Partitioning of oxygen and carbon monoxide in the three human embryonic hemoglobins.

The ratio of oxygen to carbon monoxide binding to the three fully saturated human embryonic hemoglobins has been determined. The embryonic hemoglobins exhibit significantly lower values of carbon monoxide binding than any previously reported mammalian fetal or adult hemoglobins. The possible protective role of this with regards to carbon monoxide intoxication is discussed. These data are combined with previous parameters yielding a significant correlation between oxygen affinity and carbon monoxide binding. A possible molecular origin of this correlation is discussed.

Crystal structure of a human embryonic haemoglobin: the carbonmonoxy form of gower II (alpha2 epsilon2) haemoglobin at 2.9 A resolution.

The production of recombinant embryonic haemoglobins via a yeast expression system has enabled structural and functional studies to be conducted on these proteins. As part of a programme aimed at understanding the properties of the embryonic haemoglobins we have crystallized the human alpha2 epsilon2 (Gower II) embryonic haemoglobin in its carbonmonoxy form, and determined its structure by X-ray crystallography. The structure was solved by molecular replacement and refined at 2.9 A to give a final model with R-factor=0.185 and Rfree=0.235. The Gower II hemoglobin tetramer is intermediate between the adult R and R2 states, though closer to R2. The tertiary structure of the conserved alpha subunit is essentially identical when compared to that found in the adult (alpha2 beta2) and fetal (alpha2 gamma2) hemoglobins. The embryonic epsilon subunit has a structure very similar to that of the homologous adult beta and fetal gamma subunits, although with small differences at the N terminus and in the A helix. Amino acid substitutions can be identified that may play a role in the altered response of the Gower II haemoglobin to allosteric effectors, in particular chloride ions. The reduced chloride effect is thought to be the primary cause of the higher affinity of this embryonic hemoglobin in comparison to the adult molecule.

A two-state analysis of co-operative oxygen binding in the three human embryonic haemoglobins.

The binding of oxygen to the three human embryonic haemoglobins, at pH 7.4, has been shown to occur as a co-operative process. Analysis of oxygen-binding curves obtained in the absence of organic phosphate allosteric effectors shows that the process can be described quite accurately by the two-state model of allosteric action. In the presence of organic phosphates, the binding affinity for oxygen to the T-state of the alpha 2 epsilon 2 and zeta 2 epsilon 2 haemoglobins is significantly lowered. The values of the best-fit two-state parameters determined for each of the embryonic haemoglobins together with the temperature-dependence of the overall equilibrium binding process are discussed in terms of oxygen transfer from the maternal blood supply. Fast-reaction studies have been used to determine the rate constants of the oxygen association and dissociation processes occurring in the R-state and the rate of the allosteric R > T conformational transition. Analysis of these data suggests a likely reason for the high affinity and low co-operativity of the embryonic proteins and identifies the origins of the inability of equilibrium measurements to identify chain non-equivalence in the R-state.

The control of oxygen affinity in the three human embryonic haemoglobins by respiration linked metabolites.

The effect of CO2, ATP and lactate ions on the oxygen affinity of the three human embryonic haemoglobins have been studied. CO2 lowers the affinity of both the adult and embryonic haemoglobins for oxygen, as does ATP. The ATP effect follows a simple binding process with an equilibrium constant in the mM range. Lactate ions have no effect on the oxygen equilibrium process, over the concentration range studied. These findings are discussed in terms of the likely physiological conditions experienced by human embryonic red blood cells.

Production of human embryonic haemoglobin (Gower II) in a yeast expression system.

The cDNA coding for a human embryonic globin protein has been obtained from an erythroleukaemic cell line. A plasmid expression system for human embryonic haemoglobin Gower II containing cDNA copies of the appropriate pair of globin genes coupled to synthetic galactose-regulated hybrid promoters has been engineered. Transformation of Saccharomyces cerevisiae with this plasmid yields a cellular system capable of high-level production of fully functional tetrameric embryonic haemoglobin. We have developed a purification scheme which gives high yields of pure human embryonic haemoglobin suitable for structural and functional studies. Preliminary characterization studies are reported.

The incorporation of sulphaem into recombinant adult human haemoglobin produced in a yeast expression system.

The production of adult human haemoglobin in a yeast expression system has been shown to lead to the formation of functional oxygen-binding tetrameric proteins with the incorporation of endogenously synthesized haem. Adachi et al. [(1992) Protein Engng, 5, 807-810] identified two partially resolvable forms of the expressed haemoglobin, one of which showed higher oxygen affinity and lower cooperativity than normal. We show that in contrast to the previously expressed view that the abnormal form is due to abnormal protein folding, that it represents tetrameric haemoglobin containing incorporated sulphaem. Furthermore, the incorporation of sulphaem is shown to be a time-dependent process, with no detectable sulphaem being incorporated prior to 16 h post-induction. Numerical simulation based on our analysis of sulphaem composition gives an excellent fit to oxygen binding data previously reported for samples containing mixtures of normal haemoglobin and sulphaemoglobin.