Effects of Anions on the Ligand-Linked Subunit Assembly of Human Hemoglobin: The Mutual Effects of Cl−and Edta

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Abstract
The mutual effects of Cl- and EDTA on the dimer-tetramer assembly of human deoxyhemoglobin and HbO2 were studied. These 2 anions apparently have similar but interdependent effects. In low Cl- (0.01 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 Cl-. 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 Cl- on the dimer-tetramer equilibrium constants are found to be distinctly different in deoxyhemoglobin and Hb with a major effect on the oxy form. These findings establish validity of the results from previous thermodynamic studies carried out in approximately physiological Cl- 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 Cl- ion there is no further effect of EDTA on the HbO2 dimer-tetramer equilibrium.