The stability of the heme-globin linkage in some normal, mutant, and chemically modified hemoglobins.

The rates and equilibria of heme exchange between methemoglobin and serum albumin were measured using a simple new spectrophotometric method. It is based on the large difference between the spectrum of methemoglobin and that of methemealbumin at pH 8-9. The rate of heme exchange was found to be independent of the albumin concentration and inversely proportional to the hemoglobin (Hb) concentration. Taken together with the finding that the rate was 10 times greater for Hb Rothschild, which is completely dissociated into alpha beta dimers and 10 times smaller for two cross-linked hemoglobins, the subunits of which cannot dissociate, this showed that the rate of dissociation of heme from alpha beta dimers is very much greater than from tetramers. Conditions were found for the attainment of an equilibrium distribution of hemes between beta globin and albumin. The equilibrium distribution ratio, R = methemealbumin/albumin/methemoglobin/apohemoglobin, for hemoglobin A was 3.4 with human and 0.005 with bovine serum albumin. Both the rates of exchange and the R values of HbS and HbF were the same as that for HbA. The equilibrium distribution ratio for Hb Rothschild was 7 times greater than that for HbA whereas that of one but not the other of the cross-linked hemoglobins was 10 times smaller. The structural bases for these differences are analyzed.     

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into alpha beta dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.     

Stability of asymmetrical hybrid hemoglobins. Determination by anaerobic high performance liquid chromatography

Asymmetrical hybrid hemoglobins formed in mixtures of Hb A and Hb S, Hb F and Hb S, Hb S and Hb York(beta 146 His----Pro), and Hb A and Hb York were separated by high performance liquid chromatography on cation and anion exchange columns under anaerobic conditions. The ratio of the hybrid hemoglobin to the total mixture was consistently lower than that theoretically expected and decreased with longer elution times. The hybrid tetramer appears to be unstable even under anaerobic conditions and dissociates into alpha beta dimers. The time course of dissociation of the hybrid hemoglobins was determined by varying the separation programs and thus separating the hybrid hemoglobin at different elution times. The rate of the dissociation of the hybrid hemoglobins studied follows first order kinetics. The lines representing the time course of dissociation of hybrid hemoglobins were extrapolated to time 0 to determine the fraction of the hybrid hemoglobin in the mixture prior to separation. The values obtained for equimolar mixtures of Hb A and Hb S and Hb York and Hb S or Hb A were in agreement with the expected theoretical value (50%). In contrast, the value obtained for hybrid hemoglobin FS was slightly less (about 40%). AY and SY hybrid hemoglobins dissociated into dimers at a considerably faster rate than did AS and FS hybrid hemoglobins, possibly because of the mutation at the beta 146-position in hybrid hemoglobins containing alpha beta Y dimers. This mutation hinders the formation of salt bridges that normally stabilize the "T" quaternary conformation. Since such hybrid hemoglobins have a partial "R" conformation even when deoxygenated, their rate of dissociation to dimers is expected to increase.     

Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53

The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37 °C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity.     

Tetramer-dimer dissociation in homoglobin and the Bohr effect.

The pH dependence of the apparent tetramer to dimer dissociation constant has been determined at 20 degrees for both oxy- and deoxyhemoglobins A and Kansas. These measurements were made by three different procedures: gel chromatography, sedimentation velocity, and kinetic methods in either of three buffer systems: 0.05 M cacodylate, Tris, or glycine with 1 mM EDTA and 0.1 M NaCl between pH 6.5 and 11. The tetramer-dimer dissociation constant of human oxyhemoglobin A decreases from about 3.2 X 10(-6) M at pH 6.0 to about 3.2 X 10(-8) M at pH 8.5. The slope of this line indicates that the dissociation of tetramer to dimer is accompanied by the uptake of about 0.6 protons per mol of tetramer in this region. The corresponding dissociation constant for deoxyhemoglobin in the same pH region increases apparently almost linearly from 1.0 x 10(-12) M at pH 6.5 to about 1.0 x 10(-5) M at pH 11. To dimer is associated with the release of about 1.6 protons per mol of tetramer. Comparison of these data with the known proton release accompanying the oxygenation of tetramers confirms that the pH dependence of oxygen binding by dimers must be very small. The present data predict that the overall proton release or uptake per oxygen bound by dimer should be less than 0.1. The tetramer-dimer dissociation equilibria of oxy- and deoxyhemoglobins above pH 8.5 have identical pH dependences. In this range the dissociation constant of deoxy-Hb is about one-tenth that of oxyhemoglobin. Human oxyhemoglobin Kansas is known to have an enhanced tetramer-dimer dissociation compared with that of hemoglobin A. Below pH 8.5 the tetramer-dimer dissociation constant of Hb Kansas is about 400 times greater than that of HbA in the absence of phosphate buffers. In contrast, the tetramer-dimer dissociation constants of deoxyhemoglobins A and Kansas appear to be identical. These findings are consistent with previous structural observations on these hemoglobins. The data on the tetramer-dimer dissociation of human hemoglobin were used to calculate the total free energy of binding of oxygen to the tetramer and the median oxygen pressure on the basis of fundamental linkage relations and a pH-independent estimate of the total free energy of binding oxygen to dimer. Simulated oxygen binding curves were generated with the equations of Ackers and Halvorson (Ackers, G. K., and Halvorson, H. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4312-4316) by making two assumptions: (a) that the dimers are noncooperative and pH-independent in O2 binding and (b) that the distribution of cooperative energy in the oxygenation of tetramers is independent of pH. We have compared these simulations with experimental data obtained at low protein concentrations (30 to 124 muM heme) to show that the variation in oxygen affinity with pH can be described in terms of the subunit equilibria. We conclude that an accurate analysis of the contributions of individual oxygen binding steps to the Bohr effect cannot be made without considering the contributions of the dimers to oxygen binding...     

