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J. Biol. Chem., Vol. 277, Issue 16, 13415-13420, April 19, 2002

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Assembly of Human Hemoglobin (Hb) - and -Globin Chains Expressed in a Cell-free System with -Globin Chains to Form Hb A and Hb F^*

Kazuhiko Adachi^§, Yi Zhao, and Saul Surrey^¶

From the  Children's Hospital of Philadelphia, Division of Hematology and the University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104 and the ^¶ Cardeza Foundation for Hematologic Research, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Received for publication, January 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Rates of in vitro synthesis of radiolabeled  and  chains made in a cell-free transcription/translation system were similar, but expressed globin chains were unstable. The addition of unlabeled  or  chains at the start of chain synthesis generated radiolabeled ₄ or ₂ and ₄ chains, respectively. If unlabeled -globin chains were added at the start of chain synthesis, then approximately equal amounts of radiolabeled or bands were generated. If unlabeled Hb A or Hb F was added to reactions containing radiolabeled or prior to electrophoresis, then radiolabeled Hb A or Hb F tetramers, respectively, were generated. If  chains were added after synthesis of radiolabeled  chains made in the presence of unlabeled  chains, then little radiolabeled formed. In contrast, if  chains were added after synthesis of radiolabeled  chains made in the presence of unlabeled  chains, then radiolabeled ₂₂ formed. These findings suggest that  and  chains associate with  chains during or soon after translation. This would prevent the formation of unstable monomers as well as stable ₂ dimers and suggests that  chains may bind to nascent non- chains, acting as folding catalysts to promote functional tetrameric hemoglobin formation in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Assembly in vitro of human Hb¹ subunits ( and non- chains) into stable Hb heterotetramers (e.g. ₂₂ or ₂₂) using purified globin chains has been explored and a 3-step mechanism proposed (1-8). The  chains are in monomer/dimer equilibrium-favoring monomers, whereas non- chains are in monomer/tetramer equilibrium-favoring tetramers (9, 10). It is generally assumed that dissociation of these oligomeric subunits into monomers must occur before these two different chains can combine to form or dimers, which then associate to form tetrameric Hb (₂₂ or ₂₂) (11, 12). In addition, the assembly of or dimer was postulated to be the rate-limiting step for assembly in vivo and has been theorized to be governed by electrostatic attractions between - and non- partner subunits (8, 12). Furthermore, from in vitro studies it is known that Hb F formation using purified - and -globin chains is very slow compared with Hb A using purified - and -globin chains (11). In fact, our previous studies showed approximately a 10⁵-fold slower rate of assembly in vitro for Hb F compared with Hb A (11). The slow rate of Hb F formation in vitro is caused by stable ₂ dimer formation and is unlikely to occur in erythroid precursors (11). We also found that even at low concentrations (<5 µM),  chain dimers do not dissociate readily into monomers, resulting in decreased assembly with  chains. Furthermore, our results showed that the assembly of [Ile¹¹⁶His] chains with  chains was similar to that of  chains, whereas the assembly of [Thr¹¹²Cys] with  chains was similar to wild type  chains (11). These findings indicate that amino acid differences between [Ile¹¹⁶] and [His¹¹⁶] at ₁₁ and ₁₁ interaction sites, respectively, are responsible for the different assembly rates in vitro between Hb F and Hb A. These results also indicate that dissociation of ₂ dimers to monomers limits the formation in vitro of Hb F and suggest that  chains assemble in vivo with  chains prior to forming stable ₂ dimers, possibly binding to  chains as partially folded nascent -globin chains prior to or soon after release from polyribosomes. To evaluate the assembly of - and -globin with -globin chains in vivo, we expressed  and  chains in a wheat germ-coupled cell-free transcription/translation system using cDNA expression vectors. In addition, we added  and  or  chains as well as Hb F or Hb A to reactions at different times and assessed the formation of radiolabeled assembled homo-/heterodimers and tetrameric globins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Non--Globin cDNA Expression Vectors-- The plasmids pcDNA  and pcDNA  contain the SP6 promoter and cDNAs coding for the human - or -globin chains, respectively. They were constructed from pcDNA3 and pHE2 by subcloning each cDNA (11) into the HindIII/XbaI sites of pcDNA3. Transcription in vitro by SP6 RNA polymerase generates - or -globin mRNA, which is translated in a commercially available wheat germ cell-free transcription/translation system. The sequence and insertion site of the  or  chain cDNAs in the expression vectors were confirmed by automated DNA sequence analysis using dye-tagged terminators.

