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J Clin Invest. Jul 2, 2007; 117(7): 1856–1865.
Published online Jul 2, 2007. doi:  10.1172/JCI31664
PMCID: PMC1904324

An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis


Erythrocyte precursors produce abundant α- and β-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. αHb-stabilizing protein (AHSP) binds free α subunits reversibly to maintain their structure and limit their ability to generate reactive oxygen species. Accordingly, loss of AHSP aggravates the toxicity of excessive free α-globin caused by β-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free α-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 α-globin genes (genotype Ahsp–/–α-globin*α/αα) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated α-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free α-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess α-globin, AHSP also acts as a molecular chaperone to stabilize nascent α-globin for HbA assembly. Our findings illustrate what we believe to be a novel adaptive mechanism by which a specialized cell coordinates high-level production of a multisubunit protein and protects against various synthetic imbalances.


Hemoglobin A (HbA), the major blood oxygen carrier in adult mammals, is a heterotetramer of α- and β-globin proteins, each bound to heme (1, 2). Terminal erythropoiesis involves massive synthesis of HbA, which constitutes roughly 95% of mature erythrocyte protein at a concentration of approximately 35 g/dl. Several biological and biophysical constraints can potentially interfere with this process. High protein concentrations within cells can cause misfolding and aggregation of newly synthesized polypeptides (3). Moreover, unassembled HbA components, including free α-globin, β-globin, and heme, are inherently unstable and cytotoxic. An understanding of how Hb production is coordinated and controlled to maintain the stability of individual components, balance their synthesis, and optimize assembly has remained elusive in erythroid biology.

The biological effects of unbalanced HbA synthesis are illustrated by the thalassemia syndromes, a group of common inherited anemias in which mutations in either α- or β-globin genes (also known as HBA and HBB, respectively) impair production of the corresponding protein, resulting in buildup of the unaffected subunit (4). In β thalassemia, β-globin mutations cause αHb subunits to accumulate (58). Free αHb (α-globin plus heme, or holo-α-globin) is a potent oxidant, catalyzing the production of ROSs, which damage erythroid precursors and mature erythrocytes (9). In addition, αHb is inherently unstable and tends to either precipitate, lodging in the cell membrane, or degrade, releasing toxic heme, porphyrins, iron, and apo-α-globin polypeptide (α-globin without heme). Similar effects are observed in α thalassemia, where βHb accumulates, precipitates, and damages cells. However, free αHb is more unstable than free βHb, in part because free αHb exists predominantly as monomers, while free βHb tends to self-associate into homo-tetramers, which imparts some stability (10).

Several observations indicate that erythroid cells contain adaptive mechanisms to stabilize free αHb and limit its toxicity. First, erythroid precursors contain a small pool of excess free αHb, with no deleterious effects (11, 12). Second, individuals with the β thalassemia trait, who lack approximately 50% β-globin gene output, are well compensated, with minimal erythroid pathologies (13). In general, cells cope with potentially damaging unstable proteins through 2 interrelated mechanisms (reviewed in refs. 3, 14, and 15). First, proteolytic systems recognize and eliminate misfolded and/or potentially toxic proteins. Accordingly, pathways to degrade excess free αHb occur in erythroid cells, although the molecular mechanisms are not well defined (1618). Second, most cells contain molecular chaperones that bind, stabilize, and fold potentially toxic protein intermediates during their normal synthesis or upon pathologic accumulation. Recently, we discovered αHb-stabilizing protein (AHSP, also called erythroid-associated factor [ERAF] and erythroid differentiation–related factor [EDRF]), a small, abundant erythroid protein that binds multiple forms of α-globin, stabilizes their structures, and limits their pro-oxidant activities (19).

In vitro studies using purified proteins showed that AHSP binds αHb, but not βHb or HbA (19, 20). In agreement with this finding, crystallographic analysis revealed that AHSP and βHb have overlapping binding sites on αHb (21). Moreover, βHb binds αHb much more tightly than AHSP. Therefore, under normal physiological concentrations of βHb, AHSP is not expected to interfere with HbA synthesis or function. In vitro, recombinant AHSP reduces ROS production by purified αHb, increases its thermostability, and inhibits its precipitation (19, 21, 22). Ahsp–/– mice exhibit hemolytic anemia with increased levels of erythroid ROS and Hb precipitates, demonstrating an essential role in Hb homeostasis (22). Loss of AHSP also exacerbates anemia and Hb precipitation in a murine model of β thalassemia (22). Together, these studies indicate that AHSP detoxifies excess αHb that accumulates to low levels in normal erythroid cells and to a much greater extent in β thalassemia (8, 12). A mechanism for this function is provided by biochemical and structural studies showing that AHSP converts ferrous (FeII) oxygenated αHb to a more stable ferric (FeIII) bis-histidyl form (21, 23).

We reasoned that if inhibiting the toxicities of excessive free αHb is the major role for AHSP, then it should be possible to alleviate the Ahsp–/– phenotype by reducing α-globin gene dosage. To test this, we generated Ahsp–/– mice that also lack 1 of 4 α-globin alleles (genotype α-globin*α/αα, also known as Hba). By itself, this α-globin mutation, termed α thalassemia silent carrier state, causes minimal damage to erythroid cells (24). However, the Ahsp–/– and α-globin*α/αα mutations combined cause erythroid abnormalities that are substantially more severe than those resulting from either mutation alone. Most notably, both α-globin and β-globin were destabilized, even though AHSP does not bind the latter. Failure of reduced α-globin gene dosage to suppress the Ahsp–/– phenotype might indicate that AHSP not only acts to neutralize excessive αHb, but also promotes the stability of nascent α chains for subsequent incorporation into HbA. In this case, loss of viable α-globin in Ahsp–/–α-globin*α/αα mice would increase the level of free β-globin, thus promoting its precipitation. In support of this model, we show that AHSP promotes native folding of apo-α-globin and its assembly into αHb in solution and stabilizes a pool of free αHb in erythroid precursors. Together, these studies identify AHSP as a tissue-specific molecular chaperone that protects erythroid cells from various stresses and imbalances related to HbA synthesis.


The α thalassemia silent carrier state exacerbates loss of AHSP.

