Deoxy Tetramer Research Articles

Asymmetric [deoxy dimer/azido-met dimer] hemoglobin hybrids dissociate within seconds

Double mixing stopped-flow experiments have been performed to study the stability of asymmetric hemoglobin (Hb) hybrids, consisting of a deoxy and a liganded dimer. The doubly liganded [deoxy/cyano-met] hybrid (species 21) was reported to have an enhanced stability, with tetramer to dimer dissociation requiring over 100 seconds, based on a method that required an incubation of over two days. However, kinetic experiments revealed rapid ligand binding to species 21, as for triply liganded tetramers, which dissociate within a few seconds. For the present study, [deoxy dimer/azido-met dimer] hybrids are formed within 200 ms by stopped-flow mixing of dithionite with a solution containing oxyHb and azido-metHb. The dithionite scavenges oxygen, thus transforming oxyHb to deoxyHb, and the [oxy dimer/azido-met dimer] hybrid to the asymmetric [deoxy/azido-met] hybrid (species 21). After a variable aging time of the asymmetric hybrids, their allosteric state is probed by CO binding in a second mixing. As previously observed the freshly produced asymmetric hybrids bind CO rapidly as for R-state Hb. As the hybrids are aged from 0.1 to 10 seconds, the fraction of slow CO binding increases, consistent with a dissociation of the asymmetric hybrid to form the more stable deoxy Hb tetramer which reacts slowly with CO. Control experiments showed a predominantly slow phase for deoxy Hb, and fast rebinding for the symmetric hybrids. The kinetic data can be simulated with a tetramer to dimer dissociation rate for species 21 of 1.5/second at 100 mM NaCl (pH 7.2) and 1.9/second at 180 mM NaCl (pH 7.4). These values are similar to those reported for liganded Hb, as opposed to deoxy (T-state) tetramers which dissociate over four orders of magnitude more slowly. As expected from simulations of dimer exchange, the observed transition rate depends on the initial fractions of oxy- and metHb; this effect is not consistent with a slow R to T transition. These results, showing a lifetime of about one second for species 21, do not support the symmetry rule which is based on an enhanced stability of the asymmetric hybrid.

Chimeric beta-EF3-alpha hemoglobin (Psi): energetics of subunit interaction and ligand binding.

Among the numerous strategies to design an oxygen carrier, we outline in this work the engineering of a stable homotetrameric hemoglobin, expressed in Escherichia coli. The chimeric globin (Psi) consists of the first 79 residues of human beta globin (corresponding to positions NA1 --> EF3) followed by the final 67 residues of human alpha globin (corresponding to positions EF3 --> HC3). The molecular mass for beta-EF3-alpha (Psi) globin was measured using mass spectrometry to be equal to its theoretical value: 15782 Da. Correct protein folding was assessed by UV/visible and fluorescence spectra. The subunit interaction free energies were estimated by HPLC gel filtration. In the cyanometHb species, the formation of the dimer-tetramer interface is 2 kcal/mol less favorable (Delta G = -7 kcal/mol) than that of Hb A (Delta G = -9 kcal/mol), whereas the dimer-monomer interface is tightly assembled (< -10 kcal/mol) as for the Hb A alpha 1 beta 1 interface. In contrast to Hb A, oxygen binding to Psi Hb is not cooperative. The free energy for binding four oxygen molecules to a Psi homotetramer is slightly increased compared to a Hb A heterotetramer (-28 and -27.5 kcal/4 mol of O2, respectively). The intrinsic O2 affinity of a Psi homodimer is 6-fold higher than that of a homotetramer. The linkage scheme between dimer-tetramer subunit assembly and the noncooperative oxygenation of Psi Hb predicts a stabilization of the tetramer after ligand release. This protein mechanism resembles that of Hb A for which the dimers exhibit a 100-fold higher O2 affinity relative to deoxy tetramers (which are 10(5) times more stable than oxy tetramers). A potent allosteric effector of Hb A, RSR4, binds to Psi Hb tetramers, inducing a decrease of the overall O2 affinity. Since RSR4 interacts specifically with two binding sites of deoxy Hb A, we propose that the chimeric tetramer folding is close to this native structure.

