Thermodynamic analysis of the binding of glutathione to glutathione S-transferase over a range of temperatures

The binding properties of a glutathione S-transferase (EC 2.5.1.18) from Sc histosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6. 5 over a temperature range of 15-30 degreesC. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. We have also studied the effect of pH on the thermodynamics of GSH-GST interaction. The behaviour s hown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements ind icate that GSH binds to two sites in the dimer of 26-kDa glutathione S-tran sferase from Schistosoma japonicum (SjGST). On the other hand, noncooperati vity for substrate binding to SjGST was detected over a temperature range o f 15-30 degreesC. Among thermodynamic parameters, whereas DeltaG degrees re mains practically invariant as a function of temperature, DeltaH and DeltaS degrees both decrease with an increase in temperature. While the binding i s enthalpically favorable at all temperatures studied, at temperatures belo w 25 degreesC, DeltaG degrees is also favoured by entropic contributions. A s the temperature increases, the entropic contributions progressively decre ase, attaining a value of zero at 24.3 degreesC, and then becoming unfavora ble. During this transition, the enthalpic contributions become progressive ly favorable, resulting in an enthalpy-entropy compensation. The temperatur e dependence of the enthalpy change yields the heat capacity change (DeltaC (p)degrees) of -0.238 +/-0.04 kcal per K per mol of GSH bound.