Role of beta 112 Cys (G14) in Homo- (beta 4) and Hetero- (alpha 2beta 2) Tetramer Hemoglobin Formation*

Takamasa YamaguchiDagger , Jian PangDagger , Konda S. Reddy§, Saul Surrey, and Kazuhiko AdachiDagger parallel

From the Dagger  Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, the § Department of Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and the  Departments of Pediatrics and Research, the duPont Hospital for Children, Wilmington, Delaware, 19803 and the Department of Pediatrics, Jefferson Medical College, Philadelphia, Pennsylvania 19107

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In order to assess the role of beta 112 Cys in homo- and hetero-tetrameric hemoglobin formation, we expressed four beta 112 variants (beta 112Cysright-arrow Asp, beta 112Cysright-arrow Ser, beta 112Cysright-arrow Thr, and beta 112Cysright-arrow Val) and studied assembly with alpha  chains in vitro. beta 112 Cys is normally present at beta 1beta 2 and alpha 1beta 1 interaction sites in homo- (beta 4) and hetero-tetramers (alpha 2beta 2). beta 4 formation in vitro was influenced by the amino acid at beta 112. beta 112 Asp completely inhibited formation of homo-tetramers, whereas beta 112 Ser showed only slight inhibition. In contrast, beta 112 Thr or Val enhanced homo-tetramer formation compared with beta A chains. Association constants for homo-tetramer formation increased in the order of beta 112Cysright-arrow Ser, beta A, beta 112Cysright-arrow Thr, and beta 112Cysright-arrow Val, whereas the value for beta 112Cysright-arrow Asp was zero under the same conditions. These beta 112 changes also affected in vitro alpha 2beta 2 hetero-tetramer formation. Order of alpha 2beta 2 formation under limiting alpha -globin chain conditions showed Hb beta C112S > Hb A > Hb S = Hb beta C112T = Hb beta C112V >>> Hb beta C112D. Hb beta 112D can form tetrameric hemoglobin, but this beta 112 change promotes dissociation into alpha  and beta  chains instead of alpha beta dimer formation upon dilution. These results indicate that amino acids at alpha 1beta 1 interaction sites such as beta 112 on the G helix play a key role in stable alpha beta dimer formation. Our findings suggest, in addition to electrostatic interaction between alpha  and beta  chains, that dissociation of beta 4 homo-tetramers to monomers and hydrophobic interactions of the beta 112 amino acid with alpha  chains governs stable alpha 1beta 1 interactions, which then results in formation of functional hemoglobin tetramers. Information gained from these studies should increase our understanding of the mechanism of assembly of multi-subunit proteins.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Equimolar amounts of alpha - and beta -globin chains of human hemoglobin self assemble to form alpha 2beta 2 tetramer. In addition, isolated beta  chains also assemble to form beta 4 homo-tetramers (1). Extensive previous studies using naturally occurring variants and our recent studies using recombinant beta  chain variants showed that affinity between alpha  and beta  chains is promoted by negatively charged beta  chains and is independent of charge location on the surface except at the alpha 1beta 1 interaction site (2-5). Our previous studies showed that affinity is promoted by negatively charged beta  chains up to a maximum of two additional net negative charges (5). In addition, we showed that beta 112 Cys located at an alpha 1beta 1 interaction site on the G helix is critical for facilitating formation of stable alpha beta dimers which then form functional hemoglobin tetramers. We also demonstrated that beta 112Cysright-arrow Asp inhibits formation of stable alpha 1beta 1 and beta 1beta 2 interactions in alpha 2beta 2 and beta 4 tetramers, respectively (5).

X-ray diffraction analysis of beta 4 and alpha 2beta 2 showed that beta 112 Cys on the G helix is involved in beta 1beta 2 and alpha 1beta 1 subunit interactions, respectively, and that there is similarity between the quaternary structure of carbonmonoxy beta 4 and carbonmonoxy alpha 2beta 2 tetramers (6). The detailed role of alpha 1beta 1 interaction sites involving beta 112 Cys (G 14) in alpha 2beta 2 and beta 4 tetramer formation is, however, not clear. Assembly of hemoglobin tetramers at least in vitro occurs as shown in Reactions


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Reactions I.
I. (7).

