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...