Asymmetric distribution of cooperativity in the binding cascade of normal human hemoglobin. 2. Stepwise cooperative free energy

Stepwise cooperative free energies and intermediate Hill coefficients are used to assess the presence of noncooperative sequences in the database of binding free energies previously obtained for the eight partially ligated intermediates of human hemoglobin, encompassing a variety of hemesite analog substitutions. This analysis is prompted by the observed noncooperative binding of two ligands to hemoglobins that are partially substituted with Zn2+-heme, an analog of deoxy Fe2+-heme (Holt et al. (2005) Biochemistry 44, XXXXX). The results show that noncooperative binding sequences are observed in all hemesite analog studied to date. The noncooperative binding observed in (alpha2Znbeta2FeO2) and (alpha2FeO2beta2Zn) is therefore not a Zn-specific substitution artifact. One of several binding sequences from singly to triply ligated hemoglobin is also observed to occur with little or no positive cooperativity. These results demonstrate the variability possible among different ligation pathways in a highly cooperative multi-subunit system such as hemoglobin. As a direct consequence of this variability, differences among ligation pathways are not always detectable using cooperativity functions based on statistical distributions, such as the Hill coefficient n(H). The limitations of Hill coefficient analysis in evaluating cooperativity in intermediates of complex systems is contrasted with the utility of the stepwise binding parameters.     

Oxygen binding constants for human hemoglobin tetramers

High-precision studies of oxygen binding in hemoglobin (HbA0) solutions at near-physiological concentrations (2-12 mM heme; pHs 7.0-9.1; various buffers) have led to an unanticipated result: an unmeasurably low contribution from the triply ligated species. We have obtained this result from new differential oxygen-binding measurements for human hemoglobin through the use of a thin-layer apparatus, which enables study of solutions at high Hb concentrations. The effect of tetramer dissociation into dimers, which becomes significant at hemoglobin concentrations below 1 mM in heme, is avoided. The analysis of the binding reactions is thus cast in terms of tetramer-binding polynomial written with overall Adair equilibrium constants which directly reflect the contributions of intermediate ligated species. The unmeasurable contribution of the triply ligated species renders the equilibrium constants of the third and fourth stepwise reactions practically undeterminable.     

Oxygen binding and other physical properties of human hemoglobin made in yeast.

Wagenbach et al. (1991, BioTechnology, 9, 57-61) have recently developed a system for producing soluble recombinant tetrameric hemoglobin in yeast: hemoglobin begins to appear 4-5 h after induction with galactose, alpha- and beta-globin chains fold in vivo and endogeneously produced heme is incorporated into hemoglobin tetramers. We have further characterized the oxygen-binding properties, as well as the tetramer stability, of recombinant human Hb A made in yeast. After purification by ion-exchange chromatography, a single band at the same position as normal human Hb A was obtained using cellulose acetate electrophoresis. Although the oxy and deoxy forms of purified recombinant Hb A made in yeast were spectrophotometrically identical to native human Hb A, the oxygen-binding curve was shifted slightly left of that for native human Hb A. Further purification of recombinant hemoglobin by FPLC revealed two fractions: one (fraction B) with low cooperativity and high oxygen affinity, and the other (fraction A) with almost identical cooperativity and oxygen affinity compared with native human Hb A. The Bohr effect of fraction A was also identical to native human Hb A. Hemoglobin in fraction B with lowered cooperativity precipitated approximately 1.5 times faster than normal human Hb A during mechanical agitation, while hemoglobin in fraction A with normal cooperativity precipitated with kinetics identical to native human Hb A. These results suggest that some of the recombinant molecules made in yeast fold improperly, and that these molecules may exhibit decreased cooperativity for oxygen binding and decreased stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

The oxygen-binding intermediates of human hemoglobin: evaluation of their contributions to cooperativity using zinc-containing hybrids.

