Thermodynamic Analysis of the Interaction of Heparin with Lysozyme.

Glycosaminoglycan (GAG)-protein binding governs critically important signaling events in living matter. Aiming at a quantitative analysis of the involved processes, we herein present a thermodynamic study of the interaction of the model GAG heparin and lysozyme in aqueous solution. Heparin is a highly charged linear polyelectrolyte with a charge parameter of 2.9 (37°C). The binding constant Kb was determined by ITC as the function of temperature and ionic strength adjusted through the concentration cs of added salt. The dependence on salt concentration cs was used to determine the net number of released counterions. Moreover, the binding constant at a reference salt concentration of 1M Kb(1M) was determined by extrapolation. The dependence on temperature of Kb was used to dissect the binding free energy ΔGb into the respective enthalpies ΔHb and entropies ΔSb together with the specific heat Δcp. A strong enthalpy-entropy cancellation was found similar to the results for many other systems. The binding free energy ∆Gb could furthermore be split up into a part ∆Gci due to counterion release and a residual part ∆Gres. The latter quantity reflects specific contributions as e.g. salt bridges, van der Waals interactions or hydrogen bonds. The entire analysis shows that heparin-lysozyme interactions are mainly caused by counterion release, that is, ca. three counterions are being released upon binding one lysozyme molecule. Our reported approach of quantifying interactions between glycosaminoglycans and proteins is generally applicable and suitable to provide new insights in the physical modulation of biomolecular signals.