L55P transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers

Transthyretin is a tetrameric protein associated with the commonest form of systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry, we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, L55P. Wild-type subunit exchange occurs via both monomers and dimers, whereas exchange via dimers is the dominant mechanism for the L55P variant. Because patients with the L55P mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and L55P tetramers. We found that hybrid tetramers containing two or three L55P subunits dominate in the early stages of the reaction. Surprisingly, we also found that, in the presence of L55P transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidosis.     

Conformational drift and cryoinactivation of lactate dehydrogenase

Solutions of porcine lactate dehydrogenase of micromolar concentration kept at 4 degrees C for several days lose the greater part of their enzymic activity but recover it when returned to room temperature. The rate of spoiling decreases and the rate of recovery increases with the concentration of the solutions. The decrease in tetramer stability in the cold is shown by experiments of pressure dissociation at various temperatures and confirmed because isozyme hybridization occurs in parallel with the inactivation at low temperature but is absent at room temperature. Cold-inactivated solutions contain tetramers that dissociate much more readily than those of the fully active solutions. It is postulated that cryoinactivation, like pressure inactivation, takes place through a cycle of dissociation, conformational drift [King, L., & Weber, G. (1986) Biochemistry (second paper of three in this issue)] and reassociation into inactive tetramers.     

The hybridization of glyceraldehyde 3-phosphate dehydrogenases from rabbit muscle and yeast. Kinetics and thermodynamics of the reaction and isolation of the hybrid

A kinetic and thermodynamic study was made of the formation of the hybrid (R(2)Y(2)) glyceraldehyde 3-phosphate dehydrogenase from the yeast (Y(4)) and rabbit (R(4)) enzymes. The values of the thermodynamic parameters for the equilibrium between R(4), Y(4) and R(2)Y(2) suggest that the R(2)-R(2) and Y(2)-Y(2) interactions are similar. However, the failure to observe the RY(3) and R(3)Y hybrids is interpreted in terms of differences at the interfaces of the R-R and Y-Y interactions (the glyceraldehyde 3-phosphate dehydrogenase molecule being regarded as a dimer of dimers). The kinetics of formation of the R(2)Y(2) hybrid were studied and a model was proposed to account for the results. Best-fit values for the rate constants of the individual steps were evaluated by computer simulation, and the rate-limiting steps were identified as the dissociation of tetramers to dimers. It is proposed that the cleavage plane for dissociation of the tetramers corresponds to the region of low electron density through the centre of the molecule in the X-ray-crystallographic structure for human glyceraldehyde 3-phosphate dehydrogenase (Watson et al., 1972), which is probably the plane containing the Q and R axes in the lobster enzyme (Buehner et al., 1974). The R(2)Y(2) hybrid was isolated in milligram amounts by ion-exchange chromatography and its rate of reversion to the native enzyme was shown to be consistent with the kinetic model proposed from the hybrid-formation experiments.     