Previous genetic and biochemical studies indicated that AHSP protects cells from the deleterious effects of excess free αHb (2123). In this case, reducing free α-globin levels might alleviate Hb instability and anemia caused by loss of AHSP. We tested this by breeding Ahsp–/– mice to the α thalassemia silent carrier strain, termed α-globin*α/αα, which lacks 1 of 4 α-globin genes and exhibits minimal erythroid abnormalities (24). Most notably, compound mutant mice (genotype Ahsp–/–α-globin*α/αα) exhibited more severe erythroid pathologies than mice with either mutation alone. Erythrocyte morphologies of the mutant mice are shown in Figure Figure1.1. Loss of 1 α-globin allele had no apparent effects (left panel), while Ahsp–/– erythrocytes exhibited irregularities in shape, with eosinophilic inclusions in some cells, as reported previously (22) (middle panel). In contrast, the double-mutant erythrocytes had increased central pallor and more pronounced variations in size and shape (right panel). Automated hematology analysis confirmed these differences quantitatively and demonstrated that the double-mutant animals were about 10% more anemic than Ahsp–/– mice (Table (Table1).1). In summary, the erythroid abnormalities imparted by loss of AHSP were greater in mice that had lost 1 α-globin allele, as evidenced by examination of cell morphologies and erythrocyte indices. This was somewhat surprising, as we initially expected that the *α/αα genotype might alleviate the effects of AHSP loss by reducing the level of toxic free α-globin.

Figure 1
Abnormal erythrocyte morphology in Ahsp–/–α-globin*α/αα double-mutant mice.
Table 1
Erythroid indices of Ahsp and α-globin mutant mice

The erythroid abnormalities in Ahsp–/–α-globin*α/αα mice suggest that the combined mutations intensify Hb instability. Unstable Hbs tend to precipitate and lodge in the erythrocyte plasma membrane (2530). We collected equal numbers of erythrocytes from mice with various genotypes, prepared membrane fractions, and analyzed bound globins by Triton–acetic acid–urea (TAU) gel electrophoresis, which resolves α and β-globin subunits under denaturing conditions (Figure (Figure2A).2A). Wild-type erythrocytes contained no detectable membrane-associated globins, reflecting optimal chain balance and stability (lane 1). Loss of AHSP alone caused precipitation of both α- and β-globin at roughly equal ratios, as reported previously (22) (genotype Ahsp–/–, lanes 2 and 3), while the α thalassemia silent carrier state was associated with low-level β-globin precipitates, reflecting mild α-globin deficiency (genotype α-globin*α/αα, lanes 4 and 5). Importantly, precipitation of both globins, particularly the β subunit, was increased in the double-mutant mice (genotype Ahsp–/–α-globin*α/αα, lanes 6–9). This confirms and extends our previous findings that loss of AHSP destabilizes β-globin in vivo (22), likely through indirect effects, since AHSP does not bind β-globin directly (19).

Figure 2
Increased Hb instability in Ahsp–/–α-globin*α/αα double-mutant erythrocytes.

Analysis of steady-state membrane-associated globin chains (Figure (Figure2A)2A) showed that in the context of α thalassemia silent carrier state, loss of AHSP destabilizes α-globin and, to a greater extent, β-globin. To investigate this more quantitatively and assess the kinetics of globin precipitation, we pulse-labeled equal numbers of reticulocytes from α-globin*α/αα and Ahsp–/–α-globin*α/αα mice with 35S-methionine and 35S-cysteine for 15 minutes and analyzed nascent globins in cytoplasmic and membrane fractions by TAU gel electrophoresis and autoradiography. We quantified the proportions of α- and β-globins in the cytosol and plasma membrane by Phosphorimager analysis. An example of the TAU gel is shown in Figure Figure2B,2B, and quantitative analysis of globin chain distributions in reticulocytes from multiple animals is summarized in Table Table2.2. In the α thalassemia silent carrier state, approximately 25% of nascent β-globin chain precipitated into the membrane (Figure (Figure2B,2B, lane 1, and Table Table2);2); this was increased to approximately 45% by concomitant loss of AHSP (Figure (Figure2B,2B, lane 3, and Table Table2).2). In addition, approximately 11% of nascent α-globin precipitated in α thalassemia mice lacking AHSP. These findings are consistent with analysis of steady-state globin precipitates (Figure (Figure2A)2A) and further show that pathological globin precipitation occurs rapidly in the mutant animals.

Table 2
Synthesis and fate of nascent globin chains in reticulocytes

Loss of AHSP could enhance the accumulation of unstable β-globin chains by inhibiting α-globin synthesis. To investigate this possibility, we quantified total (membrane plus cytosolic) 35S-labeled globins synthesized after pulse-labeling by Phosphorimager analysis of TAU gels. In single-mutant α-globin*α/αα reticulocytes, the α/β synthetic ratio was approximately 0.76, consistent with loss of a single α-globin allele. This ratio was not significantly different in Ahsp–/–α-globin*α/αα double mutants (Table (Table2).2). Hence, in the α thalassemia silent carrier state, loss of AHSP increases both α- and β-globin precipitation without affecting the α/β-globin chain synthetic ratio. Most likely, in the double-mutant mice, rapid precipitation of newly synthesized α-globin caused by loss of AHSP aggravates α thalassemia by increasing the level of free β-globin, thereby enhancing its precipitation. This effect may also be accelerated by ROS produced by unstable free α-globin (21). It is also theoretically possible that AHSP stabilizes newly synthesized β-globin directly, although this is unlikely, as AHSP does not bind or stabilize purified β-globin (ref. 19 and data not shown).

Free Hb subunits and their breakdown products catalyze the production of ROS, causing secondary damage to cellular proteins and lipids (9). We measured ROS in erythrocytes using the dye dichlorofluorescin diacetate (DCFH-DA), which enters cells and is converted to the fluorescent product DCF upon oxidation (Figure (Figure2C).2C). Separately, Ahsp–/– and α-globin*α/αα erythrocytes both contained mildly increased ROS levels. However, ROS levels increased about 4-fold in the double mutants. This indicates further that the 2 mutations synergize to cause Hb instability.

Unstable Hbs can impair the maturation and viability of erythroid precursors in a process termed ineffective erythropoiesis (8). We used a quantitative flow cytometry–based assay to distinguish different developmental stages in cells expressing the erythroid-specific antigen Ter119 (Figure (Figure3)3) (31). This approach uses CD71 (transferrin receptor) expression and forward scatter (FSC) to distinguish 3 different Ter119+ erythroid precursors, termed Ery.A, Ery.B, and Ery.C, which represent progressive stages of maturation (Figure (Figure3A).3A). In Ahsp–/–α-globin*α/αα mice, there was altered erythroid precursor distribution with reduced transition from Ery.B to Ery.C (Figure (Figure3A,3A, compare upper and lower panels). This effect was verified in multiple animals (Figure (Figure3B)3B) and suggests impaired maturation of Ahsp–/–α-globin*α/αα erythroid precursors at a stage coincident with high-level Hb synthesis. A similar effect of less magnitude was suggested in Ahsp–/– mice (Figure (Figure3B).3B). Hence, loss of AHSP and 1 α-globin gene together impair erythroid development to a greater extent than either mutation individually. Most likely, this abnormality is due to the damaging effects of unstable Hbs on the maturation and/or survival of erythroid precursors.

