Human hemoglobin has been studied in parallel experiments on structure and function. Structure has been examined in terms of the extent of dissociation into subunits in very dilute solutions by sedimentation velocity and equilibrium experiments with a scanning ultracentrifuge. Functional properties of the hemoglobin solutions have been evaluated by two rapid kinetic approaches, stopped flow mixing experiments with deoxyhemoglobin and carbon monoxide and flash photolysis experiments with CO-hemoglobin. The experiments indicate that the dissociation of tetrameric CO-hemoglobin into dimers is accompanied by a transition in kinetic properties from a slow rate of recombination with CO following a flash (k ∼ 1.5 x 10⁵m^-1 sec^-1) to a rapid rate of recombination (k ∼ 6 x 10⁶m^-1 sec^-1). The correlation of dissociation into dimeric units with enhancement in CO recombination rate is maintained under several conditions for which the tetramer-dimer equilibrium is considerably altered by the addition of salts, such as NaCl and triethylamine-HCl. In the standard buffer, 0.1 m phosphate, pH 7.0, the tetramer-dimer equilibrium is described by a dissociation constant of about 2 x 10^-6m (in heme).

The dissociation of deoxyhemoglobin into subunits has also been studied by ultracentrifugation of dilute solutions under anaerobic conditions in the presence of dithionite. Compared to liganded hemoglobin, deoxyhemoglobin has a much lower tendency to dissociate, and the dissociation is much less sensitive to the addition of salts. In phosphate buffer alone, a dissociation constant of K_4,2 ∼ 10^-7m is found. Values for the dissociation constant of about 5 x 10^-7m are obtained under conditions which facilitate dissociation (2 m triethylamine-HCl). Companion studies, on the functional properties of these solutions by stopped flow kinetic analysis, show no transition in kinetic properties in the range of protein concentration where dissociation to dimers occurs. The slowly reacting behavior typical of strong solutions of tetrameric hemoglobin (k ∼ 1.5 x 10⁵m^-1 sec^-1) is retained even at concentrations as low as 5 x 10^-8m where dissociation to dimers is extensive.

The observation that the dissociation of liganded and unliganded tetrameric hemoglobin gives rise to dimers with different functional properties may indicate the formation of different dimers, i.e. dissociation at different planes in the parent tetrameric molecules. The markedly lower sensitivity of the dissociation of unliganded hemoglobin to salts compared to liganded hemoglobin is consistent with this possibility. Further evidence is obtained by dissociation experiments on hemoglobin reacted partially or fully with p-mercuribenzoate. The results of these experiments, evaluated in terms of Perutz's atomic models of hemoglobin obtained by x-ray crystallography, suggest that liganded hemoglobin dissociates into dimers of the α₁β₁ type, whereas deoxyhemoglobin dissociates into dimers of the α₁β₂ type.