Bohr effect of native and chemically modified hemoglobins: Quantitative analyses based on the Wyman equation

- The Wyman equation fits the Bohr effect data for mammalian hemoglobins.
- The Wyman equation fits the Bohr effect data for hemoglobin modified at Cys93β.
- His2β, His77β, His143β involved in reduction of Bohr effect of modified hemoglobins.
- α-chain terminal amino group pKas obtained by curve-fitting to Bohr effect data.

Thirteen histidines and the α-chain terminal amino group (ACTA) make all of the contributions to the Bohr effect of human hemoglobin. The pKas of the 13 histidines in carbonmonoxy- and deoxyhemoglobin are known from 1H NMR studies. Those of ACTA are not so precisely known. We employed the Wyman equation and the 13 histidine pairs of pKas to determine the pKas of ACTA by curve-fitting to hemoglobin Bohr effect data. Using all 14 pairs of pKas as preliminary data, we employed the Wyman equation to fit the Bohr data for hemoglobin chemically modified at Cys93β with cystamine, cystine and iodoacetamide. We demonstrate quantitatively that the reduction of the Bohr effect upon chemical modification is due to three negatively contributing Bohr groups: His2β, His77β and His143β. These make twice their normal contributions to the Bohr effect in unmodified hemoglobin. We also find that the ACTA pKas increase with increasing ionic strength.

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