Expression of - or -Globin Chains in a Wheat Germ Cell-free Transcription/Translation System-- Expression of - or -globin chains in a cell-free coupled transcription/translation system was performed using a TNT^® SP6-coupled wheat germ extract system kit (Promega, Madison, WI) containing [³⁵S]methionine (Amersham Biosciences). A typical 50-µl reaction contained a 2-µg DNA template and was incubated for 30-60 min at 30 °C. For tetramer assembly studies, 6 ng of human -globin chain, human Hb (Hb A or Hb F), human  chains, or recombinant  chains were added to the reaction either at zero time or 30 min after radiolabeled chain synthesis. Synthesized - or -globin chains, as well as those assembled with  chains to form radiolabeled or dimers and ₂₂ (Hb A) or ₂₂ (Hb F) tetramers, were analyzed, respectively, by SDS-PAGE and by cellulose acetate electrophoresis (CAE) on Titan III membranes at pH 8.6 with Super-Heme buffer (Helena Laboratories, Beaumont, TX). Electrophoretic mobility of newly synthesized globin chains and assembled dimers and tetramers was compared with that of authentic human hemoglobins. Human - and -globin chains were purified from human Hb A as previously reported (13). Removal of p-chloromercuribenzoate from  and  chains was accomplished using 20 mM dithiothreitol, and globin chains were isolated after gel filtration on a Superose 12 column (Amersham Biosciences). Expression and purification of recombinant -globin chains were described previously (11). Hemin was dissolved in enough 0.1 N NaOH to give about 5 × 10⁶ M. This stock was diluted with 9 volumes of 0.5 M Tris buffer, pH 7.4, in the presence of 0.2 mM KCN. Haptoglobin (Hp) 2-2 was purchased from Sigma to confirm the presence of hemoglobin heterodimers. Unlabeled Hp (0.1 µg/ml) was added after transcription/translation reactions, and the interaction of Hp with or heterodimers was assessed by cellulose acetate electophoresis followed by autoradiography (14).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Synthesis of Radiolabeled - or -Globin Chains and Assembly of Homodimers/Tetramers in a Cell-free System-- Results from in vitro transcription/translation using - or -globin chain expression vectors showed a single radiolabeled ~16-kDa band after SDS-PAGE (Fig. 1, lane 2 in panels A and B) comigrating with purified - or -globin chains, respectively, whereas plasmids lacking cDNAs showed no band (Fig. 1, lane 1 in panels A and B). However, after cellulose acetate electrophoresis, radioactivity corresponding to newly synthesized - or -globin chains was only seen at the origin (Fig. 1, lane 2 in panels C and D).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Synthesis of radiolabeled - or -globin chains in a cell-free, coupled transcription/translation system. Transcription/translation reactions in panels A and C with pcDNA3 expression vector lacking insert (lane 1) or containing -globin cDNA insert (lanes 2 and 3) were incubated with 35S-labeled methionine for 60 min in the absence (lane 2) or presence (lane 3) of unlabeled -globin chains added at zero time. Transcription/translation reactions in panels B and D contained -globin cDNA in the absence (lane 2) or presence (lane 3) of unlabeled -globin chains. Panels A and B, reactions were electrophoresed on SDS-PAGE and then subjected to autoradiography. Panels C and D, reactions were electrophoresed on cellulose acetate membranes (CAE) and then subjected to autoradiography.

When unlabeled purified  chains were added at zero time to reactions containing  cDNA, two radiolabeled bands were observed after cellulose acetate electrophoresis following a 30-min incubation with ³⁵S-labeled methionine (Fig. 1, panel C, lane 3). The major band corresponds to ₂ homodimers, whereas traces of a second band comigrating with recombinant ₄ chains was also detected. In contrast, when unlabeled  chains were added at zero time to the  cDNA reaction, a single radiolabeled band was observed after cellulose acetate electrophoresis following a 60-min incubation with ³⁵S-labeled methionine, which corresponds to ₄ tetramers (Fig. 1, panel D, lane 3). These results indicate that monomeric forms of newly synthesized - and -globin chains are unstable, precipitate, and remain at the origin during electrophoresis at room temperature; however, the addition of excess unlabeled non- chains facilitates formation of homodimers and/or tetramers and prevents precipitation of newly synthesized globin chains during electrophoresis.