Figure 3
Blocked erythroid maturation in double-mutant mice.

AHSP promotes HbA assembly from newly synthesized α-globin in vitro.

Our genetic studies demonstrate that AHSP is important for Hb stability and erythropoiesis even when α-globin is not present in excess. A likely explanation for the more severe erythroid phenotype of Ahsp–/– and α-globin*α/αα double mutants is that AHSP specifically stabilizes newly synthesized α-globin to augment its incorporation into HbA. In this scenario, loss of AHSP causes precipitation of nascent α-globin, exacerbating β-globin excess in the setting of mild α thalassemia.

We examined this possibility further by studying how purified recombinant AHSP affects HbA formation from αHb synthesized in vitro. First, we used a wheat germ–derived transcription and translation (TNT) reaction to generate αHb from its cDNA (Figure (Figure4A).4A). We used the wheat germ system because AHSP is absent, in contrast to TNT systems derived from reticulocyte lysates. Radiolabeled α-globin protein was synthesized in the presence of cyano-hemin (a stable form of heme), with or without added recombinant AHSP. Then, purified, unlabeled βHb was added and HbA formation was analyzed by cellulose acetate electrophoresis (CAE), a nondenaturing method that fractionates mainly according to charge. Incorporation of newly synthesized α-globin into HbA was assessed by autoradiography of the CAE membrane. We also measured total α-globin polypeptide synthesis by denaturing SDS-PAGE and autoradiography. Addition of recombinant AHSP augmented HbA formation from nascent αHb in a dose-dependent fashion (Figure (Figure4B,4B, CAE), without affecting the overall yield of full-length α-globin polypeptide (Figure (Figure4B,4B, SDS-PAGE). Moreover, HbA production was augmented most effectively when recombinant AHSP was present during the entire course of the TNT reaction, indicating that AHSP stabilizes apo-α-globin and/or αHb cotranslationally or immediately afterward (Figure (Figure4C).4C). AHSP containing a point mutation (D43R) that inhibits binding to αHb had no effect on HbA synthesis from nascent αHb (Figure (Figure4D).4D). In addition, AHSP had no effect on the incorporation of newly synthesized TNT-derived βHb into HbA (data not shown). Together, these findings indicate that AHSP specifically maintains newly synthesized αHb in a soluble state that is competent for assembly with βHb to form HbA.

Figure 4
AHSP stabilizes newly synthesized α-globin in vitro.

AHSP promotes native folding of nascent apo-α-globin.

Previously, we showed that AHSP binds purified apo-α-globin (32). We therefore asked whether AHSP stabilizes newly synthesized apo-α-globin prior to heme insertion and HbA assembly. To test this, we exploited the principle that native folded proteins are relatively resistant to proteolysis compared with their unfolded counterparts (33, 34). We synthesized radiolabeled α-globin with or without recombinant AHSP in the wheat germ TNT system without added heme. Next, we treated the product with trypsin and tested the extent of digestion by SDS-PAGE and autoradiography (Figure (Figure5,5, A–D). Wild-type AHSP protected nascent α-globin from proteolytic digestion (Figure (Figure5A,5A, compare first and second panels; Figure Figure5B).5B). This effect is not simply because AHSP physically inhibits access to protease, as most predicted trypsin cleavage sites on α-globin are outside of the AHSP binding surface (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI31664DS1). More likely, AHSP causes α-globin to become protease resistant by promoting proper folding into a native state.

Figure 5
AHSP facilitates α-globin folding.

Addition of hemin during the TNT reaction also conferred protease resistance to nascent α-globin (Figure (Figure5A5A compare left and right panels; Figure Figure5B),5B), consistent with previous findings that heme stabilizes α-globin folding (3537). The mutant AHSP D43R, which binds α-globin at reduced affinity (21), did not protect nascent α-globin from proteolytic digestion (Figure (Figure5,5, C and D). Together, these results demonstrate that AHSP promotes and/or stabilizes apo-α-globin folding independent of heme.

AHSP promotes refolding of denatured α-globin.

The ability of AHSP to promote folding of nascent α-globin and its assembly into HbA is consistent with the function of a molecular chaperone. Some chaperones also promote refolding of their client proteins after denaturation. We tested whether this is true for AHSP and apo-α-globin. Apo-α-globin was denatured in phosphate buffer pH 7.0 with 0.5 M guanidinium chloride (GdCl), mixed with a 1.8-fold molar excess of purified recombinant AHSP, and analyzed for secondary structure by circular dichroism (CD) spectropolarimetry (Figure (Figure5,5, E and F). Under these conditions, apo-α-globin showed minimal helical content, while AHSP retained its native helical structure (Supplemental Figure 2, B and C). Importantly, when apo-α-globin was mixed with AHSP, the measured CD spectrum revealed helical content that was significantly greater than the theoretical sum of the CD spectra of the 2 components examined separately (Figure (Figure5E,5E, double arrow, and Figure Figure5F,5F, solid line). This effect was not observed with the reduced α-globin affinity mutant AHSP D43R (Figure (Figure5F5F and Supplemental Figure 2E). The most likely interpretation is that AHSP promotes refolding of denatured apo-α-globin. Alternatively, AHSP might gain additional secondary structure after binding apo-α-globin. However, this is less likely for 2 reasons. First, AHSP mixed with native (“holo”) αHb produced a CD spectrum nearly identical to the calculated sum of the individual spectra, indicating no net change in helicity upon formation of the αHb-AHSP complex (Figure (Figure5F5F and Supplemental Figure 2F). Second, NMR and x-ray crystallographic studies indicate no appreciable changes in the α-helical content of AHSP upon binding to αHb (21, 38) (data not shown).

AHSP stabilizes a pool of nascent α-globin in erythroid cells.

To identify αHb-AHSP complexes in vivo, we pulse-labeled reticulocytes with 35S-methionine and analyzed soluble cytoplasmic fractions by isoelectric focusing under conditions that resolve different Hb complexes. For these experiments, we used mice with β thalassemia (genotype β-globin+/th-3 to favor detection of non–HbA-associated αHb (39).