Probing the α1β2 Interface of Human Hemoglobin by Mutagenesis: ROLE OF THE FG-C CONTACT REGIONS

The allosteric transition of hemoglobin involves an extensive reorganization of the alpha 1 beta 2 interface, in which two contact regions have been identified. This paper concerns at the effect of two mutations located in the "switch" (alpha C3 Thr --> Trp) and the "flexible joint" (beta C3 Trp --> Thr). We have expressed and characterized one double and two single mutants: Hb alpha T38W/beta W37T, Hb beta W37T, and Hb alpha T38W, whose structure has been determined by crystallography. We present data on: (i) the interface structure in the contact regions, (ii) oxygen and CO binding kinetics and cooperativity, (iii) dissociation rates of deoxy tetramers and association rates of deoxy dimers, and (iv) the effect of NaI on deoxy tetramer dissociation rate constant. All the mutants are tetrameric and T-state in the deoxygenated derivative. Reassociation of deoxygenated dimers is not modified by interface mutations. DeoxyHb alpha T38W/beta W37T dissociate much faster. We propose a binding site for I- at the switch region. The single mutants binds O2 cooperatively; the double one is almost non-cooperative, a feature confirmed by CO binding. The functional data, analyzed with the two-state model, indicate that these mutations reduce the value of the allosteric constant LO.

Tetrameric Hemoglobin Expressed in Escherichia coli: EVIDENCE OF HETEROGENEOUS SUBUNIT ASSEMBLY

Recombinant alpha 2 beta 2 tetrameric Hb expressed and assembled in Escherichia coli has been characterized extensively. Electrospray mass spectrometry and optical and electron paramagnetic resonance spectroscopy suggest that the overexpressed protein is identical to native human Hb. Although the functional properties of this recombinant Hb are nearly identical to native Hb, crucial differences exist between the two molecules. The recombinant Hb expressed in E. coli has a lower Hill coefficient even though oxygen equilibrium binding studies indicate cooperative binding. The most significant difference observed between the recombinant and native Hb is the loss of oxygen affinity regulation by 2,3-diphosphoglyerate and protons. CO binding to the deoxy tetramer was found to be biphasic with both phases sensitive to the presence of allosteric effectors. The recombinant chains were isolated, and the ligand binding properties demonstrated that the recombinant chains behave in a similar fashion to native alpha-sh and beta-sh. To investigate whether the chains were capable of forming a well behaved tetramer, the isolated chains were reassembled into a tetramer and purified to homogeneity. Oxygen binding properties of the reassembled recombinant Hb now show an increased Hill coefficient of 2.5, close to, but still slightly lower than, that observed for native Hb. Additionally, reassembly of recombinant Hb produces a protein that is subject to regulation by allosteric effectors. Furthermore, CO binding to the reassembled recombinant deoxy tetramer was found to be monophasic under all conditions.

Mutagenic dissection of hemoglobin cooperativity: Effects of amino acidalteration on subunit assembly of oxy and deoxy tetramers

Free energies of oxygen-linked subunit assembly and cooperative interaction have been determined for 34 molecular species of human hemoglobin, which differ by amino acid alterations as a result of mutation or chemical modification at specific sites. These studies required the development of extensions to our earlier methodology. In combination with previous results they comprise a data base of 60 hemoglobin species, characterized under the same conditions. The data base was analyzed in terms of the five following issues. (1) Range and sensitivity to site modifications. Deoxy tetramers showed greater average energetic response to structural modifications than the oxy species, but the ranges are similar for the two ligation forms. (2) Structural localization of cooperative free energy. Difference free energies of dimer-tetramer assembly (oxy minus deoxy) yielded delta Gc for each hemoglobin, i.e., the free energy used for modulation of oxygen affinity over all four binding steps. A structure-energy map constructed from these results shows that the alpha 1 beta 2 interface is a unique structural location of the noncovalent bonding interactions that are energetically coupled to cooperativity. (3) Relationship of cooperativity to intrinsic binding. Oxygen binding energetics for dissociated dimers of mutants strongly indicates that cooperativity and intrinsic binding are completely decoupled by tetramer to dimer dissociation. (4) Additivity, site-site coupling and adventitious perturbations. All these are exhibited by individual-site modifications of this study. Large nonadditivity may be correlated with global (quaternary) structure change. (5) Residue position vs. chemical nature. Functional response is solely dictated by structural location for a subset of the sites, but varies with side-chain type at other sites. The current data base provides a unique framework for further analyses and modeling of fundamental issues in the structural chemistry of proteins and allosteric mechanisms.