The alpha  chains are in monomer-dimer equilibrium and dissociation into monomers is favored, whereas the beta  chains are in monomer-tetramer equilibrium and association into tetramers is favored. It is generally assumed that dissociation of these oligomeric subunits (Reactions I and II) into monomers must occur before they can combine to form the alpha beta dimers (Reaction III). Two alpha beta dimers then associate to form tetrameric hemoglobin (Reaction IV). The dissociation of oligomeric beta  subunits is a first-order reaction, while assembly of alpha beta dimers from alpha  and beta  monomers (Reaction III) is a second-order reaction (2, 4, 7). Formation of functional hemoglobin tetramers is dependent on these reactions (7). In vitro assembly of the liganded forms of alpha - and beta -globin chains show that the rate of dissociation of beta 4 homo-tetramers is a rate-limiting step in formation of alpha beta hetero-tetramers (2). Therefore, clarification of the role of beta 112 Cys at the alpha 1beta 1 and beta 1beta 2 interaction site on alpha beta and beta 4 formation, respectively, is critical in order to understand the mechanism of hemoglobin assembly. In this report, we expressed four beta 112 variants and characterized effects of these changes on alpha 2beta 2 and beta 4 tetramer formation in vitro in order to further assess the role of beta 112 Cys on assembly of hemoglobin.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of Soluble Recombinant Human beta -Globin Chain Variants in Escherichia coli-- Four beta 112 globin chain variants (e.g. beta 112 Ser, beta 112 Val, beta 112 Thr, and beta 112 Asp) were constructed and expressed using the pHE2beta plasmid vector that contains cDNAs coding for each beta  chain variant and methionine aminopeptidase which was originally developed to express alpha  and beta  chains at the same time (8, 9). The basic strategy for generation of these variants by site-specific mutagenesis of the normal beta  chain involves recombination/polymerase chain reaction as described previously (10). Clones were subjected to DNA sequence analysis of the entire beta -globin cDNA region using site-specific primers and fluorescently tagged terminators in a cycle sequencing reaction in which extension products were analyzed on an automated DNA sequencer. Plasmids were transfected into E. coli (JM 109) (Promega Co.), bacteria were grown at 30 °C, and soluble beta -globin chain variants were isolated as described (5, 8). Expression and purification of beta -globin variants were basically as described previously (5, 8). However, cation-exchange chromatography on a Source 15 S column (Amersham Pharmacia Biotech) instead of Superose 12 gel filtration was used for purification of the beta  chain variants. Authentic human hemoglobin, alpha -, beta A (beta 6 Glu)- and beta S (beta 6 Val)-globin chains were purified from erythrocyte lysates from normal controls and patients with sickle cell disease, respectively, according to previously described methods (11). Removal of p-mercuribenzoate from beta  chains was accomplished using 20 mM dithiothreitol (DTT),1 and globin chains were isolated using gel filtration on a Superose 12 column for the final purification step.

Biochemical Characterization of Purified beta -Globin Chains-- Molecular mass and sample purity were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described (12). Mutations in each purified beta -globin chain variant were confirmed using mass spectral analysis. Electrospray ionization mass spectrometry was performed on a VG BioQ triple quadrapole mass spectrometer (Micromass, Altrincham, UK) using the multiple charged ion peaks from the alpha -globin chain (Mr = 15,126.4) as reference for mass scale calibrations (13). Data analysis employed the MassLynx® software package (Micromass).

Purified beta -globin chains were also analyzed by cellulose acetate electrophoresis, and mobilities were compared with those of authentic human globin chains. Isoelectric focusing of purified beta -globin variants, Hb A and Hb S, was performed on Ampholine PAG plates (Amersham Pharmacia Biotech), pH 5.5-8.5, using a Multiphor II system (Amersham Pharmacia Biotech). After focusing for 2 h at a constant 25 watts at 4 °C, the gel plate was cut in half, one portion was stained using the JB-2 staining system (Isolab inc., Akron, Ohio) to detect heme proteins, and the other half was stained with Coomassie Brilliant Blue R-250 (Sigma) to detect protein. Isoelectric point of each beta -globin variant was estimated using a calibration curve prepared with isoelectric focusing standards (Bio-Rad).

Absorption spectra of purified beta -globins in the CO form were recorded using a Hitachi U-2000 spectrophotometer (Hitachi Instruments, Inc. Danbury, CT). Globin concentration was determined spectrophotometrically using a millimolar extinction coefficient of 13.4 at 540 nm for carbonmonoxy hemoglobin (14). Circular dichroism (CD) spectra of beta -globin variants were recorded using an Aviv model 62 DS instrument (Varian Analytical Instruments, San Fernando, CA) employing a 0.1-cm light path cuvette at 10 µM globin concentration. CD ellipticity of beta -globin variants compared with normal beta A was monitored between 190 and 260 nm. Oxygen dissociation curves of hemoglobin tetramers were determined in 50 mM Bis-Tris buffer containing 0.1 M NaCl and 5 mM EDTA, pH 7.2, at 20 °C using a Hemox Analyzer (TCS Medical Products, Huntingdon Valley, PA) (15).

Dissociation of beta -globin homo-tetramers was studied by fast protein liquid chromatography (FPLC) on a Superose 12 gel filtration column. beta -Globin was mixed with blue dextran and vitamin B12, internal markers for determination of void volume (V0) and total column volume (Vt), respectively. The final beta -globin solution (1.5 to 75 µM) was injected into a Superose 12 column, and gel filtration was done using 0.1 M potassium phosphate buffer, pH 7.0 at 4 °C. Elution coefficients were calculated using the following equation, where Ve represents elution volume of each sample.
K<SUB><UP>av</UP></SUB>=(V<SUB>e</SUB>−V<SUB>0</SUB>)/(V<SUB>t</SUB>−V<SUB>0</SUB>) (Eq. 1)

