Transition of hemoglobin between two tertiary conformations: The transition constant differs significantly for the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica)

We demonstrate that 5,5′-dithiobis(2-nitrobenzoate) - DTNB - reacts with only CysF9[93]β and CysB5[23]β among the multiple sulfhydryl groups of the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica). Kequ, the equilibrium constant for the reaction, does not differ very significantly between the two hemoglobins. It decreases 430-fold between pH ≈ 5.6 and pH ≈ 9: from a mean of 7 ± 1 to a mean of 0.016 ± 0.003. Quantitative analyses of the Kequ data based on published X-ray and temperature-jump evidence for a tertiary structure transition in liganded hemoglobin enable the calculation of Krt, the equilibrium constant for the r ←→ t tertiary structure transition. Krt differs significantly between the two hemoglobins: 0.744 ± 0.04 for the major, 0.401 ± 0.01 for the minor hemoglobin. The mean pKas of the two groups whose ionizations are coupled to the DTNB reaction are about the same as previously reported for mammalian hemoglobins.