The inter-relationship between the interior subunit interfaces and the exterior diphosphoglycerate (DPG) binding region of the hemoglobin tetramer and the effects of a specific N-terminal acetylation on tetramer assembly have been evaluated. Tetrameric fetal hemoglobin F in the liganded state was found to dissociate to dimers much less than previously appreciated, i.e. about 70 times less than adult hemoglobin A (K d ‫؍‬ 0.01 M and 0.68 M, for HbF and HbA, at pH 7.5, respectively) over the pH range 6.2-7.5, whereas HbF 1 , in which the N termini of the ␥-chains are acetylated, dissociates like HbA. To determine whether this feature of HbF could be transferred to hemoglobin A, the single amino acid difference in their ␣ 1 ␤ 2 /␣ 1 ␥ 2 interfaces and the 4 amino acid differences in their ␣ 1 ␤ 1 /␣ 1 ␥ 1 interfaces have been substituted in HbA to those in HbF. This pentasubstituted recombinant HbA/F had the correct molecular weight as determined by mass spectrometry, the expected mobility on isoelectric focusing, the calculated amino acid composition, and normal circular dichroism properties, oxygen binding, and cooperativity. Although HbA/F has the same amino acid side chains that bind DPG as HbA, its diminished response to 2,3-DPG resembled that of HbF. However, its tetramer-dimer dissociation constant (K d ‫؍‬ 0.14 M) was between that of HbA and HbF despite the fact that it was composed entirely of HbF subunit interfaces. The results indicate that regions of the tetramer distant from the tetramer-dimer interface influence its dissociation and, reciprocally, that the interfaces affect regions involved in the binding of allosteric regulators, suggesting flexible long range inter-relationships in hemoglobin.

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The characteristic properties of hemoglobin are due to the manner in which its individual subunits bond to one another first as an ␣␤ dimer and then as an ␣ 2 ␤ 2 tetramer. These subunit interactions also control the binding of allosteric regulatory molecules because of sites they create as they interact with one another. Some of these interactions in hemoglobin change in the transition between its tetrameric oxy (R, for "relaxed") or deoxy (T, for "tense") conformational states; adult human hemoglobin A (␣ 2 ␤ 2 ) functions as the physiological carrier of O 2 between the arterial and the venous circulation in these two conformations, respectively. The transition between these quaternary states is accompanied by concerted changes in the tertiary structure of the individual subunits upon O 2 binding known as cooperativity, which is responsible for the sigmoidal shape of the O 2 equilibrium curve (1-3). Myoglobin delivers O 2 during muscle contraction, as described in a recent minireview (4), and it has a hyperbolic O 2 equilibrium profile, i.e. no cooperative interactions because it is a single subunit protein. In tetrameric hemoglobin certain sites between the subunits at the quaternary level have the precise geometry or chemical reactivity to bind 2,3-diphosphoglycerate (2,3-DPG), 1 protons, and chloride preferentially to the deoxy conformational state and hence shift the equilibrium away from the oxy conformation, thereby favoring O 2 release. In each quaternary tetramer the oxy and deoxy dimer pairs interact differently to form the two types of tetramer-dimer interfaces in the R and T states. The strength of these interactions influences O 2 binding or release in these respective states and determines how easily the tetramer dissociates to dimers. In human Hb, dimers themselves are held together by strong interactions between their ␣-and ␤-subunits that do not differ significantly for the two R and T conformations.

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 "C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP).Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb a 9 2 [(C7) F41Y/(G4) N102Yl shows a fivefold increase in K4.2 at pH 7.0,37"C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affhity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb a2p2 [(C7) F41Y/(G4) N102Al and rHb a2p2 [(C7) F41Y/(E10) K66T], show a lower oxygen afflnity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.Keywords: auto-oxidation; hemoglobin; oxyhemoglobin; recombinant hemoglobin; tetramer-dimer equilibrium Despite the vast amount of research on hemoglobin (Hb), there are no rapid and reliable methods to measure the fraction of dimers. This is due to the difficulty of obtaining a reliable signal that differenciates between the dimeric and tetrameric species. Dimers have spectral and ligand binding properties similar to fully liganded tetramers. In addition, it is difficult to obtain data for pure dimers at pH 7, because one must use protein concentrations below 100 nM-a condition where the equilibria for the globin-heme (or hemin) and the monomerization may influence the results.