Robert W. Noble

Human hemoglobin produced in the Escherichiacoli coexpression system of Hernan et al. [(1992) Biochemistry31, 8619−8628] has been transformed into a functionally homogeneous protein whose properties closely approximate those of normal hemoglobin A. Both of the α and β chains of this hemoglobin contain a valine−methionine substitution at position 1 in order to accommodate the difference in specificity of the protein-processing enzymes of procaryotes. Despite extensive purification, functional homogeneity of the E.coli expressed hemoglobin was achieved only by the complete disassembly of the hemoglobin into its component α and β globins and their reassembly in the presence of hemin. The kinetics of CO combination and the thermodynamics of O₂ binding and cooperativity of the reassembled αV1M−βV1M hemoglobin closely approximate those of HbA. The α globin obtained from the E.coli expressed hemoglobin was also combined with normal human β chains and hemin to form the αV1M variant. The α+M variant of HbA, in which the normal N-terminal valine of the α chains is preceded by a methionine residue, was prepared by the same procedure. The kinetics of the reactions of CO with the αV1M and α+M variants are similar to those for HbA. The equilibria of oxygen binding to αV1M and HbA are similar whereas α+M exhibits a significantly higher oxygen affinity. The three-dimensional structures of αV1M and α+M offer an explanation for the latter affinity difference. Although the structures of αV1M and HbA, which have been determined by X-ray crystallography, are virtually indistinguishable except at the N-terminal residues, that of α+M indicates the displacement of a solvent molecule, possibly a chloride ion, from arginine 141α. Such an alteration in an anion binding site could result in increased oxygen affinity.

Four variants of human beta 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 alpha chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of alpha chain dimers, which are cross-linked between their alpha99 lysine residues, has been developed, and these alpha dimers were combined with two of the beta globins, betaW37G and betaW37E, 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 alphabeta dimers at micromolar protein concentrations. Substitutions at beta37 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 alphabeta dimers increases in the order HbA < betaW37Y < betaW37A < betaW37G < betaW37E. Stabilizing the betaW37G or betaW37E 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 alphabeta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alphabeta dimer. Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the beta37 position.