Role of gamma 87 Gln in the inhibition of hemoglobin S polymerization by hemoglobin F.
Previous studies suggested that gamma 87 Gln in hemoglobin (Hb) F is an important site for promoting inhibition of Hb S (alpha 2 beta 2(6 Glu-->Val) polymerization by Hb F. We engineered and isolated the double mutant (Hb alpha 2 beta 2(6 Glu-->Val,87 Thr-->Gln) using a yeast expression system and characterized polymerization properties of this modified tetramer in an effort to clarify the role of Gln at position 87 in inhibiting Hb S polymerization. Electrophoretic mobility and absorption spectra of this double mutant were the same as that of Hb S, while oxygen affinity was higher, and effects of organic phosphates on oxygen affinity were reduced. The deoxy form of the double mutant showed a characteristic delay time prior to polymerization in vitro. The critical concentration for polymerization of the double mutant was about 1.5 times higher than Hb S, and delay and polymerization times were much longer than Hb S at the same hemoglobin concentrations. The logarithmic plot of delay time versus hemoglobin concentration for the double mutant showed a straight line that was intermediate between lines for AS and FS mixtures. These results and those of kinetics of polymerization of Hb S/double mutant mixtures indicate that substitution of Gln for Thr at beta 87 in Hb S prolongs delay time and inhibits polymerization, although the double mutant forms polymers like Hb S.
Adachi
K
K
Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine 19104.
Konitzer
P
P
Surrey
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D005319:Fetal Hemoglobin / Q000737:chemistry* / Q000378:metabolism*; D005973:Glutamine; D006451:Hemoglobin, Sickle / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010100:Oxygen / Q000097:blood; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013912:Threonine
D017931:DNA Primers; D006451:Hemoglobin, Sickle; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005973:Glutamine; D013912:Threonine; D005319:Fetal Hemoglobin; D010100:Oxygen