Bohr effect of human hemoglobin: Separation of tertiary and quaternary contributions based on the Wyman equation

- pKas of the 4 organic phosphate binding groups in 2,3-BPG bound hemoglobin determined.
- The β-chain terminal amino group is a negative Bohr contributor in 2,3-BPG bound Hb.
- Lys82β is a positively contributing Bohr group in 2,3-BPG bound hemoglobin
- The total Bohr contributions of histidine groups is reduced at pH 7.4 by 2,3-BPG.
- Tertiary and quaternary Bohr contributions are separated.

As a prelude to separating tertiary from quaternary structure contributions to the Bohr effect, we employed the Wyman equation to analyze Bohr data for human hemoglobin to which 2,3-bisphosphoglycerate, 2,3-BPG, is bound. Changes in the pKas of the histidine Bohr groups result in a net reduction of their contributions to the Bohr effect at pH 7.4 compared to their contributions in stripped hemoglobin. The non-histidine 2,3-BPG binding groups - the β-chain terminal amino group and Lys82β - make negative and positive contributions, respectively, to the Bohr effect. The final result is that the Bohr effect at physiological pH is higher for 2,3-BPG bound compared to stripped hemoglobin. Contributions linked to His2β, His77β and His143β enable us to separate tertiary from quaternary Bohr contributions in stripped and in 2,3-BPG bound hemoglobin. Both contributions serve to make the Bohr effect for 2,3-BPG bound hemoglobin higher than for stripped hemoglobin at physiological pH.

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