Amino acids responsible for decreased 2,3-biphosphosphoglycerate binding to fetal hemoglobin

To clarify the role of gamma N-terminal Gly, gamma 5 Glu, and gamma 143 Ser in 2, 3-biphosphosphoglycerate (BPG) binding to fetal hemoglobin (Hb F), we engineered and produced normal human Hb F and two Hb F variants (Hb F gamma G1V, gamma S143H, and Hb F gamma G1V, gamma E5P, gamma S143H) using a yeast expression system and then compared their oxygen-binding properties with those of native human Hb F and adult Hb (Hb A). Oxygen affinity of Hb F gamma G1V, gamma S143H in the absence of 2, 3-BPG was slightly higher than that of normal Hb F. The decrease in oxygen affinities for Hb F gamma G1V, gamma S143H with increasing 2, 3-BPG concentrations was larger than that of normal Hb F, but significantly less than that of Hb A. In contrast, oxygen affinities of Hb F gamma G1V, gamma E5P, gamma S143H in the absence and presence of 2, 3-BPG were much lower than those of Hb F gamma G1V, gamma S143H and were similar to those of Hb A. These results indicate that differences between Pro and Glu at the A2 position in the A helix in Hb A and Hb F, respectively, are critical for reduced binding of 2, 3-BPG to Hb F, even though beta 5 Pro does not interact directly with 2, 3-BPG in Hb A. Hb F variants such as Hb F gamma G1V, gamma E5P, gamma S143H, which exhibit reduced oxygen affinity, should facilitate design of efficient antisickling fetal Hb variants for potential use in gene therapy for sickle cell disease. (C) 1997 by The American Society of Hematology.