Double mixing stopped-flow method for the study of equilibria and kinetics of dimer-tetramer association of hemoglobins: Studies on Hb Carp, Hb A, and Hb Rothschild

A double mixing stopped-flow method is described for studying the dimer-tetramer equilibria of oxyhemoglobins and the kinetics of association of unliganded dimers. The three hemoglobins studied were: Hb Carp, Hb A, and Hb Rothschild (Trp β37(C3) → Arg). The new method reproduces the data obtained for oxyHb A by other established methods. In agreement with previous studies, the new method indicates little, if any, dissociation of oxyHb carp into dimers even in 2 m urea solutions (0.1 m Bis-Tris pH 7.0). OxyHb Rothschild, on the other hand, is extensively dissociated into dimers ( K(Hb 4L 4 lrhar2 2Hb 2) = 37.3 × 10 −6 m) and the rate constant for the association of deoxy dimers of Hb Rothschild is about one-tenth of the value for Hb A indicating that the deoxy tetramer of Hb Rothschild is at least 10 times more dissociated into dimers than deoxyHb A.

Structural, functional, and subunit assembly properties of hemoglobin Attleboro [.alpha.138 (H21) Ser .fwdarw. Pro] a variant possessing a site mutation at a critical C-terminal residue

Hemoglobin Attleboro, a new alpha-chain variant with a substitution of proline for serine at position 138 (H21), was found to be a noncooperative high-affinity hemoglobin (P50 = 0.26 mmHg at pH 7 and 20 degrees C) which lacked an alkaline Bohr effect. Addition of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP) led to a decrease in oxygen affinity but to no alteration in either Bohr effect or cooperativity. Ligand binding kinetics studies revealed an overall rate of oxygen dissociation at pH 7.0 and 20 degrees C that was 2.7-fold slower than that for Hb A. At pH 8.5, the kinetic profile was identical with that at pH 7, confirming the absence of a Bohr effect for this variant hemoglobin. Measurement of the rate of oxygen dissociation with carbon monoxide replacement indicated a lack of cooperativity. Sedimentation velocity experiments yielded s20,w values of 2.8 and 4.3 for 65 microM solutions of oxyhemoglobins Attleboro and A, respectively (indicating an enhancement in the oxy dimer population of this variant). Studies of the carbon monoxide combination of this variant revealed an association rate 20-fold faster than that for Hb A; only in the presence of a 1000-fold molar excess of IHP was there a significant reduction in the overall rate. Rapid-scan and traditional stopped-flow experiments conducted in the Soret Soret region demonstrated an alteration in the structure and rate of assembly of the deoxy tetramer of Hb Attleboro relative to that of Hb A. The abnormal properties of this hemoglobin variant can be attributed to major perturbations in the C-terminal region.