Hemoglobin tetramers [Zn/FeO(2)] containing oxygenated subunits (FeO(2)), in combination with unligated subunits containing zinc-substituted hemes (Zn), were analyzed to determine their contributions to the cooperativity of oxygen binding at the Fe sites. Energetic consequences of possible perturbation by zinc substitution were evaluated in all combinations of unligated Zn/Fe hybrid tetramers. A general thermodynamic strategy that corrects for the energetic effects of substituting a second metal for Fe showed the perturbations of Zn substitution to be negligible. This permitted cooperativity parameters of the native Fe/FeO(2) intermediates to be calculated from data on the corresponding Zn/FeO(2) molecules. These parameters, determined explicitly for all eight oxygen-binding intermediates (Fe/FeO(2)), were found to be identical to those predicted earlier from analyzing the O(2) binding data of normal hemoglobin according to the "molecular code" of hemoglobin allostery. The cooperativity parameters determined for this system showed the same distribution pattern found previously for five other oxygen analog systems (Fe/FeCN, FE/Mn(3+), Co/FECO, Co/FeCN, and Fe/FeCO).     

The contribution of the asymmetric alpha 1beta 1 half-oxygenated intermediate to human hemoglobin cooperativity.

Considerable controversy remains as to the functional and structural properties of the asymmetric alpha1beta1 half-oxygenated intermediate of human hemoglobin, consisting of a deoxygenated and an oxygenated dimer. A recent dimer-tetramer equilibrium study using [Zn(II)/Fe(II)-O(2)] hybrid hemoglobins, in which Zn-protoporphyrin IX mimics a deoxyheme, showed that the key intermediate, [alpha(Fe-O(2))beta(Fe-O(2))][alpha(Zn)beta(Zn)], exhibited an enhanced tetramer stability relative to the other doubly oxygenated species. This is one of the strongest findings in support of distinctly favorable intra-dimer cooperativity within the tetramer. However, we present here a different conclusion drawn from direct O(2) binding experiments for the same asymmetric hybrid, [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)], and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2). In this study, the O(2) equilibrium curves for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] were determined by an O(2)-jump stopped-flow technique to circumvent the problem of dimer rearrangement, and those for [alpha(Fe)beta(Zn)]( 2) and [alpha(Zn)beta(Fe)]( 2) were measured by using an Imai apparatus. It was shown that the first and second O(2) equilibrium constants for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] are 0.0209 mmHg(-1) and 0.0276 mmHg(-1), respectively, that are almost identical to those for [alpha(Fe)beta(Zn)](2) or [alpha(Zn)beta(Fe)](2). Therefore, we did not observe large difference among the asymmetric and symmetric hybrids. The discrepancy between the present and previous studies is mainly due to previously observed negative cooperativity for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2), which is not the case in our direct O(2) binding study.     

Coupling of the oxygen-linked interaction energy for inositol hexakisphosphate and bezafibrate binding to human HbA0

The energetics of signal propagation between different functional domains (i.e. the binding sites for O2, inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA0 was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to -3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO2 = 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T- and R-like tertiary conformation(s) within a quaternary R-like structure).     

Equilibrium oxygen binding to human hemoglobin cross-linked between the alpha chains by bis(3,5-dibromosalicyl) fumarate

Oxygen equilibrium curves of human hemoglobin Ao (HbAo) and human hemoglobin cross-linked between the alpha chains (alpha alpha Hb) by bis(3,5-dibromosalicyl) fumarate were measured as a function of pH and chloride or organic phosphate concentration. Compared to HbAo, the oxygen affinity of alpha alpha Hb was lower, cooperativity was maintained, although slightly reduced, and all heterotropic effects were diminished. The major effect of alpha alpha-cross-linking appears to be a reduction of the oxygen affinity of R-state hemoglobin under all conditions. However, while the oxygen affinity of T-state alpha alpha Hb was slightly reduced at physiologic chloride concentration and in the absence of organic phosphates, KT was the same for both hemoglobins in the presence of 2,3-diphosphoglycerate (or high salt) and higher for alpha alpha Hb in the presence of inositol hexaphosphate. The reduced O2 affinity arises from smaller binding constants for both T- and R-state alpha alpha Hb rather than through stabilization of the low affinity conformation. All four Adair constants could be determined for alpha alpha Hb under most conditions, but a3 could not be resolved for HbAo without constraining a4, suggesting that the cross-link stabilizes triply ligated intermediates of hemoglobin.     

Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.     

Modulation of reactivity and conformation within the T-quaternary state of human hemoglobin: the combined use of mutagenesis and sol-gel encapsulation

A range of conformationally distinct functional states within the T quaternary state of hemoglobin are accessed and probed using a combination of mutagenesis and sol-gel encapsulation that greatly slow or eliminate the T --> R transition. Visible and UV resonance Raman spectroscopy are used to probe the proximal strain at the heme and the status of the alpha(1)beta(2) interface, respectively, whereas CO geminate and bimolecular recombination traces in conjunction with MEM (maximum entropy method) analysis of kinetic populations are used to identify functionally distinct T-state populations. The mutants used in this study are Hb(Nbeta102A) and the alpha99-alpha99 cross-linked derivative of Hb(Wbeta37E). The former mutant, which binds oxygen noncooperatively with very low affinity, is used to access low-affinity ligated T-state conformations, whereas the latter mutant is used to access the high-affinity end of the distribution of T-state conformations. A pattern emerges within the T state in which ligand reactivity increases as both the proximal strain and the alpha(1)beta(2) interface interactions are progressively lessened after ligand binding to the deoxy T-state species. The ligation and effector-dependent interplay between the heme environment and the stability of the Trp beta37 cluster in the hinge region of the alpha(1)beta(2) interface appears to determine the distribution of the ligated T-state species generated upon ligand binding. A qualitative model is presented, suggesting that different T quaternary structures modulate the stability of different alphabeta dimer conformations within the tetramer.     

Heterotropic effects of chloride on the ligation microstates of hemoglobin at constant water activity.

Dimer-tetramer assembly reactions of the 10 CN-met ligation microstates of hemoglobin (Hb) were analyzed as a function of NaCl concentration while maintaining constant water activity by the addition of compensating sucrose. The assembly free energy for fully ligated cyanomet Hb and for fully oxygenated Hb becomes less favorable by 1.8 kcal when [NaCl] is increased from 0.08 to 0.7 M, whereas that of unligated Hb is practically insensitive to changes in [NaCl]. Values of 1.6 and 0.3 mol chloride release were found for the assembly of fully ligated and deoxy Hb, respectively; i.e., a net release of 1.3 mol chloride is coupled to the ligation of tetramers for both oxygen and cyanomet ligation. The ligation-linked salt component at constant water activity was evaluated to be 1.0 mol for the full oxygenation of the Hb tetramer in agreement with the overall value previously reported. When the detailed salt linkages accompanying all 16 stepwise cyanomet ligation reactions were experimentally resolved, only two "chloride" effects were found. The first chloride effect correlates with the ligation steps, which create tertiary constraint, and the second effect is coupled to the six switchpoints of quaternary T-->R transition. The distribution of these chloride effects agrees closely with predictions of the "symmetry rule mechanism." The total chloride release for CN-met ligation is in good agreement with that for oxygenation. Free energy contributions to assembly and cooperativity arising from the osmotic effects of chloride were found to be small for all ligation species.     

Effect of removal of a salt-bridge on the oxygen binding properties and the electronic structure of heme in cobalt-iron hybrid hemoglobin.

In order to clarify the role of salt-bridges in hemoglobin, the oxygen equilibrium curves and electron paramagnetic resonance (EPR) spectra of cobalt-iron hybrid hemoglobins were determined. The EPR spectra of deoxy alpha(Co)2 beta(Fe)2 could be interpreted as a mixture of two distinct paramagnetic species: one showed a maximum of the first derivative spectrum at g = 2.39 and the other at g = 2.33. The oxygen equilibrium curves of the hybrid indicated that the former is assignable to the T structure and the latter to the R structure. The cooperativity of oxygen binding of alpha(Co)2 beta(Fe)2 exhibited a maximum at g = 2.33, which is characteristic of the R structure, regardless of the pH. Addition of inositol hexaphosphate (IHP) to des-Arg alpha(Co)2 beta(Fe)2 restored the cooperativity of oxygen binding, which implies that the deoxygenated form of des-Arg alpha(Co)2 beta(Fe)2 is converted to the T structure upon addition of IHP. However, the EPR signal at g = 2.39 was not restored upon conversion to the T structure by addition of IHP. It is therefore concluded that the EPR spectrum of the deoxy alpha(Co) subunit depends both on the quaternary structure and on the localized strain at the heme.     

