The kinetics of the alkaline Bohr effect of human hemoglobin.

Liganded hemoglobin is a stronger acid than ligand-free hemoglobin. The kinetics of the transformation between these states was studied in the pH range 6.9 to 9.0 in 0.3 M NaCl with a pH indicator (phenol red or m-cresol purple) to follow the pH changes accompanying flash photolysis of human carbon monoxide hemoglobin. At least three distinct processes involving protons were detected kinetically: a rapid uptake of H+ presumably reflecting the shift from the ligand-bound to ligand-free comformation, followed by a slower biphasic release of protons accompanying the dark reaction of CO and hemoglobin. The fast proton uptake occurred with a rate constant k = 8000 set-l (ZOO, pH 7.8), whereas the rate of the transition was greater both at pH 6.9 and 9.0 (k > 10,000 set-l). The transition state parameters were also obtained: AH* = 11.2 f 0.4 kcal per mole and AS* = -3.2 f 1.5 e.u. The biphasic CO-binding and H+ release reactions following the flash were analyzed by treating the results as the sum of two simple exponential expressions; this procedure revealed that 27% of the reaction involving protons occurred at the higher rate. The rapidly reacting hemoglobin observed (50%) under these conditions (21 pM Hb, pH 7.6, 0.3 M NaCl, 60 pM CO, 25”) is attributed, on the basis of its dependence on carbon monoxide hemoglobin concentration, to the a& dimer. The kinetic properties of both ligand binding and proton release were identical also at pH 6.95, 0.3 M NaCl, when carbon monoxide hemoglobin was subjected to flash photolysis in the presence of O2 (rate constant = 1850 set-I). At pH 7.9, however, there was a distinct lag in release of hydrogen ions. The results are consistent with the recent structural interpretation of the Bohr effect (PERIJTZ, M. F., MUIRHEAD, H., MAZZARELLA, L., CROTHER, R. A., GREER, J., AND KILMARTIN, J. V., Nature, 222, 1240 (1969)), as well as the hypothesis that the alfll dimer is devoid of cooperative behavior.