The measurement of the intrinsic alkaline Bohr effect of various human haemoglobins by isoelectric focusing

We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the ΔpI(deox.–ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are `stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (ΔlogP₅₀/ΔpH) in solutions, the ΔpI(deox.–ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the ΔpI(deox.–ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A₀ (α₂β₂) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5°C and very low concentration of KCl. The variation of the ΔpH(deox.–ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of ΔpI(deox.–ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A₀. In haemoglobin A₀ the ΔpI(deox.–ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The ΔpI(deox.–ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the α-chains are decreased by 30% compared with the ΔpI value measured in haemoglobin A₀. When the C-terminus of the β-chains is altered, as in Hb Nancy (α₂β^{Tyr-145→Asp}₂), we observed a 70% decrease in the ΔpI value compared with that measured in haemoglobin A₀. These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1α and His-146β, at the origin of the intrinsic alkaline Bohr effect Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem. 248, 7039–7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol. 138, 649–670]. In other mutant haemoglobins it is demonstrated also that the ΔpI(deox.–ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980), J. Mol. Biol. 138, 649–670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.