Thermodynamic studies on subunit assembly in human hemoglobin. Temperature dependence of the dimer-tetramer association constants for oxygenated and unliganded hemoglobins.

Equilibrium constants for dimer-tetramer association of oxygenated human hemoglobin have been determined as a function of temperature, by analytical molecular sieve chromatography, yielding a van't Hoff enthalpy of 3.8 +/- 1.6 kcal/mol, and a unitary entropy of 48.4 +/- 5.7 e.u. Under the same conditions (0.1 M Tris/HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4) the equilibrium constants for dimer-tetramer association of unliganded hemoglobin have been determined as a function of temperature from kinetic studies of the forward and reverse rate constants. The derived enthalpy and unitary entropy for association of unliganded hemoglobin are 0deltaH2 = 28.9 +/- 0.5 kcal/mol and 0deltaS2 = -41.8 +/- 1.7 e.u., respectively. Thus the oxygenation-linked enthalpy of subunit association is 32.7 +/- 1.7 kcal. The corresponding entropic coupling is 90.2 +/- 5.9 e.u. The thermodynamic parameters are consistent with an increased role of hydrophobic interactions within the dimer-dimer contact region upon oxygenation, or a decreased role of hydrogen bonds and ion pair interactions. The opposite signs for association entropies in unliganded and oxygenated states suggest the existence of two distinct subunit interaction processes during the course of oxygenation with a reversal in sign at some particular binding step. The results provide a necessary basis for interpretation of calorimetric results on reconstitution of hemoglobin, described in an accompanying paper.