Thermodynamics of binding interactions between divalent copper and chitin fragments by isothermal titration calorimetry (ITC)

Herein we report the degree of binding of chitin fragments to divalent copper cation using isothermal titration calorimetry (ITC). In a previous study we have reported the kinetic and thermodynamic binding constants for chitin polymer interactions with a range of metal cations including copper (Camci-Unal & Pohl, 2009). In order to better determine the molecular basis for metal binding to these carbohydrate polymers, a series of chitin fragments were tested for their ability to bind to divalent copper. Based on ITC data, the binding strength of N-acetyl-d-glucosamine (GlcNAc) is weakest among the tested substrates with a binding constant of 3.8 × 103 M−1. N,N′-diacetylchitobiose (GlcNAc)2 and N,N′,N′′-triacetylchitotriose (GlcNAc)3 provided binding constants of 5.1 × 103 M−1 and 13.3 × 103 M−1, respectively. Penta-N-acetylchitopentaose (GlcNAc)5 demonstrated the strongest metal interactions with a binding constant of 22.1 × 103 M−1. For comparison, a binding value of 24.8 × 103 M−1 was found for d-glucosamine, which is the deacetylated analog of GlcNAc, with the divalent copper. All experiments showed enthalpically driven interactions. Free energy of reaction values are all determined to be negative indicating spontaneous reactions. Our results indicate that increasing numbers of GlcNAc residues increase the binding strength towards divalent copper cation. However, the effect of adding sugars to the polymer chain only modestly increases the binding affinity, thereby ruling out any chelation multivalency effects.