A recombinant sickle hemoglobin triple mutant with independent inhibitory effects on polymerization.
As part of a comprehensive effort to map the most important regions of sickle hemoglobin that are involved in polymerization, we have determined whether two sites previously shown to be involved, Leu-88(beta) and Lys-95(beta), had additive effects when substituted. The former site is part of the hydrophobic pocket that binds Val-6(beta), the natural mutation of HbS, and the latter site is a prominent part of the hemoglobin exterior. A sickle hemoglobin triple mutant with three amino acid substitutions on the beta-chain, E6V/L88A/K95I, has been expressed in yeast and characterized extensively. Its oxygen binding curve, cooperativity, response to allosteric effectors, and the alkaline Bohr effect showed that it was completely functional. The polymer solubility of the deoxy triple mutant, measured by a new micromethod requiring reduced amounts of hemoglobin, was identical to that of the E6V(beta)/K95I(beta) mutant, i.e. when the K95I(beta) substitution was present on the same tetramer together with the naturally occurring E6V(beta) substitution, the L88A(beta) replacement had no additive effect on polymer inhibition. The results suggest that Lys-95(beta) on the surface of the tetramer and its complementary binding region on the adjoining tetramer are potential targets for the design of an effective antisickling agent.
Himanen
J P
JP
The Rockefeller University, New York, New York 10021, USA.
Mirza
U A
UA
Chait
B T
BT
Bookchin
R M
RM
Manning
J M
JM
D000495:Allosteric Site; D003001:Cloning, Molecular; D006451:Hemoglobin, Sickle / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010108:Oxyhemoglobins / Q000378:metabolism; D010449:Peptide Mapping; D017354:Point Mutation; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae; D018629:Spectrometry, Mass, Secondary Ion
D006451:Hemoglobin, Sickle; D046911:Macromolecular Substances; D010108:Oxyhemoglobins; C029775:oxyhemoglobin S