Interactions of Nicotinamide Adenine Dinucleotides with Varied States and Forms of Hemoglobin

Spectrofluorometric techniques were used to quantify NADPH-hemoglobin interactions based on the quenching of NADPH fluorescence upon binding to hemoglobin. Fluorometric titrations were carried out with hemoglobin in varied states and with hemoglobins in which the beta-chain anion site is altered. At pH 6.5 in 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, NADPH binds with high affinity, Kd = 1.03 microM, to deoxy human hemoglobin tetramers. Lower affinity binding of NADPH occurs as the beta-chain anion-binding site is discharged by increasing the pH. Moreover, the cofactor binds in a 1:1 ratio to deoxy tetramers, inositol hexaphosphate binds competitively, and binding is decreased in hemoglobins whose structural alterations result in decreased effects of 2,3-diphosphoglycerate. The cofactor binds to oxidized (met) hemoglobin with an estimated Kd of 33.3 microM but has little or no affinity for the oxy form. These results indicate that NADPH binds at the beta-chain anion-binding site and can be considered as a fluorescent analog of 2,3-diphosphoglycerate. Fluorescence measurements gave no indication of NADPH binding to deoxygenated ferrous or ferric myoglobin. Reductive processes within the erythrocyte, such as reduction of met hemoglobin and hemoglobin-catalyzed enzymatic reactions, may be affected by the significant binding of the reduced cofactor to both deoxygenated and oxidized hemoglobin. Cofactor-hemoglobin interactions predict a shift in redox potential as red cells become oxygenated, which may account for unexplained oxygen-linked shifts in red cell metabolism.

Hemoglobin tertiary structural change on ligand binding its role in the co-operative mechanism

Analysis of the tertiary structural alterations in hemoglobin induced by ligand binding demonstrates that an allosteric core composed of the heme, histidine F8, the FG corner and part of the F-helix plays an essential role in co-operativity. This conclusion is based on structural and spectroscopic data and theoretical studies of hemoglobin chains. The methodology employed in the calculations is presented with details of the empirical energy function. Energy minimized structures of the unliganded hemoglobin chains, which serve as reference systems for the analysis, are described. To determine the structural changes induced by ligand binding, the effects of FeN bond shortening and of heme translation and tilting perturbations are examined. Energy minimization in the presence of the perturbations serves to provide information concerning the globin structural modifications produced by them. The validity of the results is supported by comparisons with the X-ray data of Anderson, Pulsinelli, Baldwin and Chothia on tertiary changes in the hemoglobin subunits. Internal to the allosteric core, there appear to be two stable positions for its elements: one of these corresponds to the liganded and the other to the unliganded species. The unliganded geometry fits without strain into the deoxy tetramer, while the liganded one fits without strain into the oxy tetramer. On ligation of a subunit in the deoxy tetramer, the structural changes within the allosteric core are in the direction of those found in going from the unliganded deoxy to the liganded oxy system, although they are reduced by the presence of constraints due to the other subunits in the deoxy tetramer. In addition, the quaternary constraints in the deoxy tetramer prevent the large overall displacement of the allosteric core that occurs in the transition to the liganded oxy tetramer. The coupling between the changes internal to the allosteric core, produced on ligation and the overall displacement of the core that accompanies the quaternary transition, is an essential element of the co-operative mechanism. As shown in previous work (Gelin & Karplus, 1977), the proximal histidine serves as the link between the position of the heme and the F-helix; the asymmetric orientation of the histidine in the deoxy structure, coupled with contributions from other heme-protein interactions, appears to initiate the tertiary structural changes induced by ligand binding. The reduced oxygen affinity of hemoglobin results not from tension on the heme in the unliganded structure (there is none) but instead from strain in the liganded subunit of the tetramer within the deoxy quaternary structure. Further, the changes in the allosteric core provide a relatively localized reaction path for transmitting information concerning ligand binding from the heme group to the surface of the subunit; particularly in the α-chain, the residue Val FG5 appears to play an important role in the reaction path. The present analysis has important implications for realistic statistical thermodynamic models of hemoglobin co-operativity. It suggests that the previously formulated model (Szabo & Karplus, 1972) should be generalized by the introduction of two different subunit tertiary structures in the deoxy and in the oxy tetramer; they would be associated with the unliganded and the liganded allosteric core, respectively, and would take account of steric constraints that reduce the ligand affinity of the deoxy tetramer.