alpha 2beta 2 tetramer formation was assessed by mixing purified beta  chain variants (75 µ M) with varying amounts of alpha -globin chain in the CO form in 0.1 M potassium phosphate buffer, pH 7.0 at 25 °C (2, 5), and tetramer formation was monitored by FPLC using a Mono-S column. Assembled tetramers were characterized after separation from excess, free globin chains by FPLC using a Source 15 S column. Dissociation of in vitro assembled alpha 2beta 2 tetramer into subunits was assessed at a flow rate of 0.5 ml/min at 4 °C by large zone chromatography using a Superose 12 gel filtration column (1 × 31 cm) as described previously (16, 17). Sample volumes of alpha 2beta 2 variant tetramers (25 ml in 0.1 M potassium phosphate buffer, pH 7.0) were equal to the column bed volumes. Elution was monitored at 405 nm using a 5-mm flow cell. Elution volume (Ve) was measured at the centroid of the ascending leading boundary (16, 17). Dissociation constants of alpha 2beta 2 tetramers to alpha beta dimers were estimated by evaluation of the change in Ve as a function of hemoglobin concentration (17, 18).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression and Purification of Soluble beta -Globin Chain Variants-- We previously reported expression of beta 112Cysright-arrow Asp chains in bacteria (5). In order to further define the role of beta 112 Cys in alpha 2beta 2 and beta 4 tetramer formation, three additional beta -globin chain variants (e.g. beta 112Cysright-arrow Ser, beta 112Cysright-arrow Thr, and beta 112Cysright-arrow Val) were expressed and characterized. Ser is similar in size to Cys, whereas Thr and Val are slightly larger. In addition, Ser and Thr are hydrophilic like Cys, whereas Val is highly hydrophobic. After DNA sequence confirmation, the three beta -globin chain variants were expressed in bacteria and purified by a combination of two anion-exchange chromatography steps using DEAE-cellulose and Mono-Q columns (9). Further purification was achieved using cation-exchange chromatography on a Source 15 S column. Purified beta A chains migrate predominantly as 32-kDa dimers (9), whereas the four beta 112 variants migrate mainly as 16-kDa monomers with small traces of dimers on SDS-PAGE (Fig. 1). Addition of 20 mM DTT to the chains in solution prior to SDS-PAGE converts the dimers to monomers, indicating that small amounts of beta 93 Cys oxidize and form disulfide-linked dimers.


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Fig. 1.   SDS-PAGE of beta 112 variants. Purified preparations of each beta 112 variant (~2 µg) were subjected to SDS-PAGE following incubation with (+) or without (-) 200 mM DTT for 30 min at 25 °C. Samples were heated in 3% (w/v) SDS in a boiling water bath for 3 min and applied to a 15% (w/v) polyacrylamide gel, and electrophoresis was performed at a constant voltage of 100 V. The gel was stained with Coomassie Brilliant Blue R-250 to detect proteins. A, molecular weight standards; B, beta A chains (expressed in bacteria (5)); C, beta 112Cysright-arrow Asp chains; D, beta 112Cysright-arrow Ser chains; E, beta 112Cysright-arrow Thr chains; and F, beta 112Cysright-arrow Val chains.

Mass spectral analysis of the four beta  chain variants using electrospray ionization mass spectrometry resulted in values of 15,878.9, 15,850.3, 15,865.8, and 15,863.5 for beta 112Asp, beta 112Ser, beta 112Thr, and beta 112Val, respectively, which were in agreement with expected masses for the variant beta -globin chains. Carbonmonoxy forms of all four variants showed typical absorption spectral characteristics of human hemoglobin chains with peaks at 568, 540, 419, 344, and 276 nm (14), indicating correct heme insertion into the variant beta -globin chains. CD spectra were also measured to examine structures of each beta -globin variant compared with beta A-globin chains (Fig. 2). Ellipticities between 190 and 260 nm for the beta -globin variants were almost identical to that of normal human and recombinant beta -globin, but there are small differences in the region of the peak around 213 nm that are likely to be characteristic of these different beta  chains. Interestingly, the region of the peak of the spectrum for beta 112Val chains around 213 nm was slightly left shifted compared with those of beta A, beta 112Asp, and beta 112Ser chains (Fig. 2). These CD results indicate that beta -globin chain variants made in the E. coli were properly folded and had secondary structures similar to authentic beta -globin. In addition, the differences in the spectrum for the beta 112Val chains may be a reflection of increased conformational changes.


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Fig. 2.   Circular dichroism spectra of purified beta -globin chain variants. CD spectra of beta A globin purified from human red cells (4) compared with those of the four beta 112 variants (beta 112Cysright-arrow Asp (3), beta 112Cysright-arrow Ser (5), beta 112Cysright-arrow Thr (2), and beta 112Cysright-arrow Val (1)). All chains were in the CO form in 0.01 M phosphate buffer, pH 7.0, at 10 °C. Inset represents CD spectra between 205 and 230 nm of each chain normalized to values at 222 nm after subtraction of the buffer spectrum.

Characterization of Purified beta -Globin Chain Variants-- We previously reported that electrophoretic mobility on cellulose acetate of beta 112Cysright-arrow Asp chains was identical to beta s chains (5), indicating that these chains migrate as monomers (charge of -2) rather than tetramers (charge of -4) like beta 4A chains (Fig. 3). In contrast, mobility of beta 112Cysright-arrow Val and beta 112Cysright-arrow Thr chains was identical to authentic human beta A. These results indicate that conversion from Cys to Val or Thr at beta 112 does not cause significant changes in surface charge while conversion to Asp appears to increase net positive charge of the beta -globin molecule because formation of monomers is favored rather than tetramers. Effect of beta 112 amino acid on beta -globin charge was studied further by isoelectric focusing on polyacrylamide gel plates. beta 112Cysright-arrow Ser and beta 112Cysright-arrow Asp chains focused as sharp bands with pI values of 6.68 and 6.35, respectively, whereas beta 112Cysright-arrow Val and beta 112Cysright-arrow Thr chains had pI values of 6.30 and 6.68, respectively, but focused as diffuse bands possibly because they exist as mixtures of beta chain monomer, dimer, and tetramer during electrophoresis (19).