Assembly of Radiolabeled - or -Globin Chains in a Cell-free System with Unlabeled  Chains-- When unlabeled human -globin chains were added at zero time to the  cDNA reaction, a single radiolabeled band was seen after cellulose acetate electrophoresis that migrated close to the Hb F marker (Fig. 2, panel B, lane 1). When unlabeled human -globin chains were added at zero time to the  cDNA reaction, a single radiolabeled band was observed after cellulose acetate electrophoresis that migrated close to the Hb S marker (Fig. 2, panel B, lane 3). The single radiolabeled bands generated in these reactions with -globin chains added at zero time that migrate less than Hb A and Hb F probably correspond to heme-inserted or heterodimers, respectively.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Assembly of radiolabeled - or -globin chains with unlabeled -globin chains in a cell-free coupled transcription/translation system. Transcription/translation (panel B) in the presence of unlabeled -globin chains added at zero time to reactions containing  (lanes 1 and 2) or -globin cDNA vectors (lanes 3 and 4). Unlabeled Hb A (lane 2) or Hb F (lane 4) was added after the 30 min incubation period, and reactions were subjected to cellulose acetate electrophoresis. Hemoglobin A, F, S, and C markers are shown following cellulose electrophoresis (CAE) and staining with Ponceau S (panel A).

Assembly of Radiolabeled - or -Globin Chains with Unlabeled  Chains to Form Heterotetramers-- Our contention that the single radiolabeled bands in Fig. 2 (panel B, lanes 1 and 3) are radiolabeled and heterodimers, respectively, is supported by studies in which unlabeled Hb A or Hb F is added after the 30 min chain synthesis just prior to electrophoresis. Both radiolabeled bands comigrated with the corresponding heterotetramers after the addition of unlabeled Hb A or Hb F just prior to electrophoresis, respectively, (Fig. 2, panel B, lanes 2 and 4), indicating that the addition of Hb A and Hb F shifted the equilibrium from newly synthesized radiolabeled heterodimers to heterotetramers. Mobility of the radiolabeled and bands on cellulose acetate electrophoresis depended on amounts of unlabeled Hb A and Hb F added; the higher the amount, the closer the mobility to the corresponding tetrameric hemoglobins A and F (Fig. 3). The concentration of unlabeled hemoglobins A and F needed to generate band mobility midway between radiolabeled heterodimer and heterotetramer was about 0.9 and 0.4 µM, respectively, which corresponds to the dissociation constants of dimeric to tetrameric hemoglobin. These results are consistent with known properties of hemoglobin whereby tetrameric hemoglobins are in equilibrium while undergoing dissociation to dimers then re-association to form tetramers (1) and are consistent with these radiolabeled intermediates being and heterodimers.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Effects of increasing amounts of unlabeled Hb F or Hb A added after chain synthesis in the presence of unlabeled  chains on equilibration of dimers/tetramers of radiolabeled Hb F or Hb A, respectively. Transcription/translation reactions contained -globin cDNA (panel B) or -globin cDNA (panel C) reactions with unlabeled -globin chains added at zero time. Increasing concentrations of either unlabeled Hb F (panel B) or Hb A (panel C) were added after the 30 min incubation period, and reactions were subjected to cellulose acetate electrophoresis followed by autoradiography. Hb F concentrations in lanes 1-7 in panel B were 0, 0.11, 0.21, 0.42, 0.85, 1.7, and 3.4 µM, whereas those for Hb A in panel C were 0, 0.13, 0.27, 0.53, 1.1, 2.1, and 4.3 µM. Migration positions for hemoglobin A, F, S, and C are shown after staining with Ponceau S (panel A).

In addition, support for the contention that the radiolabeled bands observed after cellulose acetate electrophoresis are and heterodimers in the  and  cDNA reactions after  chain addition at zero time comes from studies of Hp binding. Hp preferentially binds hemoglobin dimers but not tetramers (1, 14). Hp affinity for the newly synthesized radiolabeled and heterodimers made in the presence of  chains added at zero time is shown in Fig. 4. The addition of Hp 2-2 to reactions after chain synthesis and heterodimer assembly followed by an additional 10-min incubation resulted in almost all of the radiolabeled heterodimer bands changing electrophoretic mobility and migrating more to the positive pole on cellulose acetate electrophoresis (Fig. 4, lane 2, panels A and B). This change in mobility is expected to result from the irreversible interaction of Hp with heterodimers (14). In contrast, newly synthesized radiolabeled heterotetramers made from reactions containing unlabeled  chains and Hb A or Hb F added at zero time showed that the addition of Hp generated two radiolabeled bands: one comigrating with the corresponding tetrameric hemoglobin and the other with the Hp heterodimer band (Fig. 4, lane 4, panels A and B). These studies collectively indicate that radiolabeled bands generated in the  and  cDNA reactions in the presence of unlabeled  chains are predominantly heterodimers.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effects of Hp binding on electrophoretic mobility of radiolabeled heterodimers and heterotetramers. Panel A, unlabeled Hp was added after the 30 min incubation period to transcription/translation reactions containing -globin cDNA in the presence of unlabeled -globin chains, in the absence (lane 2) or presence of Hb A (lane 4). Reactions were then subjected to cellulose acetate electrophoresis followed by autoradiography. Radiolabeled dimers formed in the presence of unlabeled  chains (lane 1) or radiolabeled Hb A heterotetramers formed in the presence of unlabeled  chain plus Hb A (lane 3) are shown without Hp addition. Panel B, same as panel A except  cDNA vector was used and unlabeled Hb F instead of Hb A added to form radiolabeled Hb F heterotetramers. Radiolabeled dimers (lane 1) and radiolabeled Hb F heterotetramers (lane 3) are also shown without Hp addition.

