The binding of carbon monoxide by human hemoglobin. Proof of validity of the spectrophotometric method and direct determination of the equilibrium.

Stoichiometric titrations of human hemoglobin with carbon monoxide show that the interaction of hemoglobin with ligand is accompanied by proportional changes in both the Soret and visible absorption bands. This linearity has been observed in neutral solutions, both in the presence and absence of 2 M NaCl, and in alkaline solutions at pH 9.2. It is concluded that the liganded intermediates taking part in the equilibrium do not differ significantly in spectra from the equivalent hypothetical mixtures of reduced and fully liganded hemoglobin. The CO equilibrium determined in dilute hemoglobin solutions (4.5 X 10dl M to 2.34 X lo-’ M) is characterized by a relatively high Hill constant (2.3) and a free ligand concentration of 4.3 to 5.6 x lo-* M at 50% saturation. The results are of special interest in light of the sedimentation studies and kinetic experiments which indicate that a strongly ligand-linked equilibrium between dimeric and monomeric (or single chain) hemoglobin predominates in this concentration range.