The kinetics of ligand binding and of the association-dissociation reactions of human hemoglobin. Properties of deoxyhemoglobin dimers.

Dissociation of both human deoxyhemoglobin (deoxy-Hb) and carboxyhemoglobin (HbCO) at alkaline pH exposes previously buried tyrosine residues to the environment and alters their PK. Subunit association and dissociation of deoxy-Hb and HbCO can be followed through spectral changes which arise from these changes in tyrosine ionization above pH 10.0. The difference spectrum of the dissociation reaction contains a contribution from both tyrosine and tryptophan. Ultracentrifuge results confirm that a tetramerdimer equilibrium exists at these high pH values. The tetramer-dimer dissociation constant, K4,2, of both HbCO and deoxy-Hb increases dramatically above pH 10.0 and the value of K4,2 for HbCO is significantly greater than the value of K4.2 for deoxy-Hb in the pH range of 7 to 11. The groups responsible for the spectral change on dissociation have been tentatively identified with Tyr C7(42)0( and Trp C3(37)/3, which lie in the CQ-~? interface. Substantial quantities of deoxy-Hb dimers are produced at high pH. The properties of these dimers have been examined both at high pH and at pH 7.0 after rapid pH drop in the stopped flow apparatus. Association of deoxy-Hb dimers to the tetramer is accompanied by a spectral change in the Soret region, which has allowed us to follow the association reaction at pH 7.0. Deoxy-Hb dimers react rapidly with CO at a rate characteristic of noncooperative species (2’ = 6.5 X lo6 M-’ see-I). Further, human haptoglobin l-l binds these deoxy-Hb dimers at pH 7.0 at a rate similar to that observed with HbCO dimers (6.3 X lo5 M-l set-l and 5.5 X lo5 M-' set-I, respectively). We conclude that the deoxy-Hb dimer, the unliganded dimer derived from liganded hemoglobin, and the HbCO dimer are identical in conformation, c&; the fust two are noncooperative in their ligand-binding properties. The hemoglobin tetramer is therefore the minimum unit capable of full cooperativity in ligand binding. Models in which the free hemoglobin dimer is assumed to possess basically the same functional properties as the tetramer are untenable. * This research was supported by United States Public Health Service Grant GM-14276-06 to Dr. Quentin H. Gibson and National Science Foundation Grant. GB-8773 to Dr. S. J. Edelstein. Recent s-ray studies on human and horse hemoglobins have shown that the conformation of the penultimate tyrosine residues of the Q and fl chains differs in HbOz and deoxy-Hb; Tyr HC2(145)/3’ and Tyr H23(140)(-r are more strongly hydrogen bonded to peptide carbonyl groups in deoxy-Hb than in HbOz (l-3). X-ray and functional studies by Moffat, Simon, and Konigsberg (4) indicated that cooperativity in ligand binding depends in some way on the conformation of Tyr HC2(145)P since the irreversible displacement of this tyrosine or its removal by digestion with carboxypeptidase ,4 abolished cooperativity. Spectral evidence, apparently related to these differences in tyrosine conformation in HbOz and deoxy-Hb, comes from the HbOz-deoxy-Hb difference spectrum at pH 10.55 reported by Nagel, Ranney, and Kucinskis (5). This spectrum contained a contribution which resembled a tyrosine ionization difference spectrum (6), with a peak at 245 nm. Nagel et al. suggested that this contribution was due to a shift in the pK of 1 tyrosine residue on ligand binding, from a pK of > 12, a value typical of a tyrosine completely inaccessible to solvent, to a pK of -10.6, a value typical of tyrosine in an aqueous environment. They tentatively identified this tyrosine residue as Tyr HC2(145)P. We initially intended to examine the kinetic characteristics of changes in tyrosine conformation within the tetramer on ligand binding to deoxy-Hb, and to relate these to changes in heme conformation. However, at an early stage it became apparent that the spectral change at 245 nm was not a direct consequence of ligand binding to the tetramer, but arose from dissociation of hemoglobin tetramers to dimers. This dissociation subsequent to ligand binding converts the equivalent of 1 tyrosine per dimer with an abnormally high pK to a normally ionizing tyrosine. Thus, the change in tyrosine ionization is not a measure of a change in protein conformation within the tetramer, but of the association-dissociation reactions of hemoglobin at these pH values. Furthermore, since the tetramer-dimer dissociation constants, K4.2, of both forms of hemoglobin increase markedly at pH values above 10.0 (7, 8), substantial quantities of stable dimers of deoxy-Hb and HbCO can be produced at these high pH values. The examination of the properties of the stable