Time Course for Synthesis and Assembly of Radiolabeled Globin with Unlabeled  Chains-- Time courses for incorporation of ³⁵S-labeled methionine into radiolabeled  and  chains were the same in the presence or absence of unlabeled  chain addition at zero time (data not shown) (as assessed by relative band intensity after SDS-PAGE) and plateaued after about 40 min (Fig. 5, panel A). In addition, rates of radiolabeled or dimer formation were similar in reactions containing unlabeled  chains measured by relative intensity after cellulose acetate electrophoresis (Fig. 5, panel B). Rates of formation of and dimers (panel B) were also similar to rates of globin chain synthesis (panel A).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Time course for chain synthesis and heterodimer formation. Time course is shown for radiolabeled chain synthesis assessed by SDS-PAGE (panel A) and for radiolabeled heterodimer formation assessed by cellulose acetate electrophoresis (panel B). Unlabeled -globin chains were added at zero time only in experiments in panel B. Results are expressed as relative formation (%) where 100% represents maximum values at the plateau of each reaction.

Effects of Unlabeled -Globin Chain Addition on Radiolabeled Heterodimer Formation after a 30-min Synthesis Period in the Presence of Unlabeled Non--Globin chains-- To test the effects of newly synthesized and assembled  chain monomers and dimers on assembly with  chains, reactions containing unlabeled -globin chains added at zero time were stopped after a 30-min  chain synthesis period by the addition of puromycin. Unlabeled  chains or Hb F were then added, and samples were electrophoresed on SDS-PAGE or cellulose acetate membranes followed by autoradiography. Results from cellulose acetate electrophoresis of  cDNA reactions containing unlabeled  chains at zero time followed by the addition of unlabeled  chains just prior to electrophoresis showed a single radiolabeled band comigrating with ₂ dimers with no evidence for formation of radiolabeled dimers (Fig. 6, panel A, lane 2). If reactions were incubated in the presence of  chains and Hb F for an additional 30 min prior to cellulose acetate electrophoresis, then only trace amounts of radiolabeled Hb F were seen (Fig. 6, panel C, lane 2). These results suggest that the formation of or ₂₂ requires the addition of -globin chains at zero time prior to initiation of the transcription/translation reaction. In contrast, radiolabeled ₄ homotetramers formed in  cDNA reactions containing unlabeled  chains at zero time (Fig. 6, panel B, lane 1). The addition of unlabeled  chains just prior to electrophoresis resulted in the formation of radiolabeled ₂₂ heterotetramers and no ₄ bands (Fig. 6, panel B, lane 2). These results indicate that during synthesis of radiolabeled -globin chains the unlabeled -globin chains form unstable monomers and/or stable ₂ dimers that do not dissociate readily to monomers and, therefore, do not form heterodimers following the addition of unlabeled  chains after chain synthesis. In contrast, stable ₄ tetramers formed during chain synthesis dissociate readily into monomers and assemble with  chains, resulting in the formation of Hb A as shown in in vitro studies (6, 8, 11). These findings are consistent with our previous results of in vitro assembly showing only trace amounts of Hb F formation after an additional 30-min incubation using purified  and  chains. In contrast, we showed that  chains associate readily with  chains to form functional Hb A (11).