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Fig. 3.   Cellulose acetate electrophoresis of purified beta -globin chain variants and their corresponding alpha 2beta 2 tetramers. Purified beta -globin variants (A) and their corresponding in vitro assembled alpha 2beta 2 tetramers (B) were analyzed by electrophoresis on cellulose acetate membranes. A, lanes 1 and 2, beta A and beta S globins, respectively (purified from human red blood cells); lane 3, beta 112Cysright-arrow Val chains; lane 4, beta 112Cysright-arrow Thr chains; lane 5, beta 112Cysright-arrow Ser chains; lane 6, beta 112Cysright-arrow Asp chains. B, lane 1, Hb A; lane 2, Hb S; lane 3, Hb beta C112V; lane 4, Hb beta C112T; lane 5, Hb beta C112S; and, lane 6, Hb beta C112D.

beta 4 tetramer and beta -monomer levels for each of the beta  chain variants were assessed by size-exclusion chromatography (17, 20, 21). beta A-Globin chains in solution exist as homo-tetramers (beta 4) rather than monomers in the absence of alpha -globin chains (1). In contrast, we found previously that beta 112Cysright-arrow Asp chains exist as monomers rather than beta 4 tetramers (5). In order to further characterize effects of the different beta 112 amino acids on tetramer formation, each beta -globin variant was chromatographed on a Superose 12 gel-filtration column equilibrated with 0.1 M phosphate buffer, pH 7.0, at 4 OC in order to separate tetrameric and monomeric species. The gel-filtration pattern of beta A globin, which contains beta 112 Cys, depended on concentration and temperature. At 75 µM, beta A chains eluted mainly as tetramers (86%) with a minor shoulder of monomers (14%) (Fig. 4a). The patterns of the variants depended on the beta 112 amino acid. When the same concentration as beta A globin for each beta  chain variant was applied to the column, results showed that the beta 112Cysright-arrow Ser variant contained 51% tetramer and 49% monomer (Fig. 4c), whereas the beta 112Cysright-arrow Thr variant contained a slightly higher percentage of tetramer than that of beta A (Fig. 4d). The beta 112Cysright-arrow Val chains eluted only as tetramers when the concentration was 75 µM. Dilution of samples of the other beta  chain variants and beta A chains decreased the amounts of tetramer while monomer amounts increased. In contrast, beta 112Cysright-arrow Asp chains eluted only as monomers at all hemoglobin concentrations used. The ratio of tetramer/monomer decreased as a function of beta 112 amino acid in the following order: beta 112Cysright-arrow Val > beta 112Cysright-arrow Thr > beta A(112Cys) > beta 112Cysright-arrow Ser > beta 112Cysright-arrow Asp. Values for beta 112Cysright-arrow Thr and beta 112Cysright-arrow Val were 2- and 600-fold, respectively, higher than that for beta A(112Cys) chains. Association constants for beta 4 homo-tetramer formation for the beta 112 variants was calculated using results from gel-filtration chromatography and are summarized in Table I. The beta A-globin chain value (6 × 1016 M-3) was similar to results reported previously (22). The association constant for the beta 112Cysright-arrow Asp variant was 0 since this variant existed only as a monomer. It is interesting to note that dissociation of beta S chains to monomers with decreasing concentrations was significantly less compared with that of beta A. The tetramer association constant for beta S chain monomers was about 30-fold higher than that of beta A, indicating that the beta 6 Val mutation in beta S also affects tetramer-monomer equilibrium and promotes homo-tetramer assembly.


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Fig. 4.   Superose 12 gel-filtration chromatogram of purified beta -globin chains. Purified beta -globin variants were mixed with internal markers (blue dextran for identification of void volume (V0) and vitamin B12 for total bed volume (Vt)) in 150 µl (final globin concentration = 75 µM) and applied to a Superose 12 column (1 × 30 cm). Gel filtration was accomplished at a flow rate of 0.5 ml/min using 100 mM potassium phosphate buffer, pH 7.0. a, beta A chains; b, beta S chains; c, beta 112Cysright-arrow Ser chains; d, beta 112Cysright-arrow Thr chains; e, beta 112Cysright-arrow Asp chains; and f, beta 112Cysright-arrow Val chains.

                              
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Table I
Homo-tetramer/monomer association constants of purified beta -globin variants
Association constants were calculated from monomer/tetramer ratios according to the equation, K = [beta 4]/[beta ]4 (20). Values were calculated from three to five independent experiments and represent the mean ± S.E.

Hetero-tetramer Formation (alpha 2beta 2) in Vitro Using beta -Globin Variants and Native alpha -Globin-- beta -Globin readily forms hetero-tetramers (alpha 2beta 2) in vitro in the presence of alpha -globin. Tetramers were formed in vitro, purified and separated from unreacted globins by chromatography, and then were analyzed by cellulose acetate electrophoresis (Fig. 3B). We previously reported that beta 112 Asp chains did not form beta 4 tetramers but did form alpha 2beta 2112Asp tetramers. All three new recombinant beta -globin variants formed alpha 2beta 2 tetramers in vitro. Cellulose acetate electrophoresis showed cathodic shifts in migration of tetramers compared with beta  chain variants upon binding of alpha -globin chains. It is interesting to note that, although individual beta -globin chain variants migrated differently, all alpha 2beta 2 tetramers containing the different beta 112 variants co-migrated to the same position as that of Hb A (Fig. 3B).

We also performed functional studies of the variant tetramers in 50 mM BisTris buffer, pH 7.2, containing 0.1 M NaCl at 20 °C in the presence and absence of 2,3-biphosphoglycerate and compared results with those of Hb A. Results of oxygen-binding properties for the beta 112 variant alpha 2beta 2 tetramers are summarized in Table II. Hb beta C112S, Hb beta C112T and Hb beta C112V exhibited similar oxygen affinities and n values to those of Hb A tetramers. Oxygen affinity of recombinant Hb A was previously reported to be the same as that of normal human Hb A (5, 8), while oxygen affinity of Hb beta C112D (P50 = 2.2) was higher than that of Hb A with a similar cooperativity.

