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

The heme-globin and dimer-tetramer equilibria of ferric recombinant human h emoglobins with site-specific beta chain mutations at the heme pocket or at either the alpha (1)beta (1) or the alpha (1)beta (2) interfaces have been determined, The heme pocket mutation V67T leads to a marked stabilization of the beta chain heme and does not affect the dimer-tetramer association c onstant, K-2,K-4. In the C112 mutants, the intrinsic rate of beta chain hem e loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the alpha chains. Gel filtr ation experiments indicate that the K-2,K-4 value is essentially unaltered in C112G and C112L, but is increased in C112V and decreased in C112N. Subst itution of cysteine 93 with A or M leads to a slight decrease of the rate o f beta chain heme release, whereas the observed K-2,K-4 value is similar to that obtained for HbA-wt, Modifications in oxygen affinity were observed i n all the mutant hemoglobins with the exception of V67T, C93A, and C112G. T he data indicate that there is no correlation between tetramer stability, b eta chain heme affinity, and hemoglobin functionality and therefore point t o a separate regulation of these properties. (C) 2001 Academic Press.