Structural and functional properties of human hemoglobins reassembled after synthesis in Escherichia coli.

Human hemoglobin produced in the Escherichia coli coexpression system of Hernan et al. [(1992) Biochemistry 31, 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...