Isothermal titration microcalorimetric studies of the effect of salt concentrations in the interaction between proteins and hydrophobic adsorbents

The effect of (NH₄)₂SO₄ concentrations on the interaction mechanism between myoglobin and two hydrophobic adsorbents, butyl- and octyl-Sepharose was investigated by the equilibrium binding analysis and by directly measured adsorption enthalpies. The result obtained from the isotherms demonstrated that the affinities of myoglobin adsorption onto the both adsorbents were increased with salt concentrations. Furthermore, the adsorption enthalpies measured by using isothermal titration calorimetry (ITC) were decreased with an increment of salt concentrations, and these findings were explained by the reduction in the dehydration heat and the enhancement of heat released by the hydrophobic interaction as salt concentrations increase. Additionally, the adsorption enthalpies of myoglobin with both the resin increased as the amount of bound protein, and the increment in the variation of enthalpy value at 0 M appeared to be steeper than that at 1.0 M (NH₄)₂SO₄. This result implies that protein-protein repulsion was stronger in the absence of the salt in these experiments. The thermodynamic parameters presented herein have important implication, both for providing further insight into the binding mechanism of protein adsorption and for improving theoretical approaches to HIC.