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, K2,4. 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 K2,4 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 K2,4 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.