A novel mechanism of hemoglobin cooperativity

Perutz has proposed that the cooperative effect of oxygen binding in tetrameric hemoglobin arises from an equilibration between two quaternary structures: the liganded or relaxed (R) structure with oxygen affinity comparable to the isolated subunit affinity and the unliganded, tensed (T) structure with oxygen affinity lowered by constraining salt bridges [1–3]. Perutz argues that the equilibrium between these two structures is primarily governed by displacement of the iron and proximal histidine from the mean porphyrin plane and that much of the free energy of heme–heme interaction is stored in salt bridges which break upon oxygenation. This theory has received support from other workers [4–6]. On the other hand EXAFS studies have shown that ironpyrol nitrogen bond distances do not differ between deoxyhemoglobin A and deoxyhemoglobin Kempsey (β99 asp → asn) which is a high affinity mutant essentially devoid of cooperativity [7]. Thermodynamic studies of the temperature dependence of αβdimer–tetramer equilibrium point out that the free energy of heme–heme interaction is stored in hydrogen bonds between dimers and not salt bridges [8]. Replacement of iron by cobalt, which remains low spin even in the unliganded tetramer, only slightly diminishes n, the empirical measure of cooperativity [9–]. Finally, the displacement of iron from the porphyrin plane in deoxyhemoglobin A seems to differ little from iron displacement in the noncooperative monomer deoxymyoglobin [12]. We wish to propose a truly alternative mechanism for cooperative ligand binding by hemoglobin which does not necessitate metal movement or salt bridge energetics. The important molecular movement for an increase in oxygen affinity is porphyrin sliding from the hydrophobic protein interior to a position with increased porphyrin exposure to solvent. In hemoglobin A, this movement is rigidly coupled to breaking the hydrogen bond between β99 asp and α42 tyr as the porphyrin moves towards the protein exterior, and upon oxygenation formation of a new hydrogen bond between β102 asn and α94 asp. Protein crystallographic studies report that the pophyrins of both α and β subunits are more exposed to water in the met-form than for deoxyhemoglobin [2]. The porphyrin is more exposed to solvent in the β chain of the high affinity mutant deoxyhemoglobin Yakima (β99 asp → his) than A [13]. Studies on model cobaltoporphyrins report that oxygen affinity is more dependent upon porphyrin–solvent interactions than upon 2,4-substituents or ligand trans to oxygen [14, 15]. Stellwagen has pointed out that the redox potential of hemoproteins is dependent upon the degree of porphyrin exposure to solvent, with the redox potential decreasing as porphyrin exposure increases [16]. That oxygen affinity increases with increasing porphyrin exposure to solvent is consistent with all the above facts and is tied together by the experiments of Basolo and co-workers who have shown that an inverse linear correlation exists between th logK of oxygenation and cobalt(II/III) redox potential for a series of equatorially substituted cobalt complexes [17, 18]. Recent work upon energetics across the α 1β 2 interface of hemoglobin shows that amino acid mutations in this region drastically alter cooperative energetics while substitutions at other parts of the molecule have little effect upon cooperativity [19]. Substitution of β99 asp by his, gln or gly destroys the lone hydrogen bond which connects the deoxy-dimers and abolishes all cooperative energy, and the resultant hemoglobins have oxygen affinities close to those of isolated subunits. The NMR resonance position of this hydrogen bond (−14.2 ppm) is in the range of resonance positions of ‘strong’ hydrogen bonds while the resonance position of the β102 asn−α94 asp proton (−10.3 ppm) appears normal [10, 21]. So energy storage for the deoxy tetramer is primarily localized in this β99 asp−α42 tyr hydrogen bond. Information between subunits is transferred through those amino acids involved in hydrogen bonding to heme pyrrole II and the innate porphyrin rigidity used to modulate porphyrin exposure to solvent which in turn controls oxygen affinity. Porphyrin sliding can also account for Feimidazole bond rupture in hemoglobin NO in the presence of IHP [22].

Kinetics of deoxyhemoglobin subunit dissociation determined by haptoglobin binding: estimation of the equilibrium constant from forward and reverse rates.

