Effect of water on the allosteric equilibrium and kinetics of hemoglobin

Jie Jiang

doi:10.17918/00008037

Hemoglobin has long been studied as a model for understanding the thermodynamic and kinetic behavior of allosteric proteins. All proteins interact within an environment that is at least partially aqueous. Therefore, investigation of the effect of water in protein function is very important. In this work, the effect of water on the kinetics and equilibrium of hemoglobin was studied by modulated excitation and equilibrium binding measurements. Water activity was changed by adding a small neutral solute (sucrose). From equilibrium binding experiments, we found that ligand binding affinity is reduced and p50 (oxygen pressure at half saturation) is increased by the presence of 1.1M sucrose. For oxyhb, [delta]logp50 = 0.03 ± 0.02, and this gives that 10 more water molecules are bound to hemoglobin in R state than in T state. For HbCO, [delta]logp50 = 0.01 ± 0.01. These values are in reasonable agreement with predictions based on buried surface calculations. (Lesk, A.M., Janin, J., Wodak, S. and Chothia,C., J.Mol. Biol. 183, 267-270, 1985). In kinetic experiments, the effect of water on transition rates of structure change R₃ <-> T₃ was studied. We found that the equilibrium constant L₃ was increased 74% by the presence of 1.1M sucrose. From the change of kinetic rate, the free energy contribution to the transition state by bound water molecules was studied. If both internal and external viscosity effects are considered and internal viscosity is much larger than the external viscosity, we found that the difference of the number of water molecules bound to hemoglobin between the R and the transition states (n_R - n_[TS]) = 0, and between the T and the transition states (n_T - n_[TS]) = -20 ± 17. The free energy contribution by difference of bound water between R and transition states [delta]G_[R,TS] = 0, and between T and transition states [delta]G_[T,TS] = - 0.3 ± 0.3 kcal Mol⁻¹. Using only the external viscosity, we got (n_R - n_[TS]) = 48 and (n_T - n_[TS]) = 29. In that case, the free energy [delta]G_[R,TS] = 0.8 kcal Mol⁻¹ and [delta]G_[T,TS] = 0.5 kcal Mol⁻¹. Either interpretation contradicts the transition state model of Janin and Wodak (Janin, J. and Wodak, S. Biopolymers, Vol. 24, 509-526, 1985) and argues that hemoglobin dynamics cannot be viewed as simple rigid body motion of the subunits.

Effect of water on the allosteric equilibrium and kinetics of hemoglobin

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Effect of water on the allosteric equilibrium and kinetics of hemoglobin

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