The only protein detected at steady state was HbA, as evidenced by a single band in the unstained gel (Figure (Figure6,6, bottom). However, autoradiography to visualize nascent proteins revealed 3 additional prominent bands (Figure (Figure6,6, top, lane 1). The identity of the most slowly migrating band, labeled with an asterisk, is unknown. Based on our previous studies of recombinant proteins, we predicted that the other 2 bands represent free αHb and αHb-AHSP, labeled accordingly (19). In agreement with these assignments, addition of excess purified human βHb drove both bands into a faster-moving species that migrated at the position of murine αHb/human βHb heterotetramers (mα22, lane 2). Also, addition of purified recombinant murine AHSP drove free αHb into the presumed αHb-AHSP complex (lane 3). We obtained similar results in Ahsp+/– heterozygotes (lanes 4–6). Importantly, free αHb and αHb-AHSP complex were not detected in Ahsp–/– mice (lanes 7 and 8), and less mα22 heterotetramer was formed after addition of hβhb, consistent with decreased assembly-competent free αHb. Both free αHb and αHb-AHSP complexes were best visualized in the β-globin+/th-3 background, which is associated with elevated α/β-globin synthetic ratio and excess free αHb. We obtained similar results in mice with different Ahsp genotypes and wild-type globin alleles, although the signals for free αHb and αHb-AHSP were less prominent (data not shown). Together, these findings demonstrate that αHb-AHSP complexes exist in vivo and that AHSP is required for formation and/or stabilization of a nascent free αHb pool that can be used for production of HbA.

Figure 6
Identification of transient free αHb and αHb-AHSP complex in erythroid cells.


New insights into how erythroid cells regulate Hb synthesis have been revealed through studies of AHSP, an erythroid-specific α-globin–binding protein (reviewed in ref. 40). Given the instability of free globin chains in erythroid precursors and the extremely high rate of HbA synthesis, it is predicted that molecular chaperones might participate in this process (41). Molecular chaperones are a diverse group of proteins that guide the assembly of other proteins but are not associated with the final product (3). At the time of its discovery, AHSP was identified as a candidate molecular chaperone for α-globin (42). Here we present genetic and biochemical evidence to prove this hypothesis.

Previously, we interbred mutant mice to show that β thalassemia is exacerbated by loss of AHSP, indicating that one of its functions is to limit the damaging effects of excess free αHb (22). Supporting this model, structural studies demonstrated that AHSP converts oxygenated αHb to a bis-histidyl form in which the pro-oxidant activity of heme-bound iron is reduced through interactions with 2 different histidine residues of the α-globin polypeptide, His87 and His58 (21, 23, 43). Through this mechanism, excessive free αHb that is not incorporated into HbA is sequestered into a complex with AHSP and subsequently converted into a relatively inert form.

In the current study, we bred mice with Ahsp-null alleles onto an α thalassemic background to show that AHSP is also important for Hb homeostasis when α-globin is not present in excess. In particular, Ahsp–/–α-globin*α/αα double-mutant mice showed worsened erythroid pathologies compared with mice with either mutation alone. These included greater anemia, abnormalities in erythrocyte morphology, and precipitation of both α- and β-globins. To explain these findings, we propose that AHSP acts as a molecular chaperone to stabilize newly synthesized α-globin and facilitate its incorporation into HbA. According to this model, loss of AHSP destabilizes a fraction of nascent α-globin, rendering it nonviable for HbA synthesis. This aggravates chain imbalance during concomitant α thalassemia because less α-globin is available for binding to free β-globin. Consequently, β-globin precipitation increases, contributing to cellular damage.

In addition to abnormalities in mature erythrocytes, Ahsp–/–α-globin*α/αα mice exhibit increased ineffective erythropoiesis, most likely because there is greater accumulation and precipitation of free globin proteins in the double mutants. This is consistent with the general concept that increased levels of unstable free globin chains induce ineffective erythropoiesis in erythroid precursors (44, 45). Overall, the current study shows that AHSP is important in the context of mild, asymptomatic α-globin deficiency. In the future, it will be interesting to analyze Ahsp–/– and Ahsp+/– mice with 2 α-globin genes deleted. Such studies will better establish the importance of AHSP in relation to the extent of globin chain imbalance.

Together, our past and current findings indicate that AHSP has at least 2 functions: to detoxify excess αHb and to act as a molecular chaperone that facilitates folding of newly formed α chains for assembly into HbA (Figure (Figure7).7). The relative importance of each function depends on the state of globin chain balance. One interesting question relates to how various conformational changes in α-globin that are mediated by AHSP contribute to its different functions. For example, it is likely that formation of the oxidized bis-histidyl αHb-AHSP complex is involved in detoxification of excess αHb, particularly in β thalassemia. In contrast, formation of this structure might be counterproductive to the role of AHSP as a molecular chaperone for HbA synthesis because the oxidized αHb must be enzymatically reduced at an energetic cost (43).

Figure 7
AHSP stabilizes multiple forms of α-globin at different stages of HbA synthesis and homeostasis.

As a molecular chaperone for HbA synthesis, AHSP acts on at least 2 forms of α-globin: the apo form, which lacks heme, and holo-α-globin (αHb). Apo-α-globin is extremely unstable in solution and in cells. In vitro studies and analysis of globin mutations in patients demonstrate that heme insertion stabilizes the native structure of apo-globins (37, 4648). Previous studies demonstrated that AHSP binds apo-α-globin (32). Here we show that like heme, AHSP also promotes in vivo and in vitro apo-α-globin folding into a native structure. This property could explain, at least in part, how AHSP enhances α-globin expression in Escherichia coli and in nonerythroid eukaryotic cells (19, 49). In general, protein folding occurs sequentially through discrete structural intermediates that guide formation of the native protein (50). AHSP binds αHb at the last 2 α helices, termed G and H, which are distant from the heme-binding pocket (21). Presumably, AHSP recognizes a similar protein fold in apo-α-globin. Studies of myoglobins indicate that a semifolded intermediate containing the G and H helices may represent an early step toward folding into a native structure in all apo-globins (51). By binding and stabilizing this region, AHSP could promote formation of α-globin tertiary structure. Of note, AHSP does not appear to bind or stabilize β-globin (19). This is consistent with the considerable amino acid sequence variation between α- and β-globin in their G-H helical folds.