Deoxyhemoglobin tetramers dissociate into dimers very slowly, with half-times on the order of several hours. It is demonstrated that absorbance changes in the Soret region which accompany this dissociation and persist upon binding of haptoglobin 1-1 to the dissociated dimers can be used for accurate kinetic determinations over the necessarily long periods required for study. This method of study for the slow reactions depends upon long-term spectral integrity of the reaction mixtures and upon accurate measurement. The variation in rate constants determined by this procedure has been correlated with variations in structural constraints at the dimer-dimer contact region. In the presence of 2,3-diphosphoglycerate the rate constant is decreased, consistent with the role of this effector in binding to both beta chains and stabilizing the constrained deoxy tetramer against dissociation into alphabeta dimers. With hemoglobin specifically modified (des-Arg-141alpha) to eliminate half the constraining salt links within the dimer-dimer contact region, the dissociation rate is increased by approximately three orders of magnitude. In hemoglobin S where the amino acid substitution is not directly in the intersubunit contact region of interest, the dissociation rate is found to be approximately the same as that for hemoglobin A. Combination of the dissociation rate constants determined by haptoglobin binding with stopped-flow determinations of the rate constant for reassociation of dissociated dimers provides an estimate of the equilibrium constant, 0K2, for the deoxyhemoglobin dimer-tetramer equilibrium. This estimate is independent of any assumptions regarding other energetic quantities, and yields a value of 2.54 +/- 0.7 X 10(10)M-1 (heme) in 0.1 M Tris-HCl, 0.1 M NaCl, and 1 mM EDTA, pH 7.4, 21.5 degrees C. Thus the intersubunit contact energy is -14.0 +/- 0.2 kcal/mol of heme. The stabilization energy between deoxy and oxy tetramers is found to be approximately 6.4 kcal/mol, under these conditions.

The Effect of 2,3-Diphosphoglycerate on the Tetramer-Dimer Equilibrium of Liganded Hemoglobin

Abstract The effect of 2,3-diphospho-d-glycerate (P2-glycerate) on the kinetic behavior of deoxyhemoglobin generated by the rapid dissociation of gaseous ligand (either by flash photolysis of carbon monoxide hemoglobin (HbCO) or by deoxygenation of oxyhemoglobin (HbO2) in the presence of sodium dithionite) is consistent with a stabilization of the tetrameric state of liganded hemoglobin by the organic phosphate. The fraction (α) of rapidly reacting hemoglobin produced by pulsed laser photolysis of phosphate-free HbCO in 0.05 m 2,2'-bis(hydroxymethyl)-2,2',2''-nitriloethanol-0.1 m NaCl, pH 7.0, was independent of CO concentration below about 500 µm, but increased systematically with dilution of the hemoprotein. The apparent tetramer-dimer dissociation constant (Kl4,2) calculated from the dependence of α on [HbCO] was 3.6 ± 1.0 µm in the absence of phosphates and decreased to 1.4 ± 0.3 µm when 1 mm P2-glycerate was added. These values of Kl4,2 are similar to estimates for liganded hemoglobin obtained in sedimentation experiments conducted at pH 7.0 in 0.1 m phosphate (Edelstein, S. J., Rehmer, M. J., Olson, J. S., and Gibson, Q. H. (1970) J. Biol. Chem. 245, 4372–4381) and in 0.1 m Tris-0.09 m NaCl (Kellett, G. L. (1971) J. Mol. Biol. 59, 401–424). The magnitude of the Soret absorbance drift accompanying deoxygenation of dilute solutions of HbO2 in the presence of sodium dithionite was decreased by added 1 mm P2-glycerate, and the second order rate constant characterizing the drift phase was increased at 20° from 0.54 ± 0.09 µm-1 s-1 to 1.57 ± 0.18 µm-1 s-1 by the organic phosphate. Since the drift has been shown to result from the formation of deoxy tetramers from deoxy αβ dimers (Kellett, G. L., and Gutfreund, H. (1970) Nature 227, 921–926), this result is also consistent with the proposed phosphate inhibition of dimer formation by liganded hemoglobin.

