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The chloride effect in the human embryonic haemoglobins.

ABSTRACT: The interactions of the three human embryonic haemoglobins with chloride ions have been investigated. Each of the three embryonic haemoglobins exhibits a unique pattern of oxygen-affinity-dependence on chloride ion concentration. Human embryonic haemoglobin Portland (zeta 2 gamma 2) is found to be completely insensitive to chloride ion concentration. Haemoglobin Gower I (zeta 2 gamma 2) shows a small concentration dependence, whilst haemoglobin Gower II (alpha 2 epsilon 2) exhibits a dependence approaching that of the adult protein. The degree of co-operativity for each protein is essentially chloride concentration independent. The chloride-dependent and -independent components of the alkaline Bohr effects have been measured for each of the embryonic haemoglobins and compared with that of the adult protein. Both the chloride-binding data and the Bohr effect have been analysed in terms of the recently developed allosteric model proposed by Perutz [Perutz, Fermi, Poyart, Pagnier and Kister (1993) J. Mol. Biol. 233, 536-545].

SUBMITTER: Hofmann O

PROVIDER: S-EPMC1135724 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Project description: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 DeltapI(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 (DeltalogP(50)/DeltapH) in solutions, the DeltapI(deox.-ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the DeltapI(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 A(0) (alpha(2)beta(2)) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5 degrees C and very low concentration of KCl. The variation of the DeltapH(deox.-ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of DeltapI(deox.-ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A(0). In haemoglobin A(0) the DeltapI(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 DeltapI(deox.-ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the alpha-chains are decreased by 30% compared with the DeltapI value measured in haemoglobin A(0). When the C-terminus of the beta-chains is altered, as in Hb Nancy (alpha(2)beta(Tyr-145-->Asp) (2)), we observed a 70% decrease in the DeltapI value compared with that measured in haemoglobin A(0). These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1alpha and His-146beta, 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 DeltapI(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.

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The chloride effect in the human embryonic haemoglobins.

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