Indefinite noncooperative self-association of chicken deoxy hemoglobin D

The minor tetrameric hemoglobin (Hb), Hb D, of chicken red blood cells self-associates upon deoxygenation. This self-association enhances the cooperativity of oxygen binding. The maximal Hill coefficient is greater than 4 at high Hb concentrations. Previous measurements at low Hb concentrations were consistent with a monomer-to-dimer equilibrium and an association constant of ～1.3-1.6 × 10(4) M(-1). Here, the Hb tetramer is considered as the monomer. However, new results indicate that the association extends beyond the dimer. We show by combination of Hb oligomer modeling and sedimentation velocity analyses that the data can be well described by an indefinite noncooperative or isodesmic association model. In this model, the deoxy Hb D associates noncooperatively to give a linear oligomeric chain with an equilibrium association constant of 1.42 × 10(4) M(-1) at 20°C for each step. The data are also well described by a monomer-dimer-tetramer equilibrium model with monomer-to-dimer and dimer-to-tetramer association constants of 1.87 and 1.03 × 10(4) M(-1) at 20°C, respectively. A hybrid recombinant Hb D was prepared with recombinant α(D)-globin and native β-globin to give a Hb D tetramer (α(2)(D)β(2)). This rHb D undergoes decreased deoxygenation-dependent self-association compared with the native Hb D. Residue glutamate 138 has previously been proposed to influence intertetramer interactions. Our results with recombinant Hb D show that Glu138 plays no role in deoxy Hb D intertetramer interactions.     

Oxygen binding to partially nitrosylated hemoglobin

Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb–NO). Many aspects of the formation and persistence of Hb–NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O₂. Partially nitrosylated Hb samples had predominantly hexacoordinate NO–heme geometry and resisted oxidation when exposed to O₂ in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO–heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO–heme geometry also affected O₂ binding equilibria. O₂-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O₂ affinity T-state of the Hb by increasing percentages of NO–heme, much as occurs with increasing levels of CO–heme. Samples containing IHP showed small decreases in O₂ affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/β-met tetramers. Most remarkably, O₂-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO–heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O₂ transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Effects of S-nitrosation on oxygen binding by normal and sickle cell hemoglobin.

S-Nitrosated hemoglobin (SNO-Hb) is of interest because of the allosteric control of NO delivery from SNO-Hb made possible by the conformational differences between the R- and T-states of Hb. To better understand SNO-Hb, the oxygen binding properties of S-nitrosated forms of normal and sickle cell Hb were investigated. Spectral assays and electrospray ionization mass spectrometry were used to quantify the degree of S-nitrosation. Hb A(0) and unpolymerized Hb S exhibit similar shifts toward their R-state conformations in response to S-nitrosation, with increased oxygen affinity and decreased cooperativity. Responses to 2, 3-diphosphoglycerate were unaltered, indicating regional changes in the deoxy structure of SNO-Hb that accommodate NO adduction. A cycle of deoxygenation/reoxygenation does not cause loss of NO or appreciable heme oxidation. There is, however, appreciable loss of NO and heme oxidation when oxygen-binding experiments are carried out in the presence of glutathione. These results indicate that the in vivo stability of SNO-Hb and its associated vasoactivity depend on the abundance of thiols and other factors that influence transnitrosation reactions. The increased oxygen affinity and R-state character that result from S-nitrosation of Hb S would be expected to decrease its polymerization and thereby lessen the associated symptoms of sickle cell disease.     

