Resolution of the Equilibrium Constant for the T State → RState Conformational Change of Human Hemoglobin into Endothermic and Exothermic Component Reactions

The dimensionless equilibrium constant for the allosteric conformation change, K_ΔC = 0.02602 (Knowles & Magde, linked ms 2) following binding of O₂ by α-chains in ^Tstate Hb₄/BPG (whole blood under standard conditions) is shown to be comprised of: (i) an endothermic change in conformation, from ^Tstate to ^Rstate, of 24.3 kJ/mol; (ii) exothermic conversion of ^Tstate ^TαO₂-chains to ^Rstate ^RαO₂-chains of -13.8 kJ/mol; (iii)exothermic binding of BPG by R-states. Eq. (1) defines the component steps whereby the ^Tstate conformation is converted to the ^Rstate conformation.

ΔG^o(^R(Hb₄), BPG) describes the endothermic decomposition of the binary complex, ^THb₄/BPG into ^RHb₄ and BPG, equal to + 33.7 kJ/mol (DeBruin et al. (1973). J. Biol. Chem. 248, 2774-2777). ΔG^o for the equilibrium constant for ΔG^O(K_ΔC) and Σ ΔG^o for binding of O₂ by the pair of equivalent ^Tstate α-chains, ΔG^O(^Tα^*O₂), + 9.41 kJ/mol and – 49.6 kJ/mol, respectively, are determined by fitting of O₂ equilibrium binding data to the Perutz-Adair equation. ΔG^o for reaction of a pair of equivalent ^Rstate α-chains with O₂, ΔG^O(^RαO₂), was estimated from the known affinity of myoglobin for O₂ at 37^oC (Theorell H. (1936). Biochem. Z., 268, 73-81), -63.4 kJ/mol. The unknown quantity, ∆G^O(^R(HbO₂)₄/BPG), was obtained by solving Eq. (1), being -10.5 kJ/mol, K (^R(HbO₂)₄/BPG) = 58.4 L/mol. The value of the equilibrium constant for binding BPG to R-state conformations represents 0.0073% of the value of the binding constant of BPG to ^Tstate conformations: 800,000 L/mol. The value of K_ΔC; (i) accounts for the ability of O₂ to escape, virtually unhindered from rbcs and (ii) provides a biophysical basis for manifestation of high resting rates of metabolism in warm blooded species.