Since AHSP and heme bind α-globin at different sites, they probably synergize to stabilize the native structure. Accordingly, α-globin bound to both heme and AHSP (αHb-AHSP) is more thermodynamically and chemically stable than free αHb alone (19, 21). Moreover, αHb-AHSP can interact with βHb to form HbA and free AHSP. In vivo, it is possible that AHSP stabilizes both the apo and heme-bound forms of α-globin successively toward formation of HbA tetramers (Figure (Figure7).7). These functions are well served by the high concentration of AHSP protein (~0.1 mM) in erythroid precursors undergoing active Hb synthesis (19). By contrast, the concentration of HbA tetramer in a mature erythrocyte is about 5 mM. The levels of various forms of free α-globin in erythroid precursors are not known but presumably are much lower, as indicated in Figure Figure6.6. Therefore, sufficient AHSP is probably present during erythropoiesis to bind most or all nascent α-globin before it interacts with heme and/or β-globin to form stable HbA. It is also possible that AHSP facilitates the assembly of monomeric α- and β-globin subunits into αβ dimers, which is believed to be the rate-limiting step for HbA synthesis (52). In this case, loss of AHSP combined with reduced α-globin dosage, which also slows monomer assembly, could impair this process to a point where individual unstable α and β subunits are more prone to precipitate.

Our findings indicate that in erythroid cells AHSP stabilizes newly formed α-globin transiently and then passes it off to βHb to form HbA. Analogous mechanisms in eukaryotes exist for other multisubunit proteins. For example, in B cells, Ig heavy chains are stabilized in the ER by the molecular chaperone BiP (53). Binding of light chains to heavy chains triggers BiP release to form mature Ig. However, compared with most chaperones, including BiP, AHSP recognizes a much more limited repertoire of “client” proteins. Specifically, AHSP recognizes a specific helix-loop-helix fold on a surface of αHb that normally binds β-globin. Indeed, α-globin is the only known client protein for the molecular chaperone activities of AHSP. Similarly, single protein–specific molecular chaperones function in the assembly of tubulin (54, 55) and collagen (56, 57), multisubunit proteins that are produced at relatively high levels. One interesting open question is whether AHSP–α-globin heterodimers form higher-order complexes with ubiquitously expressed chaperones that further facilitate α-globin folding and/or degradation.

In mice, AHSP protects erythroid cells from the deleterious effects of both α- and β-globin deficiencies and from oxidant stress (22). The clinical significance of these findings for humans is not fully known. Mutations that ablate AHSP production or alter its protein structure are rare (refs. 58, 59, and M.J. Weiss et al., unpublished observations), but some reports indicate that reduced AHSP expression correlates with more severe thalassemic phenotypes (60, 61). One preliminary report suggests that reduced AHSP expression associated with specific gene haplotypes contributes to infection or drug-induced hemolytic anemia (62). In principle, it is also possible that stabilization of apo-α-globin by AHSP becomes particularly important when heme availability for this function is limited, for example, during iron deficiency or genetic porphyrias. This potential function for AHSP would synergize with that of heme-regulated kinase (HRI), a cytosolic signaling protein that limits translation of apo-globins during heme-deficiency states (63). These possibilities can be investigated further by analyzing the effects of genetic porphyrias or iron deprivation in Ahsp–/– mice.

Our overall view is that by binding multiple forms of α-globin, AHSP acts as a versatile homeostatic regulator to buffer against various imbalances and stresses that perturb Hb synthesis. Analogously, the chaperone heat-shock protein 90 (HSP90) can buffer against phenotypic manifestations of genetic variation in Drosophila and Arabidopsis (64, 65). Accordingly, it is possible that one evolutionary effect of AHSP is to increase tolerance to potentially deleterious globin mutations that confer resistance to malaria and environmental insults such as iron deficiency and oxidative stress.



Ahsp+/– mice were generated in our laboratory (19). α Thalassemia silent carrier mice (genotype α-globin*α/αα) were provided by Y. Kan (UCSF, San Francisco, California, USA) (24). β Thalassemic mice (genotype β-globin+/th-3) were provided by Oliver Smithies (University of North Carolina, Chapel Hill, North Carolina, USA) (39). Mice were analyzed at age 3–6 months. The mice used were of mixed genetic background (129/C57BL/6). For the data shown in Table Table1,1, littermates of various genotypes were analyzed to minimize any effects that might be due to their mixed genetic backgrounds. Animal protocols were approved by the Institutional Animal Care and Use Committee of The Children’s Hospital of Philadelphia.

Hematology analysis.

Blood was collected from the retro-orbital sinus or tail vein and then analyzed on a Bayer ADVIA 120 Hematology System. Photographs of blood smears were taken using a Axioskop 2 microscope with an Axiocam color digital camera (Zeiss).


Recombinant AHSP proteins and purified Hb chains were prepared as described previously (19, 32). Apo-α-globin (>95% heme-free) was prepared by high-performance liquid chromatography (Supplemental Figure 2A) (32). Apo-α-globin was dissolved in 6 M GdCl overnight and then dialyzed against 0.5 M GdCl. The concentration of apo-α-globin was calculated using an extinction coefficient of 9.97 cm–1 at 280 nm (ProtParam tool; http://www.expasy.ch/tools/protparam.html). Oxygenated αHb concentration was calculated using an extinction coefficient of 13.5 cm–1 at 541 nm.

In aqueous buffer, apo-α-globin tended to precipitate and also exhibited variable folding, as determined by CD (data not shown). In 0.5 M GdCl, apo-α-globin was more soluble and remained consistently denatured, while holo-αHb and AHSP retained their native helical structures under these conditions (Supplemental Figure 2, C and D). Therefore, protein-refolding experiments were performed in 10 mM sodium phosphate buffer (pH 7. 0) with 0.5 M GdCl.

CD spectropolarimetry.

α-Helical content was monitored using the JASCO J-810 CD system using a 1-mm-path-length cell. The temperature was maintained at 4°C using a water-jacketed cell holder. Spectra were collected over the wavelength range of 200 nm to 250 nm using a scanning speed of 20 nm/min and a bandwidth of 1 nm. GdCl produced excessive background signal at wavelengths less than 208 nm; therefore, data obtained at these wavelengths are not shown. Spectra were analyzing using SigmaPlot software (version 10.0; Systat).

Analysis of membrane-associated and cytosolic erythrocyte globins.

Equal numbers of erythrocytes (adjusted by hematocrit) were lysed with hypotonic buffer and washed 5 times (66). Cytosolic protein fractions were collected by pooling the supernatant and first 2 washes after cell lysis. Membrane skeleton fractions were prepared as described previously (66). Globin proteins were fractionated by TAU gel electrophoresis (67) and stained with Coomassie brilliant blue.

