Thermodynamic Characterization of the Interaction between the C-Terminal Domain of Extracellular Superoxide Dismutase and Heparin by Isothermal Titration Calorimetry

摘要

Extracellular superoxide dismutase (ECSOD) interacts with heparin through its C-terminalndomain. In this study we used isothermal titration calorimetry (ITC) to get detailed thermodynamicninformation about the interaction. We have shown that the interaction between ECSOD and intestinalnmucosal heparin (Mw 6000-30000 Da) is exothermic and driven by enthalpy at physiological saltnconcentration. However, the contribution from entropy is favorable for binding of small isolated heparinnfragments. By studying different size-defined heparin fragments, we also concluded that a hexasaccharidenmoiety is sufficient for strong binding to ECSOD. The binding involves proton transfer from the buffer to thenECSOD-heparin complex, and the results indicate that the number of ionic interactions made betweennECSOD and heparin upon binding varies from three to five for heparin and an octasaccharide fragment,nrespectively. Surprisingly and despite themany charges found on both the protein and the polysaccharide, ournresults indicate that the nonionic contribution to the binding is large. From the temperature dependence wenhave calculated the constant pressure heat capacity change (ΔCp) of the interaction to -644 J K-1nmoln-1nandn-306 J K-1nmoln-1nfor heparin and an octasaccharide, respectively