Transition of Hemoglobin Between Two Tertiary Conformations: Determination of Equilibrium and Thermodynamic Parameters from the Reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9βsulfhydryl Group
The equilibrium constant of the reaction of 5,5′-dithiobis(2-nitrobenzoate) with the CysF9β sulfhydryl group of hemoglobin decreases by 2 to 3 orders of magnitude between pH 5.6 and 9. The reaction is coupled to the ionizations of two groups on the protein. At 25 °C one group has a pKa of 5.31 ± 0.2 when hemoglobin is in its (tertiary) r conformation, typified by the thiolate anion form of CysF9β; this changes to 7.73 ± 0.4 in the (tertiary) t conformation, typified by the mixed disulfide form of the sulfhydryl. The second group ionizes with a pKa of 7.11 ± 0.4 in the r conformation; this changes to 8.38 ± 0.2 in the t conformation. Krt, the equilibrium constant for the r ←→ t isomerization process, is 0.22 ± 0.06. The standard enthalpy and entropy changes for the isomerization are ΔHort = 24.2 kJ mol− 1 and ΔSort = 68.8 JK− 1mol− 1, respectively.