Dissociation and in vitro reconstitution of bovine liver uridine diphosphoglucose dehydrogenase. The paired subunit nature of the enzyme

Uridine diphosphoglucose dehydrogenase (EC 1.1.1.22: UDPglucose dehydrogenase) at pH 5.5-7.8 is a stable homohexamer of 305 +/- 7 kDa that does not undergo concentration-dependent dissociation at enzyme concentrations greater than 5 micrograms/mL. Chemical cross-linking of the native enzyme at varying glutaraldehyde concentrations yields dimers, tetramers, and hexamers; at greater than 2% (w/v) glutaraldehyde, plateau values of 21% monomers, 16% dimers, 5% tetramers, and 58% hexamers are obtained. Dissociation at acid pH (pH 2.3) or in 4-6 M guanidine hydrochloride leads to inactive monomers (Mr 52,000). Denaturation at increasing guanidine hydrochloride concentration reveals separable unfolding steps suggesting the typical domain structure of dehydrogenases holds for the present enzyme. At greater than 4 M guanidine hydrochloride complete randomization of the polypeptide chains is observed after 10-min denaturation. Reconstitution of the native hexamer after dissociation/denaturation has been monitored by reactivation and glutaraldehyde fixation. The kinetics may be described in terms of a sequential uni-bimolecular model, governed by rate-determining folding and association steps at the monomer level. Trimeric intermediates do not appear in significant amounts. Reactivation is found to parallel hexamer formation. Structural changes during reconstitution (monitored by circular dichroism) are characterized by complex kinetics, indicating the rapid formation of "structured monomers" (with most of the native secondary structure) followed by slow "reshuffling" prior to subunit association. The final product of reconstitution is indistinguishable from the initial native enzyme.     

Hemoglobin equilibrium analysis by the multiangle laser light-scattering method

Dimer-tetramer and monomer-dimer-tetramer equilibria of tetrameric hemoglobins and their single chains in the CO form, respectively, were evaluated using the microbatch multiangle light-scattering (MALS) analysis system. The molecular weights of human Hb A and Hb F in the CO form were dependent on concentration. The dissociation constants to dimers of Hb A and Hb F were 2.58 x 10(-6) and 0.66 x 10(-6), respectively. Equilibration of single globin chains, including alpha, beta, and gamma chains, was also evaluated by the same method. The dissociation constants of alpha-chain dimers to monomers, of beta-chain tetramers to monomers, and of gamma-chain tetramers to dimers were 14 x 10(-6), 25 x 10(-17), and 6.86 x 10(-6) M, respectively. These results indicate that the MALS analysis system can not only determine molecular weight but also characterize protein-protein interactions of multi-subunit proteins.     

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.     

Different Pressure Resistance of Lactate Dehydrogenases from Hagfish is Dependent on Habitat Depth and Caused by Tetrameric Structure Dissociation

The effects of high hydrostatic pressure on lactate dehydrogenase (LDH) activities from two species of hagfish were examined. LDH from Eptatretus okinoseanus, a deep-sea species, retained 67% of the original activity even at 100 MPa. LDH activity from Eptatretus burgeri, a shallow-sea species, was completely lost at 50 MPa but recovered to the original value at 0.1 MPa. The tetrameric structure of LDH-A₄ from E. okinoseanus did not change at 50 MPa. In contrast, almost all LDH tetramers from E. burgeri dissociated to dimers and monomers at 50 MPa but reverted to tetramers at 0.1 MPa. These results show that the dissociation of tetramers caused the inactivation of E. burgeri LDH. The difference depends on the number 6 and 10 amino acids. The mechanism of the slight, gradual inactivation of E. okinoseanus LDH at high pressure differs and is probably due to the metamorphosis of its inner structures.     

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 beta 37 (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(Hb4L4 in equilibrium with 2Hb2) = 37.3 x 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.     

Dissociation of dimers of human hemoglobins A and F into monomers

Dissociation of alpha beta and alpha gamma dimers of human hemoglobins (Hb) A and F into monomers was studied by alpha chain exchange (Shaeffer, J. R., McDonald, M. J., Turci, S. M., Dinda, D. M., and Bunn, H. F. (1984) J. Biol. Chem. 259, 14544-14547). Unlabeled carbonmonoxy-Hb A was incubated with trace amounts of preparatively purified, native, 3H-alpha subunits in 10 mM sodium phosphate, pH 7.0, at 25 degrees C. At appropriate times, free alpha monomers were separated from Hb A tetramers by anion exchange high performance liquid chromatography. Transfer of radioactivity from the alpha chain pool into Hb A was measured, yielding a first order dimer dissociation rate constant, k2 = (3.2 +/- 0.3) X 10(-3) h-1. The Arrhenius plot of k2 was linear between 7 and 37 degrees C, yielding an enthalpy of activation of 23 kcal/alpha beta dimer. As the chloride concentration was raised from 0 to 0.2 M, the dissociation rate increased 3-fold; with higher salt concentrations, however, the rate gradually returned to baseline. This rate was not altered by raising the pH from 6.5 to 7.2, but as pH was further raised to 8.4, kappa 2 increased about 3-fold. Hb F, which has an increased stability at alkaline pH, dissociated into alpha and gamma monomers 3 times more slowly than Hb A. Moreover, the dimer-monomer dissociation of Hb F was characterized by a significantly reduced pH dependence. These results demonstrate that both alpha beta and alpha gamma dimers of Hb A and Hb F dissociate reversibly into monomers under physiologic conditions. The differential pH dependence for dimer dissociation between Hb A and Hb F suggests that specific amino acid replacement at the alpha 1 gamma 1 interface confers increased resistance to alkaline denaturation.     

