Mutational analysis of sickle haemoglobin (Hb) gelation

The use of recombinant Hb has provided the advantage that any amino acid su bstitution can be made at sites not represented by natural mutants or that cannot be modified by chemical procedures, We have recently reported the ex pression of human sickle Hb (HbS) in the yeast Saccharomycescerevisiae that carries a plasmid containing the human alpha- and beta-globin cDNA sequenc es; N-terminal nascent protein processing is correct and a soluble correctl y folded Hb tetramer is produced. The yeast system produces a recombinant s ickle Hb that is identical by about a dozen biochemical and physiological c riteria with the natural sickle Hb purified from the red cells of sickle-ce ll anaemia patients. Most importantly, the gelling concentration of this re combinant sickle Hb is the same as that of the HbS purified from human sick le red cells, The misfolding of Hb reported for the Escherichia coli-expres sed protein is not apparent for Hb expressed in yeast by any of the criteri a that we have used for characterization. These findings indicate that this system is well suited to the production of HbS mutants to explore those ar eas of the HbS tetramer whose roles in the gelation process are not yet def ined and to measure quantitatively the strength of such interactions at cer tain inter-tetrameric contact sites in the deoxy-HbS aggregate. This articl e reviews our studies on a number of sickle Hb mutants, including polymeriz ation-enhancing HbS mutants and polymerization-inhibiting HbS mutants.