Non‐equivalence of Human: Hemoglobin Chains in the Oxidation‐Reduction and Heme‐Transfer Reactions

Abstract

13C NMR spectroscopy was applied to the investigation of chain non-equivalence for 2 reactions of human Hb: oxidation-reduction and hemetransfer. The method is based on previous observations that in the carbonyl region, Hb13CO gives 2 well-resolved resonances which arise from 13C of carbonyls bound, respectively, to the .alpha. and .beta. chains; moreover, integration of spectra allows estimation of their relative abundance. A mixture of ferrous and ferric Hb in dye-mediated oxidation-reduction equilibrium can be formally considered to be equivalent to 2 redox couples in equilibrium, namely .alpha.III/.alpha.II and .beta.III/.beta.II; from a knowledge of these ratios, one can conclude whether the chains are equivalent or not in their oxidation-reduction properties. In this work, these ratios were evaluated by reacting the redox systems with 13CO and integrating the 13C NMR spectra. The results showed differences in the intrinsic oxidation-reduction potentials of the chains in Hb tetramer, E1/2 (.beta.) being higher than E1/2 (.alpha.) in neutral solution but not at pH 9 and above. The binding of inositol hexakisphosphate did not modify the difference between .beta. and .alpha. though substantially increasing the overall potential. The results were discussed in the light of current hypotheses to account for the change of Hill coefficient with pH for the reaction studied. The non-equivalence of chains was shown also for heme transfer from methemoglobin. For the phosphate-free protein, the .beta. chains lost heme more rapidly than the .alpha. chains; the addition of inositol hexakisphosphate resulted in the decrease of overall heme transfer as well as of chain heterogeneity.