Stability of quaternary structure and mode of dissociation of fructosediphosphate aldolase isoenzymes

Using a highly sensitive "subunit exchange" assay, we have studied the relative strengths of interactions between different subunit types (A and C) of fructosediphosphate aldolase and have determined the mode of dissociation of aldolase tetramers in vitro. Interactions between C subunits within C4 tetramers were found to be considerably more resistant to disruption than were interactions between A subunits in A4 tetramers with regard to increasing concentrations of H+, OH-, or urea. Slight dissociation of A4 was also observed in 1.2 M magnesium chloride. These observations suggest that the quaternary structure of aldolase C4 is inherently more stable than that of aldolase A4. Also, the symmetrical heterotetramer A2C2 was found to be more resistant to urea-mediated dissociation than was the aldolase A4 homotetramer; this observation suggests that, even when in heteromeric combination, C subunits have a stabilizing influence on the quaternary structure of aldolase tetramers. In no case did we find evidence for a stable dimeric intermediate in the dissociation of aldolase tetramers to monomers. These observations are considered in terms of the tetrahedral arrangement of subunits in the aldolase tetramer. The general applicability of the subunit exchange assay described here for studying the subunit structure and mode of dissociation of oligomeric enzymes is discussed.     

Structure-function relationship of mitochondrial malate dehydrogenase at high dilution and in multicomponent systems

The structure-function relationship of mitochondrial malate dehydrogenase was investigated at low enzyme concentration, as well as in the presence of polyethylene glycol (PEG 6000) and structure making ions. Previous reports claimed the dimeric enzyme to undergo dissociation in dilute solution, and PEG-induced pairing of dimers in the crystalline state. Sedimentation analysis and gel filtration in 0.1 M sodium phosphate pH 7.6 plus 1 mM EDTA and 1 mM dithioerythritol prove the enzyme to be a stable dimer at c greater than or equal to 0.2 microgram/ml (5 nM). In the presence of 8-20% (w/v) PEG 6000, association of the dimer to tetramers and higher aggregates is observed. At 20% (w/v) polyethylene glycol, ultracentrifugal analysis yields up to 50% tetramers; chemical cross-linking by glutaraldehyde confirms the association in a qualitative way. The enzymatic properties of mMDH (specific activity, Km for oxaloacetate and NADH) in the absence and in the presence of PEG 6000 are indistinguishable. At high polyethylene glycol concentrations (greater than or equal to 20%), the thermal stability of the enzyme is found to be increased. The fluorescence emission, as well as the far-UV and near-UV circular dichroism remain unaffected. Accumulated evidence from equilibrium experiments at low enzyme concentration and reconstitution kinetics (after dissociation at acid pH) proves the active species of mMDH to be the dimer.     

Mechanism of actin filament nucleation

We used computational methods to analyze the mechanism of actin filament nucleation. We assumed a pathway where monomers form dimers, trimers, and tetramers that then elongate to form filaments but also considered other pathways. We aimed to identify the rate constants for these reactions that best fit experimental measurements of polymerization time courses. The analysis showed that the formation of dimers and trimers is unfavorable because the association reactions are orders of magnitude slower than estimated in previous work rather than because of rapid dissociation of dimers and trimers. The 95% confidence intervals calculated for the four rate constants spanned no more than one order of magnitude. Slow nucleation reactions are consistent with published high-resolution structures of actin filaments and molecular dynamics simulations of filament ends. One explanation for slow dimer formation, which we support with computational analysis, is that actin monomers are in a conformational equilibrium with a dominant conformation that cannot participate in the nucleation steps.     

Immobilized hybrids of glyceraldehyde-3-phosphate dehydrogenase.

Yeast glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) immobilized on CNBr-activated Sepharose 4-B has been subjected to dissociation to obtain matrix-bound dimeric species of the enzyme. Hybridization was then performed using soluble glyceraldehyde-3-phosphate dehydrogenase isolated from rat skeletal muscle. Immobilized hybrid tetramers thus obtained were demonstrated to exhibit two distinct pH-optima of activity characteristic of the yeast and muscle enzymes, respectively. The results indicate that under appropriate conditions the activity of each of the dimers composing the immobilized hybrid tetramer can be studied separately.