The Reaction of n-Butyl Isocyanide with Human Hemoglobin: II. THE LIGAND-BINDING PROPERTIES OF THE α AND β CHAINS WITHIN DEOXYHEMOGLOBIN

Abstract At pH 7.0, 20°, both in the absence and presence of phosphates, the time course of n-butyl isocyanide binding to deoxyhemoglobin is biphasic, and the two kinetic phases can be isolated spectrally and identified with the properties of the α and β chains. Under all conditions at this pH, both the association and dissociation rate constants of the β chains are at least 10 times larger than the corresponding α chain rate constants, which indicates that the β heme site is much more accessible to this isocyanide than the α heme site. However, the spectral and functional heterogeneity observed in the kinetic and equilibrium data at pH 7.0 is absent at pH 9.1. This would appear to indicate that the chains possess similar ligand-binding properties at alkaline pH. For stripped hemoglobin in 0.05 m 2,2-bis(hydroxymethyl)2,2',2''-nitriloethanol (bis-tris), pH 7.0, 20°, the affinities of the chains within deoxyhemoglobin (defined by the reaction Hb4 + X ⇌ Hb4X) are roughly equivalent, the affinity of the deoxy α chains being slightly greater than that of the β chains. For unstripped hemoglobin in 0.1 m Pi, pH 7.0, the affinity of the deoxy α chains appears to be slightly lower than that of the β chains. At pH 7.0, the addition 2,3-diphosphoglycerate or inositol hexaphosphate to samples of stripped hemoglobin allows the generation of populations of partially saturated intermediates which contain n-butyl isocyanide bound preferentially to β heme sites. Thus, the most striking effect of phosphates is a lowering of the ligand affinity of the α chains relative to that of the β chains within the deoxy tetramer. A detailed analysis has been performed on both equilibrium and kinetic data obtained with stripped hemoglobin in 0.05 m bis-tris, pH 7.0. In order to fit both types of data with a single set of rate constants, the tetramer had to be considered as the minimum unit of function, and within this unit, the structural transition from deoxy-like to oxy-like hemoglobin was postulated to occur only after three ligand molecules had been bound. From the resultant values assigned to this set of rate constants, the total free energy of interaction per binding site was calculated to be ∼2.0 kcal per mole which is 0.3 kcal per mole smaller than the value which has been reported for the oxygen reaction. This result, coupled with the remarkable effects of phosphates and pH on the manifestation of chain differences, suggests the need for caution not only in comparing results obtained under different experimental conditions but also in using results obtained with one ligand to predict the behavior of hemoglobin with another.

Noncooperativity of the dimer in the reaction of hemoglobin with oxygen (human-dissociation-equilibrium-sulfhydryl-absorption-x-ray analysis).

The theory that the alphabeta dimer is the functional unit of cooperativity in hemoglobin has been tested by determination of the oxygen equilibrium curve of stable deoxy dimers, obtained by the addition of 0.9 M MgCl(2) to human des-Arg 141alpha-hemoglobin. Cooperativity was absent in this medium, but was regained on transfer of the hemoglobin to a dilute phosphate buffer, where tetramers reformed. X-ray analysis of crystals of oxy- and deoxy-des-Arg hemoglobins showed that the removal of Arg 141alpha would leave the structure of alphabeta dimers unchanged. Nonreactivity of the sulfhydryl groups at 112beta proved that the subunits in deoxy dimers form the same contact as in oxy dimers, namely alpha(1)beta(1), and that no significant dissociation into free subunits occurs in 0.9 M MgCl(2). The absorption spectrum of the deoxy dimers corresponded to the sum of the spectra of the free deoxy alpha and beta subunits, and was different from that of the deoxy tetramer, showing the constraining salt bridges formed by the C-terminal residues in the tetramer to be necessary for the spectral changes normally observed on association of the deoxy subunits.