Effects of anions on the ligand-linked subunit assembly of human hemoglobin: the mutual effects of Cl- and EDTA.

We have studied the mutual effects of chloride ion and EDTA on the dimer-tetramer assembly of human deoxyhemoglobin and oxyhemoglobin. It is found that these two anions have similar but interdependent effects. In low C1- (.01 M) increasing concentrations of EDTA are found to decrease both forward and reverse rate constants for deoxyhemoglobin, whereas no effect is observed at 0.1 M C1-. These results suggest that binding of anions at the alpha 1 beta 2 intersubunit contact may stabilize both the dimeric and tetrameric forms of the deoxy molecule, thus inhibiting both the dissociation and reassociation reactions. The overall effects of EDTA and low C1- on the dimer-tetramer equilibrium constants are found to be distinctly different in deoxy and oxyhemoglobins with a major effect on the oxy form. These findings establish validity of the results from previous thermodynamic studies carried out in approximately physiological C1- concentrations along with the small amounts of EDTA which are used to minimize artifacts of oxidation. As observed for deoxyhemoglobin, it is found that in 0.1 M C1- ion there is no further effect of EDTA on the oxyhemoglobin dimer-tetramer equilibrium.