                              
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Table II
Oxygen-binding properties of beta 112-variant hemoglobin tetramers
Oxygen equilibrium curves of hemoglobins were determined using 35 µM Hb concentration in 50 mM BisTris/HCl buffer, pH 7.2, containing 100 mM NaCl and 5 mM EDTA at 20 °C. P50 is partial oxygen pressure required to give 50% oxygen saturation of hemoglobin. nmax values were calculated from the Hill plot of oxygen-equilibrium curves. Concentration of 2,3-biphosphoglycerate (BPG) when present (+) is 2 mM. Mean P50 values (n = 5) for Hb A with and without BPG are shown with a mean ± S.E. of 0.1 and 0.3, respectively. K4,2 values for Hb A, Hb beta C112T, and Hb beta C112V were calculated as shown in Fig. 6. However, the K4,2 values for Hb beta C112T and Hb beta C112V were not calculated since these two tetramers dissociated directly to monomers instead of dimers upon dilution as shown in Fig. 6.

Tetramer Formation in Vitro with Limiting Amounts of alpha  Chains-- Competition for alpha 2beta 2 hetero-tetramer formation between two different beta -globin chains can be assessed by monitoring tetramer assembly in the presence of limiting amounts of alpha  chains using 1:1 mixtures of the two different beta -globin chains (3, 5). Previous studies in vitro showed that variant hemoglobin percentages were higher when using more negatively charged beta  chains like J-Baltimore (beta 16Glyright-arrow Asp) and N-Baltimore (beta 95Lysright-arrow Glu) (4, 5). These studies suggested that more negatively charged beta  chains bind positively charged alpha  chains more readily than beta A chains (2-5). Furthermore, Hb A tetramers formed twice as readily as Hb S, suggesting that beta A chains interact with alpha  chain more readily than beta S under limiting alpha -globin conditions and that this was due to the positive net charge of Val instead of Glu at the beta 6 position (3-5). In addition, our previous subunit competition studies with equimolar mixtures of beta S and beta 112Cysright-arrow Asp chains in the presence of limiting alpha  chains suggested that beta 112Cys (G14) is a key amino acid in formation of stable alpha beta dimers (5). The beta 112 position is known to be localized at the alpha beta subunit interface after hetero-tetramers assemble (6).

In order to further clarify the effect of beta 112 amino acid side chain on assembly, tetramer formation in vitro was evaluated for each of the purified beta -globin variants after addition of alpha -globin chain isolated from human red blood cells. Competition experiments were done in which varying amounts of alpha  chains were added to equimolar mixtures of beta S and either beta 112Cysright-arrow Val, beta 112Cysright-arrow Thr, beta 112Cysright-arrow Ser, or beta A chains, and assembled tetramers were separated from free chains by ion-exchange chromatography (Fig. 5). Our previous competition experiments in vitro using mixtures of purified alpha  and beta  chains showed that alpha beta A dimers form about twice as readily as alpha beta s dimers when the concentration of alpha  chains becomes limiting (5). This results in assembly of less Hb S relative to Hb A when equimolar amounts of beta A and beta S chains compete for limiting amounts of alpha -globin (Fig. 5). Similar amounts of variant tetramers (Hb beta C112T and Hb beta C112V) and Hb S were formed in mixtures at all alpha -globin concentrations tested. These results indicate that levels of Hb beta C112T and Hb beta C112V would be less than that of Hb A in competition studies with the beta A chain. Total amounts of Hb beta C112S were always more than that of Hb S, with the Hb beta C112S/Hb S ratio approaching 3.5 when alpha chain/total beta  chain approaches 0.1. On the other hand, as shown previously, much less Hb beta C112D formed in mixtures of beta 112Cysright-arrow Asp and beta S, and almost all alpha -globin chains assembled with beta S chains to form Hb S, when the ratio of alpha /total beta  chains was < 0.5 (5).


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Fig. 5.   Effect of beta 112 amino acid on relative amounts of in vitro assembled tetramers as a function of varying amounts of input alpha -globin. Equimolar mixtures (75 µM) of the beta  chain variant (beta X) and beta S were added to varying amounts of alpha -globin chain in 0.1 M potassium phosphate buffer, pH 7.0, at 25 °C, and assembled tetramers were analyzed by FPLC. The relative ratio (y axis) is shown for Hb A to Hb S (open circle ) in (beta A + beta S)/alpha mixtures, Hb beta C112D to Hb S (square ) in (beta S + beta 112Cysright-arrow Asp)/alpha mixtures, Hb beta C112S to Hb S (black-triangle) in (beta S + beta 112Cysright-arrow Ser)/alpha mixtures, Hb beta C112T to Hb S (triangle ) in (beta S + beta 112Cysright-arrow Thr)/alpha mixtures, and Hb beta C112V to Hb S (black-square) in (beta S + beta 112Cysright-arrow Val)/alpha mixtures. Each value represents the mean ± S.E. from three-five independent experiments.

