THERMODYNAMIC STUDIES ON THE EQUILIBRIUM PROPERTIES OF A SERIES OF RECOMBINANT BETA-W37 HEMOGLOBIN MUTANTS

In human hemoglobin (Hb) the beta 37 tryptophan residue (beta W37), lo cated at the hinge region of the alpha(1) beta(2) interface, forms man y contacts with alpha subunit residues of the opposite dimer, in both the T and R quaternary structures. We have carried out equilibrium O-2 binding studies on a series of recombinant Hbs that have mutations at this residue site: beta W37Y, beta W37A, beta W37G, and beta W37E. Bi nding isotherms measured at high concentrations of these mutants were found to be shifted toward increased affinity and decreased cooperativ ity from that of the normal HbA(0) tetramer. Analysis of these binding isotherms indicated that amino acid substitutions at the beta 37 posi tion could both destabilize the tetrameric form of the mutants relativ e to their constituent dimers and also alter cooperativity of the inta ct tetrameric species. These alterations from wild-type function are d ependent on the particular side chain substituted, with the magnitude of change increasing as Trp is substituted by Tyr, Ala, Gly, and Glu. The dimer to tetramer assembly free energy of deoxy-beta W37E, the mos t perturbed mutant in the series, were measured using analytical gel c hromatography to be 9 kcal/tetramer less favorable than that of deoxy HbA(0). Stabilizing the beta W37E tetramer by addition of IHP, or by c ross-linking at the alpha K99 positions, does not restore normal O-2 b inding behavior. Thermodynamic parameters of all the mutants were foun d to correlate with their CO binding rates and with their high-resolut ion X-ray crystal structures (see accompanying papers: Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335; Peterson & Friedman (1998) Bioc hemistry 37, 4346-4357; Kavanaugh et al. (1998) Biochemistry 37, 4358- 4373].