The heme-globin and dimerization equilibria of recombinant human hemoglobins carrying site-specific β chains mutations

The heme-globin and dimer-tetramer equilibria of ferric recombinant human hemoglobins with site-specific β chain mutations at the heme pocket or at either the α₁β₁ or the α₁β₂ interfaces have been determined. The heme pocket mutation V67T leads to a marked stabilization of the β chain heme and does not affect the dimer-tetramer association constant, K_2,4. In the C112 mutants, the intrinsic rate of β chain heme loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the α chains. Gel filtration experiments indicate that the K_2,4 value is essentially unaltered in C112G and C112L, but is increased in C112V and decreased in C112N. Substitution of cysteine 93 with A or M leads to a slight decrease of the rate of β chain heme release, whereas the obvserved K_2,4 value is similar to that obtained for HbA-wt. Modifications in oxygen affinity were observed in all the mutant hemoglobins with the exception of V67T, C93A, and C112G. The data indicate that there is no correlation between tetramer stability, β chain heme affinity, and hemoglobin functionality and therefore point to a separate regulation of these properties.