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Effects of unlabeled -globin chain addition on radiolabeled heterodimer formation after a 30-min synthesis period in the presence of unlabeled non- chains. In panel A, reactions containing -globin cDNA vector were incubated with 35S-labeled methionine for 30 min in the presence of unlabeled -globin chains added at zero time (lanes 1 and 2). Reactions were stopped by the addition of puromycin, and unlabeled -globin chains (lane 2) were added just prior to electrophoresis on cellulose acetate membranes and then subjected to autoradiography. In panel B, -globin cDNA and unlabeled  chains were used. Reactions were stopped by the addition of puromycin, and -globin was added (lane 2) just prior to cellulose acetate electrophoresis. In panel C, reactions contained  cDNA vector and unlabeled  globin at zero time. Reactions were stopped after 30 min by the addition of puromycin, and unlabeled -globin chains and Hb F were added just prior to electrophoresis (lane 1) or incubated for an additional 30 min prior to electrophoresis (lane 2).

Effects of Unlabeled -Globin Chains and/or Hb F Addition at Zero Time on Radiolabeled Heterodimer, ₂ Homodimer, and Hb F Formation-- Because  chains form stable ₂ homodimers in vitro, which stabilize newly synthesized -globin chains in the cell-free system, it was important to know how unlabeled  and  chains compete for assembly with newly synthesized and/or nascent  chains to form ₂ homodimers and/or heterodimers. Therefore, we studied the effects of unlabeled Hb F added at zero time to  cDNA reactions on the formation of radiolabeled Hb F. It is known that dimers are in equilibrium with ₂₂ tetramers (Hb F) that can dissociate into  and  monomers (15). Dissociated monomers should be able to associate with newly synthesized radiolabeled -globin chains in this cell-free system to form radiolabeled or radiolabeled ₂ dimers. As shown earlier (Fig. 2, lane 3), the addition of unlabeled -globin chains at zero time resulted in newly synthesized -globin chains forming radiolabeled dimers (Fig. 7, panel A, lane 1). When unlabeled human Hb F was added at zero time in the absence of added  chains, a single radiolabeled band was detected that comigrated with Hb F after cellulose acetate electrophoresis (Fig. 7, panel A, lane 3). No radiolabeled ₂ band was generated, and the intensity of the Hb F band was less than that of the heterodimer band generated by the addition of  chains at zero time (Fig. 7, panel A, compare band intensity in lane 3 versus lane 1). In addition, after adding Hb F at zero time in the absence of  chain addition (lane 3) radiolabeled material was present at the origin, indicating that some of the radiolabeled unassociated  chain monomers precipitated. This may be caused by the limited amounts of - and -globin chains generated from dissociation of Hb F even though both chains can stabilize newly synthesized -globin chains. Increasing the concentration of Hb F 10-fold had no effect on increasing the formation of radiolabeled ₂ homodimer bands (data not shown). In contrast, when unlabeled -globin chains plus Hb F were added at zero time to reactions containing  chain expression vector, a major radiolabeled Hb F band appeared with no ₂ dimer band or material at the origin (Fig. 7, panel A, lane 2). Amounts of  chain synthesized in reactions containing  chains with Hb F or just Hb F alone were the same as assessed by SDS-PAGE (Fig. 7, panel B, compare intensity in lanes 2 and 1). These results indicate that if  and  chains are present as a result of Hb F dissociation only dimers form. These findings suggest that unlabeled  chains have a lower affinity than  chains for newly synthesized -globin chains or that the slow dissociation of unlabeled Hb F to  and  monomers was rate-limiting, therefore generating less radiolabeled Hb F than that observed in the  plus Hb F reaction. We believe that in the absence of  chains, the newly synthesized  chains (after translation) are unstable and readily precipitate during electrophoresis because of their low concentration. However, when unlabeled  and/or  chains are added at zero time, they assemble with newly synthesized radiolabeled  chains to form heterodimers or ₂ homodimers, respectively. These more stable forms prevent precipitation during electrophoresis.

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Effects of unlabeled -globin chain and/or Hb F addition at zero time on radiolabeled heterodimer or 2 homodimer and Hb F formation. Reactions containing -globin cDNA expression vector were incubated for 30 min in the presence of unlabeled -globin chains (lane 1), unlabeled Hb F (lane 3), or both (lane 2). Reactions were subjected to cellulose acetate electrophoresis (panel A) or SDS-PAGE (panel B) followed by autoradiography.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Studies on the in vitro assembly of Hb A show that the ₂₂ tetramer assembles via a stable dimer intermediate as follows in Equation 1.