To analyze newly synthesized globin chains in reticulocytes, 20 μl of freshly collected blood was washed in methionine- and cysteine-free medium (Iscove’s Modified Dulbecco Medium [IMDM] MetCysGlu media, 2 mM l-glutamine, 10% dialyzed FBS, and 0.1 mM nonessential amino acids) and then incubated at 37°C for 1 hour in the same medium. 35S-labeled methionine/cysteine (50 μCi) was added for 15 minutes, then the cells were lysed and fractionated as described in the preceding paragraph. The entire radiolabeled membrane fraction and 5% of the cytosolic fraction for each sample were fractionated on TAU gels and analyzed by autoradiography. Phosphorimager (GE Healthcare) was used to quantify radiolabeled globins identified on the TAU gels. Cell culture reagents were obtained from Invitrogen or Cellgro (Mediatech Inc.).

Flow cytometry analysis.

Flow cytometry was performed using a FACSCalibur System (BD Biosciences) and analyzed with CellQuest Pro software (version 5.1.1; BD Biosciences). Mouse spleens were disaggregated into single-cell suspensions and stained with PE-conjugated anti-Ter119, FITC-conjugated anti-CD71, and 7-AAD as described previously (31). For detection of intracellular ROS, 2 × 106 peripheral blood cells were incubated with 25 μg/ml 2′,7,-DCFH-DA (Sigma-Aldrich), and fluorescence emitted by oxidized DCF was detected on FACS channel FL-1.

In vitro synthesis of HbA.

In vitro TNT was performed using the wheat germ TNT kit (Promega) according to published protocols (68, 69). Plasmids containing human α- or β-globin cDNAs (pcDNA3-α, T7 promoter or pcDNA3-β, Sp6 promoter) were added to the TNT mixture with CN-hemin. After the reaction, purified human αHb or βHb were added to complex with the newly synthesized radiolabeled globin chain. Additionally, purified unlabeled HbA was added to facilitate the formation and subsequent analysis of radiolabeled HbA tetramers (68, 69). HbA assembly was monitored by electrophoresis on Titan IIIH cellulose acetate membranes (Helena Laboratories) according to the manufacturer’s instructions. After electrophoresis, the membranes were washed in 5% acetic acid for 1 hour and subjected to autoradiography.

Limited proteolysis.

α-Globin was generated by TNT reaction and mixed with an equal volume of buffer containing 10 mM Tris-HCl pH 7.6, 100 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, and 0.5 mM cycloheximide. After addition of 10 μg/ml trypsin, aliquots were withdrawn at various time points, added to an equal volume of 95°C 2× SDS sample buffer (Sigma-Aldrich), and maintained at 95°C for 10 minutes in preparation for electrophoresis. Image analysis was performed using ImageJ software (version 1.34; http://rsb.info.nih.gov/ij/) and plotted using SigmaPlot software.

Isoelectric focusing of nascent reticulocyte proteins.

Twenty microliters of freshly collected blood was incubated in methionine-free IMDM, 2 mM l-glutamine, 10% dialyzed FBS, and 0.1 mM nonessential amino acids at 37°C for 1 hour, then 35S-labeled methionine was added for 10 minutes. Then the cells were washed with PBS and exposed to carbon monoxide to stabilize ferrous heme groups and inhibit their oxidation. The cells were lysed in water and clarified by centrifugation, and soluble fractions were loaded onto precast agarose gels (FR9120 Hemoglobin Test Kit; PH6-8; PerkinElmer). Electrophoresis was performed on an LKB 2117 Multiphor at 3,000 V for 50 minutes, and labeled proteins were analyzed by autoradiography.


Data in Figure Figure33 and Table Table11 were analyzed by 2-tailed Student’s t test. A P value of less than 0.05 was considered significant.

Supplementary Material

Supplemental data:


We thank Yuet Wei Kan and Judy Chang for providing α thalassemic mice. Kazuhiko Adachi and Yi Zhao provided technical advice and reagents for the wheat germ TNT reactions. Kelley Capobianchi assisted with hematology analysis. CD experiments were performed with the assistance of the protein core facility at The Children’s Hospital of Philadelphia. We thank David Nathan, Gerd Blobel, and Yair Argon for comments on the manuscript. This work was funded by NIH grant R01 DK69692-01.


Nonstandard abbreviations used: AHSP, αHb-stabilizing protein; CAE, cellulose acetate electrophoresis; CD, circular dichroism; GdCl, guanidinium chloride; Hb, hemoglobin; TAU, triton–acetic acid–urea; TNT, transcription and translation.

Conflict of interest: The authors have declared that no conflict of interest exists.

Citation for this article: J. Clin. Invest. 117:1856–1865 (2007). doi:10.1172/JCI31664

See the related Commentary beginning on page 1746.


