Probing the Importance of the amino-terminal sequence of the beta- and gamma-chains to the properties of normal adult and fetal hemoglobins

A recombinant mutant of human fetal hemoglobin (Hb F), named rHb Oscar, has been constructed to explore the importance of the sequence of the amino-te rminal region of the gamma -chain to the structural and functional properti es of Hb F as compared to human normal adult hemoglobin (Hb A). Substitutio ns in the N-terminal region of Hb A have shown this region to be important to its structural and functional properties. Recent studies of recombinant mutants of Hb A with gamma-chain mutations have been used to probe the sign ificance of the N-terminal sequence to the properties of Hb F. One of these mutants of Hb A, called rHb Felix, contains eight substitutions in the N-t erminal region of the beta-chain corresponding to the sequence of the gamma -chain in that region [Dumoulin et al. (1998) J. Biol. Chem. 273, 35032-350 38]. rHb Felix exhibits a 2,3-bisphosphoglycerate (2,3-BPG) response like t hat of Hb A, but its tetramer-dimer dissociation constant is similar to tha t of Hb F. In contrast, rHb Oscar contains a gamma -chain with eight mutati ons at the N-terminal end corresponding to the sequence of the beta -chain of Hb A in that region. H-1 NMR studies of rHb Oscar indicate a global stru cture like that of Hb F. rHb Oscar is not as stable against alkaline denatu ration as Hb F but is more stable than Hb A, and it exhibits a stronger res ponse to 2,3-BPG and inositol hexaphosphate as compared to Hb F. The 2,3-BP G effect in rHb Oscar also appears to be slightly enhanced compared to that in Hb A. Subzero isoelectric focusing experiments suggest that rHb Oscar d oes not have dissociation properties like those of Hb A. These results alon g with those of rHb Felix illustrate that the effects of the N-terminal reg ion on structure and function of the Hb molecule are complicated by interac tions with the rest of the molecule that are not yet well defined. However, studies of complementary mutations of Hb A and Hb F may eventually help to define such interactions and lead to a better understanding of the relatio nship between the amino acid sequence and the properties of the Hb molecule .