(Eq. 1)

This pathway reflects the relative stability of the ₂₂ tetramer and equilibration with dimer (2  ₂₂ or 2  ₂₂) and involves stable protein-protein interactions of  and  chains at ₁₁ interfaces as well as ₁₁ interfaces of Hb F (1). The ₁₁ or ₁₁ interface forms first at low subunit concentrations in vitro, implying that this interface is energetically more stable than all the other subunit interfaces combined. This equilibrium exists under physiological conditions, is an important determinant of hemoglobin function, and shifts depending on various conditions such as pH, ionic strength, and Hb concentration. Subunit dissociation to dimers upon dilution has been studied by a variety of methods, all of which involve measurement of the average molecular weight of Hb dimer and tetramer (16). Our present results show that in the presence of excess  chains, heterodimers rather than heterotetramers form because of the low concentration of newly synthesized globin chains generated in this cell-free system (~10⁸ M) compared with the dissociation constant (K_d) of tetrameric Hb (~10⁶ M) (16-18).

The biosynthesis of - and non--globin polypeptide chains to form heterodimers and tetramers in vivo is normally balanced. How the linear amino acid sequence in polypeptides promotes protein folding leading to formation in vivo of functional tetramers after transcription/translation is not completely understood. We found differences in the assembly of purified  and  chains to form Hb F and of  and  chains to form Hb A in vitro compared with expression of both tetramers in bacteria and yeast (11). The difference between in vitro and in vivo assembly can be explained by our present experimental results suggesting that nascent -globin chains assemble with free  chains to form heterotetramers during translation, or soon after, but prior to formation of stable ₂ homodimers (11). Furthermore, our previous studies in vitro showed a 10⁵-fold slower rate of stable ₂ dimer assembly with  chains to form Hb F compared with Hb A formation by - and -globin chains. This large rate difference can be explained by the fact that ₂ homodimers do not dissociate readily to monomers like ₂ or ₄ chains (11). However, if  chains are in the monomeric state, they can associate with  chains as  chains (11). Our present results using this in vitro cell-free system show that in the presence of added unlabeled  chains prior to translation, newly synthesized -globin chains do not form unstable  chain monomers or stable ₂ homodimers. The chains assemble with  chains and form radiolabeled heterodimers that can then form Hb F tetramers. Our present results also show that  or  chains can be stabilized by the formation of homodimers or homotetramers in the absence of  chains. In addition, it was expected that in the presence of unlabeled - and -globin chains that newly synthesized  chains would form not only heterodimers but also ₂ homodimers. However, our results show that the addition of unlabeled -globin chains or -globin chains and Hb F at zero time produced only radiolabeled or Hb F, respectively, and no ₂ or ₄. These results suggest that  chains associate with newly synthesized -globin chains faster than -globin chains, even though -globin chains can form stable homodimers.

In addition, Komar et al. (19) demonstrated that incomplete human -globin molecules of 140, 100, and 86 amino acid residues are capable of co-translational heme binding with an approximately equal efficiency, whereas polypeptide chains of 75, 65, and 34 amino acid residues display a significantly weaker, or just nonspecific, affinity for heme. This indicates that nascent  chains having 86 amino acids possess a structure that allows interaction with heme or that heme binding to nascent globin chains promotes formation of the proper tertiary structure of the growing polypeptide chain on polyribosomes (19, 20). It is not clear how - and non--globin genes are transcribed at nearly equal rates; however, -globin mRNA, whether by virtue of a higher rate of transcription or greater stability, accumulates in slight excess compared with -globin mRNA during normal adult erythropoiesis. In fact, -globin mRNA tends to be 25-50% more abundant than -globin mRNA in normal erythrocytes (21). Furthermore, in red cells the concentration of hemoglobin is very high (~5 mM) even though free globin chain concentrations are not known. From these results, we propose that nascent non- chains undergo folding and in the presence of  chain monomers assemble with already folded -globin chains thereby preventing the formation of ₂ or apo ₂ homodimers. This would result in the formation of stable -non- globin heterodimers, which then leads to the formation of functional hemoglobins in vivo. Current attempts are focused on demonstrating  chain interaction with nascent  chains during translation to form functional Hb F using cell-free systems.

	FOOTNOTES

^* This research was supported in part by Grant HL58879 from the National Institutes of Health and by the Cardeza Foundation for Hematologic Research and Jefferson Medical College.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.

^§ To whom correspondence should be addressed: Div. 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@email.chop.edu.

Published, JBC Papers in Press, February 4, 2002, DOI 10.1074/jbc.M200857200

	ABBREVIATIONS

The abbreviations used are: Hb, hemoglobin; Hp, haptoglobin; CAE, cellulose acetate electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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