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 hemoglobins with site-specific beta chain mutations at the heme pocket or at either the a1beta1 or the alpha1beta2 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 constant, K2,4. In the C112 mutants, the intrinsic rate of beta chain heme loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the alpha 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 beta 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, beta chain heme affinity, and hemoglobin functionality and therefore point to a separate regulation of these properties.
Gattoni
M
M
CNR Center of Molecular Biology, Department of Biochemical Sciences, University La Sapienza, Rome, Italy.
Piro
M C
MC
Boffi
A
A
Brinigar
W S
WS
Fronticelli
C
C
Chiancone
E
E
D000418:Albumins / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002850:Chromatography, Gel; D019281:Dimerization; D006418:Heme / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D000418:Albumins; D006454:Hemoglobins; D011994:Recombinant Proteins; D006418:Heme; D010100:Oxygen