Hemoglobin Little Rock (β143 (H21) His→Gln)

The amino acid substitution of hemoglobin Little Rock (HbLR) is here defined as β143 (H21) His → Gln, a site which is important for binding of 2,3-diphosphoglycerate (HbA). Studies of the effect of DPG and of chloride and phosphate on the oxygen affinities of chromatographically isolated HbLR and HbA show that HbLR has a much higher oxygen affinity than HbA at all anion concentrations. The shapes of the titration curves are consistent with binding of each species of anion studied to both the deoxy and oxy conformations of hemoglobin. Equations were developed from simple equilibrium models to calculate anion dissociation constants from the oxygen equilibrium data. The model for DPG binding assumed one oxygen-linked binding site per hemoglobin tetramer, and that for small anion binding assumed two sites. The DPG dissociation constant for deoxy HbLR (Kd = 3.3 x 10-5 m) is approximately twice that of deoxy HbA (Kd = 1.8 x 10-5 m), while the dissociation constant for oxy HbLR (Ko = 7.0 x 10-3 m) is four times that of oxy HbA (Ko = 1.7 x 10-3 m). Dissociation constants for chloride (HbLR: Kd = 2 x 10-3 m, Ko = 0.6 m; HbA: Kd = 2 x 10-3 m, Ko = 0.4 m) and phosphate (HbLR: Kd = 3 x 10-4 m, Ko = 0.1 m; HbA: Kd = 2.5 x 10-4 m, Ko = 0.1 m) are larger than the corresponding constants for DPG for both hemoglobins. However, the differences between HbLR and HbA in the binding of these anions are not significant. These data indicate the importance of the β143 histidine for the binding of DPG, but not of small anions, to HbA in solution. These findings appear to be in good agreement with crystallographic observations reported by Arnone ((1972) Nature 237, 146–149).