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

13 C nuclear magnetic resonance (NMR) spectroscopy has been applied to the investigation of chain non‐equivalence for two reactions of human hemoglobin: oxidation‐reduction and heme‐transfer. The method is based on previous observations that in the carbonyl region, Hb 13 CO gives two well‐resolved resonances which arise from 13 C of carbonyls bound respectively to the α and β chains; moreover, integration of spectra allows one to estimate their relative abundance. A mixture of ferrous and ferric hemoglobins in dye‐mediated oxidation‐reduction equilibrium can be formally considered to be equivalent to two redox couples in equilibrium, namely α 111 /α 11 and β 111 /β 11 : 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 13 CO and integrating the 13 C NMR spectra. The results show differences in the intrinsic oxidation‐reduction potentials of the chains in hemoglobin tetramer, E 1/2 (β) being higher than E l/2 (α) in neutral solution but not at pH 9 and above. The binding of inositol hexakisphosphate does not modify the difference between β and α though substantially increasing the overall potential. The results are 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 is shown also for heme transfer from methemoglobin. For the phosphate‐free protein, the β chains lose heme more rapidly than the α chains; the addition of inositol hexakisphosphate results in the decrease of overall heme ‐transfer as well as of chain heterogeneity.