Participation and strength of interaction of lysine 95(beta) in the polymerization of hemoglobin S as determined by its site-directed substitution by isoleucine.
The role of Lys-95(beta), which is on the exterior of the hemoglobin (HbS) tetramer, in the aggregation process has been addressed because there is a lack of agreement on its importance. The early studies on the aggregation of HbS in the presence of other mutant hemoglobins are consistent with the subsequent electron microscopic studies in demonstrating the participation of Lys-95(beta) in gelation; the results of the crystal structure do not agree with these conclusions. Therefore, with the objective of clarifying its role we have carried out site-directed substitution of Lys-95(beta) to an isoleucine residue. The mutation was introduced by polymerase chain reaction recombination methodology, and the absence of other mutations in the beta-globin gene was established by sequencing the gene in its entirety. The recombinant mutant hemoglobin was expressed in yeast and characterized by peptide mapping and sequencing, which demonstrated that the only different tryptic peptide had the Ile substitution at position 95(beta). The recombinant hemoglobin had the correct amino acid composition and molecular weight by mass spectrometric analysis. It was also pure as judged by isoelectric focusing. It was fully functional because it had an average Hill coefficient of 3.1 and responded normally to the allosteric regulators, chloride, 2,3-diphosphoglycerate, and inositol hexaphosphate. Of particular interest was the finding that this hemoglobin mutant aggregated at a concentration of about 40 g/dl, nearly twice that at which HbS itself aggregated (24 g/dl). Therefore, Lys-95(beta) has a very important role in the aggregation process and is a good candidate site for the design of a therapeutic agent for sickle cell anemia.
Himanen
J P
JP
Rockefeller University, New York, New York 10021, USA.
Schneider
K
K
Chait
B
B
Manning
J M
JM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D006451:Hemoglobin, Sickle / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D007532:Isoleucine; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011108:Polymers / Q000378:metabolism; D013329:Structure-Activity Relationship
D000596:Amino Acids; D006451:Hemoglobin, Sickle; D011108:Polymers; D007532:Isoleucine; D008239:Lysine