Thermodynamic studies on subunit assembly in human hemoglobin. Self-association of oxygenated chains (alphaSH and betaSH): determination of stoichiometries and equilibrium constants as a function of temperature.
The homogeneous self-association of isolated alphaSH chains and betaSH chains from human hemoglobin has been studied by analytical molecular sieve chromatography over the concentration range 0.004 to 15.2 mg/ml. Detailed studies were carried out as a function of temperature at pH 7.4 in 0.1 M Tris/HCl, 0.1 M NaCl, 1 mM Na2EDTA in order to establish stoichiometries, equilibrium constants, and enthalpies for the self-association reactions. The dissociation data best describe the alphaSH system as being a monomer-dimer equilibrium (2alpha1  in equilibrium alpha2). Under the same conditions the betaSH system is best described by a monomer-tetramer equilibrium (4beta1 in equilibrium beta4). van't Hoff enthalpies were determined from the temperature dependence of the equilibrium constants. For the 2alpha in equilibrium alpha2 reaction the molar enthalpy delta H = 4.3 +/- 0.5 kcal, and for the reaction 4beta1 in equilibrium beta4, deltaH = 23.5 +/- 1.0 kcal. Unitary entropies were determined to be: deltaSu = 40.6 e.u., deltaSu = 177.5 e.u., respectively. Thermodynamic parameters for association of the two types of chains are roughly comparable in magnitude if four bonding interactions are assumed in the beta4 tetramer. Both reactions are entropy-driven, and the overall results (including salt effects) are consistent with a dominant role of hydrophobic interactions. Increasing the NaCl concentration to 2 M at 21.5 degrees under the same buffer conditions increases the association constant for both the alphaSH and betatsh chains. This increase in the association constants with increasing salt concentration is attributable to the increased binding of salt, or the release of bound water upon formation of the association complexes, or both. The present results do not distinguish between these possibilities. The introduction of inositol hexaphosphate (IHP) was found to have no effect upon subunit association in betaSH chains. This result and the previously observed effect of IHP upon oxygenation of beta chains imply that oxygenation and self-association are completely unlinked in this system. Accurate determinations of (a) the enthalpy changes for homogeneous reactions of isolated chains, carried out in this study and of (b) the enthalpy of forming alpha2beta2 tetramers from alphabeta dimers provide a basis for the interpretation of (c) calorimetric studies on reconstitution of hemoglobin from the isolated chains, described in accompanying papers.
D006418:Heme; D006441:Hemoglobin A; D006454:Hemoglobins; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D010108:Oxyhemoglobins; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D013816:Thermodynamics
D006454:Hemoglobins; D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D006418:Heme; D006441:Hemoglobin A