A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constr ucted to determine what effects specific amino acid residues in the gamma c hain have on the biophysical and biochemical properties of the native prote in molecule. Target residues in these recombinant fetal hemoglobins were re placed with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (g amma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Th r and gamma 130Trp to the stability of Hb F against alkaline denaturation a nd in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alk aline denaturation as Hb F, suggesting that the amino acid residues mention ed above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (I EF) was employed to investigate the extent of hybrid formation in equilibri um mixtures of Hb S with these hemoglobins and with several other hemoglobi ns in the carbon monoxy form. Equimolar mixtures of Kb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer , which is in agreement with the expected binomial distribution. Similar mi xtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for t wo of our recombinant mutants of Hb F were identical to the results for mix tures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr ), produced 42% hybrid tetramer, The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb m ixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no sig nificant differences from Hb F. While these mutants of Hb F did not have te tramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gen e therapy for the treatment of patients with sickle cell anemia.