1. Dickerson, R.E., and Geis, I. 1983. Hemoglobin: structure, function, evolution, and pathology. Benjamin Cummings. Menlo Park, California, USA. 176 pp.
2. Fermi, G., and Perutz, M.F. 1981. Atlas of molecular structures in biology. Volume 2, Haemoglobin and myoglobin. Oxford University Press. Oxford, United Kingdom. Clarendon Press. New York, New York, USA. 104 pp.
3. Frydman J. Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu. Rev. Biochem. 2001;70:603–647. [PubMed]
4. Forget, B.G., and Pearson, H.A. 1995. Hemoglobin synthesis and the thalassemias. In Blood: principles and practices of hematology. R.I. Handin, S.E. Lux, and T.P. Stossel, editors. J.B. Lippincott. Philadelphia, Pennsylvania, USA. 1525–1990.
5. Nathan D.G., Gunn R.B. Thalassemia: the consequences of unbalanced hemoglobin synthesis. Am. J. Med. 1966;41:815–830. [PubMed]
6. Bank A. Hemoglobin synthesis in β-thalassemia: the properties of the free α-chains. J. Clin. Invest. 1968;47:860–866. [PMC free article] [PubMed]
7. Baglioni C. Chromosomal and cytoplasmic regulation of haemoglobin synthesis. Bibl. Haemat. 1966;29:1056–1063. [PubMed]
8. Rachmilewitz, E., and Schrier, S.L. 2001. Pathophysiology of β thalassemia. In Disorders of hemoglobin: genetics, pathophysiology and clinical management. M.H. Steinberg, B.G. Forget, D.R. Higgs, and R.L. Nagel, editors. Cambridge University Press. Cambridge, United Kingdom. 233–251.
9. Brunori M., et al. Formation of superoxide in the autoxidation of the isolated a and b chains of human hemoglobin and its involvement in hemichrome precipitation. Eur. J. Biochem. 1975;53:99–104.
10. Bunn, H.F., and Forget, B.G. 1986. Hemoglobin function. In Hemoglobin: molecular, genetic and clinical aspects. H.F. Bunn and B.G. Forget, editors. Saunders. Philadelphia, Pennsylvania, USA. 37–60.
11. Shaeffer J.R. Evidence for soluble alpha-chains as intermediates in hemoglobin synthesis in the rabbit reticulocyte. Biochem. Biophys. Res. Commun. 1967;28:647–652. [PubMed]
12. Tavill A.S., Grayzel A.I., Vanderhoff G.A., London I.M. The control of hemoglobin synthesis. Trans. Assoc. Am. Physicians. 1967;80:305–313. [PubMed]
13. Olivieri, N.F., and Weatherall, D.J. 2001. Clinical aspects of beta thalassemia. In Disorders of hemoglobin: genetics, pathophysiology and clinical management. M.H. Steinberg, B.G. Forget, D.R. Higgs, and R.L. Nagel, editors. Cambridge University Press. Cambridge, United Kingdom. 277–341.
14. Bukau B., Weissman J., Horwich A. Molecular chaperones and protein quality control. Cell. 2006;125:443–451. [PubMed]
15. Wickner S., Maurizi M.R., Gottesman S. Posttranslational quality control: folding, refolding, and degrading proteins. Science. 1999;286:1888–1893. [PubMed]
16. Shaeffer J.R., Kania M.A. Degradation of monoubiquitinated alpha-globin by 26S proteasomes. Biochemistry. 1995;34:4015–4021. [PubMed]
17. Shaeffer J.R., Cohen R.E. Enhancement by ubiquitin aldehyde of proteolysis of hemoglobin alpha-subunits in beta-thalassemic hemolysates. Ann. N. Y. Acad. Sci. . 1998;850:394–397. [PubMed]
18. Shaeffer J.R. Turnover of excess hemoglobin alpha chains in beta-thalassemic cells is ATP-dependent. J. Biol. Chem. 1983;258:13172–13177. [PubMed]
19. Kihm A.J., et al. An abundant erythroid protein that stabilizes free alpha hemoglobin. Nature. 2002;417:758–763. [PubMed]
20. Baudin-Creuza V., et al. Transfer of human alpha- to beta-hemoglobin via its chaperone protein: evidence for a new state. J. Biol. Chem. 2004;279:36530–36533. [PubMed]
21. Feng L., et al. Molecular mechanism of AHSP-mediated stabilization of alpha-hemoglobin. Cell. 2004;119:629–640. [PubMed]
22. Kong Y., et al. Loss of α-hemoglobin–stabilizing protein impairs erythropoiesis and exacerbates β-thalassemia. J. Clin. Invest. 2004;114:1457–1466. doi: 10.1172/JCI200421982. [PMC free article] [PubMed] [Cross Ref]
23. Feng L., et al. Structure of oxidized alpha-haemoglobin bound to AHSP reveals a protective mechanism for haem. Nature. 2005;435:697–701. [PubMed]
24. Chang J., et al. Inactivation of mouse alpha-globin gene by homologous recombination: mouse model of hemoglobin H disease. Blood. 1996;88:1846–1851. [PubMed]
25. Advani R., et al. Characterization and comparison of the red blood cell membrane damage in severe human alpha- and beta-thalassemia. Blood. 1992;79:1058–1063. [PubMed]
26. Joshi W., Leb L., Piotrowski J., Fortier N., Snyder L.M. Increased sensitivity of isolated alpha subunits of normal human hemoglobin to oxidative damage and crosslinkage with spectrin. . J. Lab. Clin. Med. 1983;102:46–52. [PubMed]
27. Rouyer-Fessard P., et al. A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia. J. Biol. Chem. 1989;264:19092–19098. [PubMed]
28. Schrier S.L., Rachmilewitz E., Mohandas N. Cellular and membrane properties of alpha and beta thalassemic erythrocytes are different: implication for differences in clinical manifestations. Blood. 1989;74:2194–2202. [PubMed]
29. Shinar E., Rachmilewitz E.A., Lux S.E. Differing erythrocyte membrane skeletal protein defects in α and β thalassemia. J. Clin. Invest. 1989;83:404–410. [PMC free article] [PubMed]
30. Shinar E., Shalev O., Rachmilewitz E.A., Schrier S.L. Erythrocyte membrane skeleton abnormalities in severe beta-thalassemia. Blood. 1987;70:158–164. [PubMed]
31. Liu Y., et al. Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo. Blood. 2006;108:123–133. [PMC free article] [PubMed]
32. Gell D., Kong Y., Eaton S.A., Weiss M.J., Mackay J.P. Biophysical characterization of the alpha-globin binding protein alpha-hemoglobin stabilizing protein. J. Biol. Chem. 2002;277:40602–40609. [PubMed]
33. Fedorov A.N., Baldwin T.O. Protein folding and assembly in a cell-free expression system. Methods Enzymol. 1998;290:1–17. [PubMed]
34. Frydman J., Nimmesgern E., Ohtsuka K., Hartl F.U. Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature. 1994;370:111–117. [PubMed]
35. Ascoli F., Fanelli M.R., Antonini E. Preparation and properties of apohemoglobin and reconstituted hemoglobins. Methods Enzymol. 1981;76:72–87. [PubMed]
36. Hargrove M.S., Olson J.S. The stability of holomyoglobin is determined by heme affinity. Biochemistry. 1996;35:11310–11318. [PubMed]