Effect of beta 112 Variants on Dimer (alpha beta ) and Tetramer (alpha 2beta 2) Equilibrium-- In order to evaluate effects of beta 112 amino acid on dimer-tetramer equilibrium, large-zone gel-filtration chromatography of beta 112-variant alpha 2beta 2 tetramers was performed using a Superose 12 HR column at various hemoglobin concentrations (17). Decreasing concentrations of alpha 2beta 2 variant tetramers in the CO form (25 ml in 0.1 M potassium phosphate buffer, pH 7.0), whose volumes were equal to the column bed volume, were loaded on the column in order to prevent dilution of samples during chromatography. Elution profiles of hemoglobin variants and Hb A exhibited sharp leading boundaries which then plateaued at different levels depending on the concentration applied. The elution volume (Ve), assessed by the centride of the leading boundary, depends on hemoglobin concentration below 1.3 µM; the lower the concentration then the higher the Ve. Ve values were constant above 1.3 µM hemoglobin, indicating most molecules exist as tetramers (Fig. 6). With decreasing concentrations of Hb A, the Ve values approached 15.5 ml, which corresponds to the value for alpha beta dimers. A plot of Ve as a function of hemoglobin concentration showed similar curves for Hb beta C112T and Hb A, whereas the curve for Hb beta C112V was slightly shifted to the left. These findings suggest that beta 112 Cysright-arrowVal inhibits tetramer dissociation. The tetramer-dimer dissociation constants for Hb A, Hb beta C112T and Hb beta C112V were calculated as described previously (23) and were 0.11, 0.12, and 0.07 µM, respectively (Table I). In contrast, the plots of elution volume as a function of concentration for Hb beta C112S and Hb beta C112D were right shifted from that of Hb A (Fig. 6). Furthermore, Ve values for Hb beta C112S and Hb beta C112D below 0.1 µM were greater than that for alpha beta dimers and approached 16.25 ml at lower concentrations, which corresponds to the Ve value for monomeric forms determined using beta 112Cysright-arrow Asp chains. The plot for Hb beta C112D was more right shifted than that for Hb beta C112S (Fig. 6). These results indicate that tetrameric forms of Hb beta C112S and Hb beta C112D continue to dissociate to alpha  and beta  monomers upon dilution rather than remaining as alpha beta dimers and that Hb beta C112D dissociates more than Hb beta C112S.


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Fig. 6.   Effect of beta 112 amino acid on dissociation of alpha 2beta 2 tetramers. Varying concentrations ranging from 0.01 to 5 µM hemoglobin tetramer in 25 ml of 0.1 M potassium phosphate buffer, pH 7.0, were loaded onto a Superose 12 column, and gel-filtration chromatography was performed at a flow rate of 0.5 ml/min in the same buffer at 4 °C. A, elution position (Ve) was plotted against hemoglobin concentration, and Ve of globin monomers was determined using beta 112Cysright-arrow Asp chains. Samples included Hb A from human red blood cells (open circle ) and in vitro assembled beta 112 variant alpha 2beta 2 tetramers (Hb beta C112D (square ), Hb beta C112S (black-triangle), Hb beta 112T (triangle ), and Hb beta C112V (black-square)). B, K4,2 values of Hb A (a), Hb beta 112V (b), and Hb beta C112T (c) were calculated from the results of Fig. 6A. K4,2 = [D]2/[T] is the value when log (1 -alpha ) = 2 log alpha  , where alpha  is the fraction of dimers. D (dimer) and T (tetramer) fractions were estimated from the results in Fig. 6A. K4,2 values of Hb beta C112D and Hb beta C112S could not be calculated since these tetramers dissociate directly to monomers upon dilution.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of beta 112 Amino Acids on Homo-tetramer Assembly-- X-ray analysis of beta 4 homo-tetramers at 1.8 Å resolution showed that beta 112 Cys (G14) is located at the beta 1 and beta 2 chain interface and that the side chains of beta 1112 Cys and beta 2112 Cys in the beta 4 tetramer are very close to the molecular dyad at this interface (6). These two residues exist on the surface of the beta  chains and may be involved in weak interactions with other residues. Our present results showing absence or trace amounts of disulfide dimer formation for all four of the beta 112 chain variants in contrast to beta A and other beta  chain variants, suggest that beta 1112 Cys and beta 2112 Cys are physically close together and that disulfide beta  chain dimer formation is governed in part by these two cysteine residues. Our results also show that beta 112 Val chains exist mainly as beta 4 tetramers in solution, which is in contrast to the previous results with beta 1112 Asp chains that exist as monomers (5). In addition, beta 112 Thr and beta 112 Ser chains can form beta 4 homo-tetramers, whose levels are intermediate between those of beta 112 Val and beta 112 Asp. Relative order of tetramer formation among the different beta 112 variants is a direct function of hydrophobicity of the beta 112 amino acid, the higher the hydrophobicity then the more tetramers are formed. These results suggest that beta 1 and beta 2 interactions in homo-tetramers are governed to a large extent by hydrophobic interactions involving the beta 112 amino acid and that hydrophobic interactions between beta 1112 Val and beta 2112 Val are strongest followed by beta 1112 Thr-beta 2112 Thr, beta 1112 Cys-beta 2112 Cys, and then beta 1112 Ser-beta 2112 Ser. In contrast, beta 112 Asp chains exist as monomers since beta 1112 Asp and beta 1112 Asp cannot participate in hydrophobic interactions, which results in lack of formation of homo-tetramers. The ellipticity values for the CD spectra in the far ultraviolet region (190-280 nm) are expected not to be dependent on the heme moiety but on overall protein conformation (24). The spectral difference in beta 112 Val compared with those of beta A- and other beta  chain variants may be related to the high homo-tetramer formation of the beta 112 Val variant. Detailed studies of the conformational change differences between homo-tetramer and monomer forms are now in progress.