Involvement of Glu G3(101)beta in the function of hemoglobin. Comparative O2 equilibrium studies of human mutant hemoglobins

The glutamyl residue at G3(101)beta of normal hemoglobin (Hb A) is one of the alpha 1 beta 2 subunit contacts which are vital to O2 binding properties of the molecule. The O2 equilibrium properties of the four mutants with different substitutions at this site are studied in order to elucidate the role of this residue. Under stripped conditions with minimum chloride the order of O2 affinity is: Hb A (Glu) much less than Hb Rush (Gln) less than or equal to Hb British Columbia (Lys) less than or equal to Hb Potomac (Asp) less than or equal to Hb Alberta (Gly). The first Adair constants, K1, for the mutant hemoglobins are greater than that for Hb A whereas the fourth, K4, are similar, indicating that the allosteric constants (L) of these mutants are greatly reduced. Therefore, the G3(101)beta residue contributes intrinsically to the strengthening of the structural constraints that are imposed upon the deoxy (T) forms but not the oxy (R) form. On addition of 0.1 M Cl- and further addition of 2,3-diphosphoglycerate or inositol hexaphosphate, their O2 affinities and cooperativities are altered, reflecting different responses to anionic ligands. Hb Rush exhibits a stronger chloride effect than Hb A and the other variants and, as a result, an increased Bohr effect and a smaller heat of oxygenation at pH 6.5. These changes are consistent with an increased positive net charge in the central cavity of Hb Rush and subsequent extra anion binding in the deoxy form. The tetramer to dimer dissociation constants are estimated to be greater than normal for Hb British Columbia and less than normal for Hb Alberta. This comparative study of the G3(101)beta mutants indicates that the size and the charge of this residue may influence the switching of two neighboring interchain hydrogen bonds that occurs during oxygenation of normal hemoglobin.     

Isolation and characterization of the triply oxidized derivative of a cross-linked hemoglobin.

Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperative oxygen binding, subunit assembly, and sulfhydryl reaction kinetics of the eight cyanomet intermediate ligation states of human hemoglobin.

Correlations between the energetics of cooperativity and quaternary structural probes have recently been made for the intermediate ligation states of Hb [Daugherty et al. (1991) Proc. Natl. Acad. Sci. US 88, 1110-1114]. This has led to a "molecular code" which translates configurations of the 10 ligation states into switch points of quaternary transition according to a "symmetry rule"; T-->R quaternary structure change is governed by the presence of at least one heme-site ligand on each of the alpha beta dimeric half-molecules within the tetramer [see Ackers et al. (1992) Science 255, 54-63, for summary]. In order to further explore this and other features of the cooperative mechanism, we have used oxygen binding to probe the energetics and cooperativities for the vacant sites of the cyanomet ligation species. We have also probed structural aspects of all eight cyanomet ligation intermediates by means of sulfhydryl reaction kinetics. Our oxygen binding results, obtained from a combination of direct and indirect methods, demonstrate the same combinatorial aspect to cooperativity that is predicted by the symmetry rule. Overall oxygen affinities of the two singly-ligated species (alpha +CN beta)(alpha beta) and (alpha beta +CN)(alpha beta) were found to be identical (pmedian = 2.4 Torr). In contrast, the doubly-ligated species exhibited two distinct patterns of oxygen equilibria: the asymmetric species (alpha +CN beta +CN)(alpha beta) showed very high cooperativity (nmax = 1.94) and low affinity (pmedian = 6.0 Torr), while the other three doubly-ligated species showed diminished cooperativity (nmax = 1.23) and considerably higher oxygen affinity (pmedian = 0.4 Torr). Extremely high oxygen affinities were found for the triply-ligated species (alpha +CN beta +CN)(alpha beta +CN) and (alpha +CN beta +CN)(alpha +CN beta) (pmedian = 0.2 Torr). Their oxygen binding free energies are considerably more favorable than those of the alpha and beta subunits within the dissociated alpha beta dimer, demonstrating directly the quaternary enhancement effect, i.e., enhanced oxygen affinity at the last binding step of tetramer relative to the dissociated protomers. Oxygen binding free energies measured for the alpha subunit within the isolated (alpha beta +CN) dimer and for the beta subunit within the isolated (alpha +CN beta) dimer sum to the free energy for binding two oxygens to normal hemoglobin dimers (-16.3 +/- 0.2 versus -16.7 +/- 0.2, respectively), arguing against cooperativity in the isolated dimer. Correlations were established between cooperative free energies of the 10 cyanomet ligation microstates and the kinetics for reacting their free sulfhydryl groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.