37. Waks M., Yip Y.K., Beychok S. Influence of prosthetic groups on protein folding and subunit assembly. Recombination of separated human alpha- and beta-globin chains with heme and alloplex interactions of globin chains with heme-containing subunits. J. Biol. Chem. 1973;248:6462–6470. [PubMed]
38. Santiveri C.M., et al. NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding. . J. Biol. Chem. 2004;279:34963–34970. [PubMed]
39. Yang B., et al. A mouse model for beta 0-thalassemia. Proc. Natl. Acad. Sci. U. S. A. 1995;92:11608–11612. [PMC free article] [PubMed]
40. Weiss M.J., et al. Role of alpha hemoglobin-stabilizing protein in normal erythropoiesis and beta thalassemia. Ann. N. Y. Acad. Sci. 2005;1054:103–117. [PubMed]
41. Huang, S.-C., and Benz, E.J. 2001. Posttranscriptional factors influencing the hemoglobin content of the red cell. In Disorders of hemoglobin: genetics, pathophysiology and clinical management. M.H. Steinberg, B.G. Forget, D.R. Higgs, and R.L. Nagel, editors. Cambridge University Press. Cambridge, United Kingdom. 146–173.
42. Luzzatto L., Notaro R. Haemoglobin’s chaperone. Nature. 2002;417:703–705. [PubMed]
43. Zhou S., Olson J.S., Fabian M., Weiss M.J., Gow A.J. Biochemical fates of alpha hemoglobin bound to alpha hemoglobin-stabilizing protein AHSP. J. Biol. Chem. 2006;281:32611–32618. [PubMed]
44. Fessas P. Inclusions of hemoglobin erythroblasts and erythrocytes of thalassemia. Blood. 1963;21:21–32. [PubMed]
45. Ranney, H.M. 2001. Hemoglobin: a historical perspective. In Disorders of hemoglobin: genetics, pathophysiology and clinical management. M.H. Steinberg, B.G. Forget, D.R. Higgs, and R.L. Nagel, editors. Cambridge University Press. Cambridge, United Kingdom. 1–24.
46. Dacie J.V., Shinton N.K., Gaffney P.J., Jr., Lehmann H. Haemoglobin Hammersmith (beta-42 (CDI) Phe replaced by ser). Nature. 1967;216:663–665. [PubMed]
47. Wajcman H., et al. Structure of the EF corner favors deamidation of asparaginyl residues in hemoglobin: the example of Hb La Roche-sur-Yon [β81 (EF5) Leu→His]. Biochim. Biophys. Acta. 1992;1138:127–132. [PubMed]
48. Yip Y.K., Waks M., Beychok S. Influence of prosthetic groups on protein folding and subunit assembly. I. Conformational differences between separated human alpha- and beta-globins. J. Biol. Chem. 1972;247:7237–7244. [PubMed]
49. Vasseur-Godbillon C., Hamdane D., Marden M.C., Baudin-Creuza V. High-yield expression in Escherichia coli of soluble human alpha-hemoglobin complexed with its molecular chaperone. Protein Eng. Des. Sel. 2006;19:91–97. [PubMed]
50. Maity H., Maity M., Krishna M.M., Mayne L., Englander S.W. Protein folding: the stepwise assembly of foldon units. Proc. Natl. Acad. Sci. U. S. A. 2005;102:4741–4746. [PMC free article] [PubMed]
51. Musto R., Bigotti M.G., Travaglini-Allocatelli C., Brunori M., Cutruzzola F. Folding of Aplysia limacina apomyoglobin involves an intermediate in common with other evolutionarily distant globins. Biochemistry. 2004;43:230–236. [PubMed]
52. Bunn H.F. Subunit assembly of hemoglobin: an important determinant of hematologic phenotype. Blood. 1987;69:1–6. [PubMed]
53. Vanhove M., Usherwood Y.K., Hendershot L.M. Unassembled Ig heavy chains do not cycle from BiP in vivo but require light chains to trigger their release. Immunity. 2001;15:105–114. [PubMed]
54. Archer J.E., Vega L.R., Solomon F. Rbl2p, a yeast protein that binds to beta-tubulin and participates in microtubule function in vivo. Cell. 1995;82:425–434. [PubMed]
55. Cowan N.J., Lewis S.A. Type II chaperonins, prefoldin, and the tubulin-specific chaperones. Adv. Protein Chem. 2001;59:73–104. [PubMed]
56. Nagai N., et al. Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J. Cell Biol. 2000;150:1499–1506. [PMC free article] [PubMed]
57. Tasab M., Batten M.R., Bulleid N.J. Hsp47: a molecular chaperone that interacts with and stabilizes correctly-folded procollagen. EMBO J. 2000;19:2204–2211. [PMC free article] [PubMed]
58. Cappellini M.D., Refaldi C., Bignamini D., Zanaboni L., Fiorelli G. Molecular analysis of alpha hemoglobin stabilizing protein (AHSP) in Caucasian patients with different beta-thalassemia phenotypes [abstract]. Blood (ASH Annual Meeting Abstracts). 2004;104:3770.
59. Viprakasit V., et al. Evaluation of alpha hemoglobin stabilizing protein (AHSP) as a genetic modifier in patients with beta thalassemia. Blood. 2004;103:3296–3299. [PubMed]
60. Galanello R., Perseu L., Giagu N., Sole G. AHSP expression in beta-thalassemia carriers with thalassemia intermedia phenotype [abstract]. Blood (ASH Annual Meeting Abstracts). 2003;102:1881a.
61. Lai M.I., et al. Alpha-haemoglobin stabilising protein is a quantitative trait gene that modifies the phenotype of beta thalassemia. Br. J. Haematol. 2006;133:675–682. [PubMed]
62. Kanno H., et al. Alpha hemoglobin stabilizing protein (AHSP) is a susceptibility gene to drug/infection-induced hemolytic anemia [abstract]. Blood (ASH Annual Meeting Abstracts). 2005;106:479a.
63. Han A.P., et al. Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency. EMBO J. . 2001;20:6909–6918. [PMC free article] [PubMed]
64. Queitsch C., Sangster T.A., Lindquist S. Hsp90 as a capacitor of phenotypic variation. Nature. 2002;417:618–624. [PubMed]
65. Rutherford S.L., Lindquist S. Hsp90 as a capacitor for morphological evolution. Nature. 1998;396:336–342. [PubMed]
66. Sorensen S., Rubin E., Polster H., Mohandas N., Schrier S. The role of membrane skeletal-associated alpha-globin in the pathophysiology of beta-thalassemia. Blood. 1990;75:1333–1336. [PubMed]
67. Alter B.P. Gel electrophoretic separation of globin chains. Prog. Clin. Biol. Res. 1981;60:157–175. [PubMed]
68. Adachi K., Zhao Y., Surrey S. Assembly of human hemoglobin (Hb) beta- and gamma-globin chains expressed in a cell-free system with alpha-globin chains to form Hb A and Hb F. J. Biol. Chem. 2002;277:13415–13420. [PubMed]
69. Adachi K., Zhao Y., Surrey S. Effects of heme addition on formation of stable human globin chains and hemoglobin subunit assembly in a cell-free system. Arch. Biochem. Biophys. 2003;413:99–106.. [PubMed]

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