Abstract
The normal and differential titration curves of liganded and unliganded hemoglobin were measured at various KCl concentrations (0.1 to 2.0 M). In this range of KCl concentrations, the curves for deoxyhemoglobin showed no salt-induced pK changes of titratable groups. In the same salt concentration range oxyhemoglobin showed a marked change in titration behavior which could only be accounted for by a salt-induced increase in pK of some titratable groups. These results show that the suppression of the alkaline Bohr effect by high concentrations of neutral univalent salt is not caused by a weakening of the salt bridges in deoxyhemoglobin but is due to an interaction of chloride ions with oxyhemoglobin. Measurements of the Bohr effect at various KCl concentrations showed that at low chloride ion concentration (5 times 10-3 M) the alkaline Bohr effect is smaller than at a concentration of 0.1 M. This observation indicates that at a chloride ion concentration of 0.1 M, part of the alkaline Bohr effect is due to an interaction of chloride ions with hemoglobin. Furthermore, at low concentrations of chloride ions the acid Bohr effect has almost vanished. This result suggests that part of the acid Bohr effect arises from an interaction of chloride ions with oxyhemoglobin. The dependence of the Bohr effect upon the chloride ion concentration can be explained by assuming specific binding of chloride ions to both oxy- and deoxyhemoglobin, with deoxyhemoglobin having the highest affinity.
Highlights
10e3 M) the alkaline Bohr effect is smaller than at a concentration of 0.1 M. This observation indicates that at a chloride ion concentration of 0.1 M, part of the alkaline Bohr effect is due to an interaction of chloride ions with hemoglobin
In a recent short report on the dependence of the alkaline Bohr effect on the chloride ion concentration [15], we showed that the number of Bohr protons re
Since pK shifts introduced by chloride binding will strongly affect the proton-binding behavior of hemoglobin, we present in this paper a study of hydrogen ion titration curves of Hb and HbCO at KC1 concentrations ranging from 0.1 M to 2.0 M, with additional data on the influence of chloride ions on the Bohr effect
Summary
Hydrogen ion titration curves were determined at 25” with automatic titration equipment, as described elsewhere [20]. This equipment has been improved by using a pH meter of very high stability which was built with an electrometer operational amplifier (Analog Devices type 311 K). In each experiment 4 ml of the hemoglobin solution were brought into the titration vessel. In the experiments with Hb the hemoglobin solutions were deoxygenated in a rotating tonometer, while argon was continuously passed over the solution. The solutions were brought to the desired concentration of chloride ions with KC1 (Merck, Suprapur), and KC1 was added to the titrant to ensure a constant KC1 concentration during the measurements
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