Effects of beta 112 Amino Acid on Hetero-tetramer Assembly-- beta 112 Cys is located at the interface of alpha 1beta 1 in alpha 2beta 2 hemoglobin tetramers and interacts with Val alpha 107 (G14) and Ala alpha 110 (G11), which are critical for stabilization of the alpha beta interface (6). Amino acid changes at beta 112 also affected in vitro assembly of alpha 2beta 2 variant tetramers. Order of alpha 2beta 2 formation under limiting alpha -globin chain conditions showed Hb beta C112S > Hb A > Hb S = Hb beta C112T = Hb beta C112V >>> Hb beta C112D. In addition, apohemoglobin dimers were reported previously to dissociate into monomers 170-fold faster than normal alpha beta dimers because no interactions occur between beta 112Cys and alpha 104 Cys as a result of loss of the heme moiety (25). These findings and our results indicate that relative affinity of alpha  for beta  chains is highly dependent on direct alpha 1 and beta 1 interaction sites even though surface charge of the chains also affects interactions during assembly (2-5). Amino acids at alpha 1beta 1 interaction sites such as beta 112 Cys may be critical for alpha beta assembly.

Although the precise steps in alpha 2beta 2 tetramer assembly in vivo are not completely understood, functionally intact alpha  and beta  chains can be isolated and readily reconstituted into alpha 2beta 2 tetramers by mixing equimolar amounts of alpha  and beta  chains. As noted previously, it is generally assumed that isolated alpha  and beta  chains exist in a monomer-dimer and monomer-tetramer equilibrium at least in vitro, respectively (see Reactions I and II in the Introduction). Dissociation of alpha 2 dimers and beta 4 tetramers follows kinetics of a first-order reaction, while the monomer and dimer combination steps (Reaction III in the Introduction) are second-order processes that depend on protein concentration (7). Interaction of alpha  and beta  chain monomers in Reaction III is very rapid compared with dissociation of beta 4 tetramers to monomers (7). The second-order rate constant of Reaction III for alpha  and beta A (alpha beta k5) is about 105 M-1 s-1 (20, 26), which means that alpha  monomer binds monomeric beta A chain very rapidly, roughly following second-order reaction kinetics. In contrast, dissociation of beta 4right-arrow4beta is very slow exhibiting a dissociation constant (beta k4) of about 10-3 s-1 (20). These results indicate that isolated single alpha  and beta  chains assemble quickly but that formation of tetrameric hemoglobin depends on beta 4 stability and total amounts of monomer in solution. Substitution of beta 112 Cys with Val as well as beta 6 Glu with Val in beta S chains inhibits tetramer dissociation, whereas substitution of beta 112 Cys with Ser or Asp promotes dissociation. When these beta  chain variants competed with beta S chain for assembly with limiting alpha  chains, the order of assembly of the beta 112 variant hetero-tetramers (e.g. Hb beta C112S > Hb A > Hb beta C112V) shown in Fig. 5 implies that beta 112 Val forms a weaker alpha 1beta 1 interface even though the hydrophobic substitution to Val is expected to strengthen hydrophobic interactions with alpha 107 Val (G14) and alpha 110 Ala (G11) (27). These results, however, may be reconciled by the fact that beta 112 Val facilitates increased tetramer stability compared with beta A chains and that dissociation of beta 112 Val homo-tetramers to monomers is much slower than that of beta 112 Cys homo-tetramers. This slow dissociation of beta 112 Val homo-tetramers leads to less monomer formation and therefore less Hb Cbeta 112V compared with Hb A. Furthermore, homo-tetramers containing beta 112 Ser dissociate to monomers more readily than those containing beta 112 Thr. This results in beta 112 Ser promoting alpha 2beta 2 tetrameric formation compared with beta 112 Thr under limiting alpha  chain conditions. In contrast, even though beta 112 Asp promotes formation of monomers, the results of the order of tetramer assembly (e.g. Hb beta C112T = Hb beta C112V >>> Hb beta C112D) indicates that stability of the interaction between alpha  and beta  chains plays a key role in assembly in addition to ability to form monomers. However, when beta 112 Val associates with alpha  chains, this substitution of the strong hydrophobic amino acid Val for Cys at beta 112 could stabilize alpha 1beta 1 interactions due to its ability to exclude water. This would facilitate interactions with the alpha  chain at alpha 107 Val (G14) and alpha 110 Ala (G11) (27). In contrast, a beta 112 Cys substitution to a hydrophilic amino acid like Asp would inhibit this hydrophobic interaction and result in no homo-tetramer formation and weaken alpha beta interactions. This also would destabilize formation of hetero-tetramers. In vivo, hemoglobin synthesis occurs in erythroblasts and reticulocytes (28). Even though the assembly scheme in vitro is clear, it is not clear whether these results in vitro apply to assembly in vivo. The newly synthesized beta  chains in the cytoplasm probably exist at low concentrations (<10 µM), which would favor formation of monomers rather than homo-tetramers (3). However, gel-filtration experiments using beta  chains at 1.5 µM indicate that more than 80% exist as tetramers when analyzing beta S and beta 112 Val chains. The combination of heme and apoglobin is so rapid that newly translated subunits probably bind heme prior to assembly (3, 29). Our present results on production of soluble beta -globin chains in bacteria in the presence of heme but in the absence of alpha -globin also suggest that these conditions in vivo promote correct beta -globin chain folding in the absence of alpha -globin chains. The relevance of beta 4 tetramer formation and dissociation under in vivo conditions in which alpha  and beta  chain synthesis is balanced is not clear. Hb H (beta  chain homo-tetramers) is formed in a severe form of alpha  thalassemia in which patients produce only about one-fourth of the normal amount of alpha  chains. As a result, beta  chain homo-tetramers form in cells. The ease of dissociation of beta  chain homo-tetramers to monomers in addition to direct alpha and beta  interactions may be rate-limiting for assembly in vivo.

