Preparation and kinetic characterization of a series of βW37 variants of human hemoglobin A: Evidence for high-affinity T quaternary structures

Four variants of human β globin in which the Trp at position 37 has been replaced with a Tyr, Ala, Gly, or Glu have been expressed in Escherichia coli. These globins have been combined with normal human α chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of α chain dimers, which are cross-linked between their α99 lysine residues, has been developed, and these α dimers were combined with two of the β globins, βW37G and βW37E, to form the corresponding cross-linked variants. The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as functions of pH in the presence and absence of the organic phosphate inositol hexaphosphate, IHP. The kinetic measurements indicate that replacement of the tryptophan with other residues destabilizes the hemoglobin tetramer, resulting in considerable dissociation of even the deoxygenated hemoglobins into αβ dimers at micromolar protein concentrations. Substitutions at β37 also alter the properties of the deoxygenated hemoglobin tetramer. The alteration of the functional properties of the T states of these variants as well as the tendency of the deoxygenated derivatives to dissociate into αβ dimers increases in the order HbA < βW37Y < βW37A < βW37G < βW37E. Stabilizing the βW37G or βW37E tetramers by addition of IHP or by cross- linking does not restore the normal functional properties of the T state. Measurements of the geminate rebinding of CO establish a kinetic difference between the normal R state tetramer and the αβ dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the αβ dimer. Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the β37 position.