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The thermodynamics of binding of the trivalent cations cobalt hexammine and spermidine to plasmid DNA was studied by isothermal titration calorimetry. Two stages were observed in the course of titration, the first attributed to cation binding and the second to DNA condensation. A standard calorimetric data analysis was extended by applying an electrostatic binding model, which accounted for most of the observed data. Both the binding and condensation reactions were entropically driven (TΔS∼+10 kcal/mol cation) and enthalpically opposed (ΔH∼+1 kcal/mol cation). As predicted from their relative sizes, the binding constants of the cations were indistinguishable, but cobalt hexammine had a much greater DNA condensing capacity because it is more compact than spermidine. The dependence of both the free energy of cobalt hexammine binding and the critical cobalt hexammine concentration for DNA …