Effect of beta 112 Variants on Dimer-tetramer Equilibrium-- Interactions between amino acid residues at the alpha 1beta 1 and alpha 1beta 2 interfaces are different (6, 28, 30). The alpha 1beta 1 interface remains relatively fixed during deoxygenation, and contacts between the alpha 1 and beta 1 subunits are identical in oxyhemoglobin and deoxyhemoglobin. In deoxyhemoglobin, there are about 40 contacts, including 19 hydrogen bonds. In contrast, there is considerable movement at the alpha 1beta 2 interface during oxygenation and deoxygenation. When hemoglobin is oxygenated, the total number of contacts drops to about 23, including 12 hydrogen bonds. In view of the important role that conformational isomerization plays in hemoglobin function, it is not surprising that residues at the alpha 1beta 2 interface are invariant and highly conserved throughout vertebrate evolution (28, 30).

Under physiologic conditions, liganded tetrameric hemoglobins dissociate reversibly into dimers: alpha 2beta 2 right-left-harpoons  2alpha beta . The alpha 1beta 1 interface in alpha beta dimers participates in formation of noncovalent intersubunit bonds that do not dissociate easily in the oxy and deoxy forms. In contrast, the alpha 1beta 2 contacts can dissociate upon oxygenation and exhibit large oxygenation-induced subunit interaction energies (28, 30). The reduction in contacts at the alpha 1beta 2 interface following oxygenation suggests that dissociation occurs there rather than at the alpha 1beta 1 interface. Accordingly, human hemoglobin variants with substitutions at the alpha 1beta 2 interface usually exhibit dramatic alterations in their functional properties, whereas those with substitutions at the alpha 1beta 1 interface do not. In fact, oxygen affinities and cooperativity (n value) of Hb beta C112T, Hb beta C112V, and Hb beta C112S were similar to those of Hb A, whereas oxygen affinity was higher and cooperativity was lower for Hb beta R40S (P50 = 2.2, n = 2.0), Hb beta R40A (P50 = 2.2, n = 1.6), Hb beta E101A (P50 = 3.4, n = 2.2), and Hb beta E101K (P50 = 2.5, n = 2.2), which have substitutions at the alpha 1beta 2 interface compared with Hb A (P50 = 5.1, n = 2.7) (31). Furthermore, our results show that Hb beta C112D (P50 = 2.2, n = 2.6) has similar cooperativity to Hb A and higher oxygen affinity compared with Hb A (P50 = 4.2 ± 0.1, n = 2.7), whereas Hb beta R40D (P50 = 1.2, n = 1.0) and Hb beta E101D (P50 = 0.9, n = 1.9) (31) exhibited lower cooperativity and much more increased oxygen affinities even though Hb beta C112D favors dissociation to dimer and monomer forms compared with Hb beta R40D and Hb beta E101D (31). These results reinforce the contention that allosteric transition between oxy and deoxy forms of tetrameric hemoglobin is influenced by amino acid residues located at the alpha 1beta 2 interaction sites (28, 32, 33).

Interestingly, recent studies showed that beta 112Cysright-arrow Gly chains in tetrameric hemoglobin unexpectedly stabilized alpha 1beta 2 interactions (34). Our studies also showed that beta 112Cysright-arrow Val stabilizes the alpha 1beta 2 interface without significantly changing oxygen affinity. Substitution of Cys at the beta 112 position with Val appears to stabilize alpha 1beta 2 interactions but may not affect local quaternary conformational changes induced during the oxygenation-deoxygenation process. Furthermore, even though the beta 112 Ser variant decreased alpha beta stability by promoting dissociation into monomers, the oxygen affinity of Hb beta C112S was not influenced by this mutation. These findings suggest that local quaternary conformational changes induced during oxygenation/deoxygenation are not influenced by this substitution at an alpha 1beta 1 interaction site. In contrast, the beta 112 Asp variant destabilizes the alpha 1beta 1 interaction sites and slightly reduced oxygen affinity. Furthermore, this substitution also increased dimer dissociation from hetero-tetramers compared with normal Hb A. These results indicate that the beta 112 amino acid plays a critical role in tetramer-dimer stability and suggest that structural changes at the alpha 1beta 1 contact can be propagated through the protein and lead to stability at the alpha 1beta 2 interface. We are now investigating the relationship between mutation at the alpha 1beta 1 interface and dimer-tetramer stability of hemoglobin. These studies should aid in our understanding of hemoglobin assembly as well as in how changes at one site influence tertiary and quaternary structure at a distant location.

    ACKNOWLEDGEMENTS

We thank Dr. Eric Rappaport and members of the Nucleic Acid/Protein Core at the Children's Hospital of Philadelphia for automated DNA sequence analysis. We are grateful to Dr. H. E. Witkowska for mass spectral analysis of the beta  chain variants performed at the Children's Hospital Mass Spectrometry Facility in Oakland, CA. (Dr. C. Shackleton, director), which is supported in part by NIH Grant HL20985 and a Shared Instrumentation Grant RR06505.

    FOOTNOTES

* This research was supported in part by grants from the National Institutes of Health (P60 HL38632 and DK 16691), the American Heart Association, the Joseph Stokes, Jr. Research Institute, UNICO National Inc., and by the Nemours Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Division of Hematology, the Children's Hospital of Philadelphia, 34th St. & Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-3576; Fax: 215-590-4834; E-mail: adachi{at}email.chop.edu.

1 The abbreviations used are: DTT, dithiothreitol; Hb, hemoglobin; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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