ASSEMBLY OF HUMAN HEMOGLOBIN - STUDIES WITH ESCHERICHIA COLI-EXPRESSED ALPHA-GLOBIN

The alpha-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of nati ve beta-chains, The functional and structural properties of the expres sed alpha-chains were assessed in the isolated state and after assembl y into a functional hemoglobin tetramer, The recombinant and native he moglobins were essentially identical on the basis of sensitivity to ef fecters (Cl- and 2,3-diphosphaglycerate), Bohr effect, CO binding kine tics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the alpha(1) beta(1) and alpha(1) beta(2) interfaces. However, the nu clear magnetic resonance revealed subtle differences in the heme regio n of the expressed alpha-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relativ e to native HbA, These results indicate that subtle conformational cha nges in the heme pocket can alter hemoglobin cooperativity in the abse nce of modifications of quaternary interface contacts or protein dynam ics, In addition to incorporation into a HbA tetramer, the alpha-globi n refolds and incorporates heme in the absence of the partner beta-cha in. Although the CO binding kinetics of recombinant alpha-chains were the same as that of native alpha-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics reve aled an additional faster component. These results show that recombina nt alpha-chain assumes alternating conformations in the absence of bet a-chain and indicate that the isolated alpha-chain exhibits a higher d egree of conformational flexibility than the alpha-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility o f the expressed alpha-globin as a tool for elucidating the role of thi s chain in hemoglobin structure-function relationships.