial binding"), and, very importantly, strong chemical bonding (not a covalent bonding, though) of Ca²⁺ on conformationally well-defined sites on the polymer, with local charge annihilation. Intrinsic viscosity and number-average molar mass data as a function of R_j showed that calcium bonding brings about chain association right from the beginning of addition to pectate. The analysis of the microcalorimetric curve using the modified CCT revealed two types of bonding. In the order of development as a function of R_j, the first mode (type 1) could be reconciled with the "tilted egg-box" type, recently proposed for Ca²⁺ binding to alginate and the second mode (type 2) with the "shifted egg-box" proposed for calcium pectate on the basis of conformational analysis investigation. Likewise, the two types of bonding turned out to be superimposable with similar bonding categories proposed for alginate and low-methoxyl pectin (LMP), on the one side, and for the association of semiflexible polyelectrolytes, on the other. The analysis allowed us to obtain standard Gibbs free energy, enthalpy, entropy, and volume molar values both for the affinity and the chemical bonding processes. Interestingly, the analysis of the dependence of the gelation temperatures, T_g, of LMP upon increasing additions of calcium ions provided the values of T_g and standard Gibbs free-energy of calcium-to-pectate association coinciding with those obtained from calorimetry for the type-2 bonding process. This finding corroborated previously reported evidence on the enthalpic nature of the elasticity of Ca²⁺-pectate gels. Finally, comparative analysis of different techniques, but of CD in particular, enabled proposing a "loose-2₁-helix" as the starting conformation of sodium pectate in aqueous solution.