Effects of NaCl on the linkages between O2 binding and subunit assembly in human hemoglobin: titration of the quaternary enhancement effect.

Oxygen binding by human hemoglobin (Hb) and the coupled reactions of dimer-tetramer assembly were studied over a range of NaCl concentrations (from 0.08 M to 1.4 M) at pH 7.4 and 21.5 degrees C. A strategy of multi-dimensional analysis was employed [G.K. Ackers and H.R. Halvorson, Proc. Natl. Acad. Sci. U.S.A., 91, (1974) 4312] to optimize the resolution of the contributions to cooperativity and their heterotropic salt linkages at each stoichiometric degree of O2 binding. A wide range of Hb concentration was utilized at each [NaCl] in which O2-linked subunit assembly reactions contributed significantly to the positions and shapes of the binding isotherms. Kinetic determinations yielded forward and reverse rate constants for assembly of the unligated species. Amplitudes for the assembly rate data had concentration dependences in agreement with the independently determined dimer-tetramer assembly constants of oxyhemoglobin. Concentration-dependent binding isotherms were analyzed, in combination with the kinetically determined equilibrium constants, to yield salt-linked components of cooperativity at the four stages of oxygenation. The principal results of this study were as follows. (i) Assembly of fully oxygenated Hb tetramers is opposed by NaCl: the dimer-to-tetramer equilibrium constant becomes two orders of magnitude less favorable over the [NaCl] range 0.08 M to 1.4 M. By contrast, for deoxy-Hb the assembly equilibrium constant is reduced only two-fold. (ii) Oxygen binding to dimers is non-cooperative over the entire salt range, whereas dimer affinity is slightly favored by increasing the NaCl concentration. (iii) Overall affinity of tetramers for O2 is opposed by NaCl, becoming an order of magnitude less favorable over the range employed. Most of this decrease occurs at the fourth binding step, which shows a large, salt-mediated quaternary enhancement effect; i.e., the assembly of dimers into tetramers at 0.08 M NaCl is accompanied by an eight-fold increase in O2 affinity. (iv) The quaternary enhancement effect at the last O2-binding step is titrated progressively by salt until it reaches a negligible value near the highest [NaCl] of this study. The lowest [NaCl] condition (0.08 M) elicits the greatest tetramer cooperativity with the largest maximal Hill coefficient and the greatest suppression of intermediates. Possible origins and mechanistic implications of these phenomena are considered.