Experimental Evidence of Counterion Affinity in Alginates: The Case of Nongelling Ion Mg 2+ Ivan Donati,* ,† Fioretta Asaro, ‡ and Sergio Paoletti † Department of Life Sciences, and Department of Chemical Sciences, UniVersity of Trieste, Via Licio Giorgieri 1, I-34127 Trieste, Italy ReceiVed: March 31, 2009; ReVised Manuscript ReceiVed: July 23, 2009 The present contribution aims at testing experimentally the theoretical model previously devised (Donati, I.; Cesa `ro, A.; Paoletti, S.; Biomacromolecules 2006, 7, 281-287) for the description of the interaction between alginate and nongelling Mg 2+ ions. The model, based on an extension of the counterion condensation theory, introduces a contribution of free energy of affinity, ΔG aff,0 , which depends on the monomer composition of the polyuronate. In the present work, three different alginates separately mimicking the mannuronan (polyM), the guluronan (polyG), and the polyalternating (polyMG) components of alginate, together with a natural alginate isolated from Laminaria hyperborea (L. hyperborea), were examined. They were treated with Mg 2+ ions, and relative variations in scattered light intensity, isothermal calorimetry (4H mix ), specific viscosity, and 23 Na NMR longitudinal relaxation rates were monitored with respect to samples at the same ionic strength but containing only Na + ions. The fraction of condensed magnesium counterions was found to be strongly dependent on alginate composition, increasing along the series mannuronan < polyalternating ∼ L. hyperborea < guluronan, thus in good agreement with the theoretical predictions. Introduction Long-range electrostatic interactions profoundly affect the structure, solubility, and stability of polyelectrolytes. The concentration and valence of the small ions in solution determine the overall conformation of a polyelectrolyte and, under certain circumstances, can induce its large scale aggregation (e.g., hydrogel formation). Conformation of DNA was shown to strongly depend both on the counterion species and on its valence, e.g., including supercoiling around globular octamers of histone proteins. 1-3 As to the effect of small ions, a marked ion selectivity was reported for DNA; 4 the competition between Na + and Mg 2+ ions for the polynucleotide was experimentally demonstrated and theoretically tackled by means of a Poisson- Boltzmann approach. 5 Recently, sequence-specific binding of monovalent counterions in the minor groove of AT tract was demonstrated by several authors. 6-8 Alginate is a collective term for a family of algal polysac- charides composed of 1-4 linked -D-mannuronic (M) acid and R-L-guluronic (G) acid arranged in a blockwise pattern with homopolymeric regions of G residues (G-blocks, guluronansor polyGssequences) and M residues (M-blocks, mannuronansor polyMssequences) interspersed by regions of strictly alternating sequence (MG-blocks, polyalternatingsor polyMGssequences). This polysaccharide is very well-known for its selectivity toward different divalent cations. For example, Ca 2+ (as well as Ba 2+ ) induces instantaneous hydrogel formation, whereas Mg 2+ does not. The strong and very fast interaction between alginate and the former ions has been largely exploited for biotechnological applications. In fact, encapsulation of several different cells types and tissues, among which Langerhans islets represent the most well-known example, has been exploited in research and preclinical studies. 9-12 A marked compositional dependence of the ion binding of alginate has been reported since the late 1970s, as it was recognized that G-blocks do possess structural features allowing the strong coordination of calcium ions. 13-15 More recently, this overall picture has been extended owing to the availability of compositionally homogeneous alginates prepared by an enzymatic approach. 16,17 With respect to the great amount of work performed on Ca 2+ (and to a lower extent on Ba 2+ ), relatively little attention has been devoted to the behavior of this polysaccharide in the presence of Mg 2+ . Although a limited amount of experimental data on Mg 2+ -alginate are available, the lack of strong polymer-ion interactions and chain-chain associations have been unquestionably demonstrated. 18 Therefore, despite the less appealing industrial and biotechnological application of such system, Mg 2+ ion can represent the best candidate to test the applicability of the counterion condensation (CC) theory, as originally developed by Manning. 19 The latter approach, al- though derived in the limit of zero salt concentration, has proved to describe fairly well the experimental behavior of biopolyelectro- lytes 20-23 and in particular of ionic polysaccharides. 24-27 The case of counterion systems of mixed valence has been consis- tently tackled 28 in the framework of the so-called “chemical model” of the CC theory developed by Manning himself. 22,29 Recently, extensions of Manning theory which take into account finite salt concentration have been proposed. 30,31 Some of us have previously dealt with the description of the interaction between Mg 2+ ions and alginates of different compositions on the basis of heat-of-mixing (microcalorimetric) data and by means of an extension of the CC theory to include a specific affinity between the divalent ion and the polyelec- trolyte(s). 32 However, it is important to stress that such affinity does not imply an inner-sphere “bonding” of the ion; on the contrary, the condensed counterions are still considered as free to move within the condensation volume (“territorial binding”). * Corresponding author. Tel.: +39 040 558 3682. Fax: +39 040 558 3692. E-mail: idonati@units.it. † Department of Life Sciences. ‡ Department of Chemical Sciences. J. Phys. Chem. B 2009, 113, 12877–12886 12877 10.1021/jp902912m CCC: $40.75  2009 American Chemical Society Published on Web 09/08/2009