Subunit interacting surfaces of human hemoglobin in solution: Localizationof the alpha-beta subunit interacting surfaces on the alpha-chain by a comprehensive synthetic strategy

By using synthetic overlapping peptides encompassing the entire alpha-chain of adult human hemoglobin (HbA), we have mapped on the alpha-chain the reg ions responsible for its binding to the beta-chain in solution. These bindi ng surfaces were, in general, in good agreement with those expected from th e crystal structure (peptides alpha 81-95, alpha 101-115, alpha 111-125, an d alpha 131-141). However, we observed some significant differences in the levels of binding found here in solution and those expected from the crysta l structure. Peptide alpha 31-45, which in the crystal had the highest numb er of contact residues of all the alpha-chain peptides, did not bind the be ta-chain in solution. Similarly, peptide alpha 91-105, with seven contact r esidues in the crystal, showed low binding with the beta-chain in solution. On the other hand, peptides alpha 41-55 and alpha 121-135 possessed much h igher binding activity in solution than would be expected from their contri bution to subunit association in the crystal. In fact, peptide alpha 121-13 5 had the highest binding activity of the alpha-chain peptides. These studi es and our previous findings, which localized on the beta-chain the regions that bind to the alpha-chain in solution, have shown that the regions of s ubunit association in solution are close to, but not identical with, those in the crystal. The approach should be quite useful for mapping subunit ass ociation in oligomeric proteins and could even be applied to proteins that are isolated only in traces or whose three-dimensional structure is not yet known.