Chapter 7 Hemoglobin: Portrait of a Protein in Action

Hemoglobin gives blood its red color responsible for the transport of oxygen

Hemoglobin was one of the first proteins to have its structure determined

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The transition from anaerobic to aerobic life was a major step in evolution because it uncovered a rich reservoir of energy. Fifteen times as much energy is extracted from glucose in the presence of oxygen than in its absence. For single-celled and other small organisms, oxygen can be absorbed into actively metabolizing cells directly from the air or surrounding water. Vertebrates evolved two principal mechanisms for supplying their cells with an adequate supply of oxygen. The first is a circulatory system that actively delivers oxygen to cells throughout the body. The second is the use of the oxygen-transport and oxygen-storage proteins, hemoglobin and myoglobin. Hemoglobin, which is contained in red blood cells, is a fascinating protein, efficiently carrying oxygen from the lungs to the tissues while also contributing to the transport of carbon dioxide and hydrogen ions back to the lungs. Myoglobin, located in muscle, provides a reserve supply of oxygen available in time of need.

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These two evolutionarily related proteins employ nearly identical structures for oxygen binding (Chapter 6). Hemoglobin is able to use as much as 90% of its potential oxygen-carrying capacity. Myoglobin would be able to use only 7% of its potential capacity. What accounts for this dramatic difference? Myoglobin exists as a single polypeptide, Hemoglobin comprises four polypeptide chains. The four chains in hemoglobin bind oxygen cooperatively, binding of oxygen in one chain increases the likelihood that the remaining chains will bind oxygen. Furthermore, the oxygen-binding properties of hemoglobin are modulated by the binding of hydrogen ions and carbon dioxide in a manner that enhances oxygen-carrying capacity. Both cooperativity and the response to modulators are made possible by variations in the quaternary structure of hemoglobin when different combinations of molecules are bound.

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Hemoglobin and myoglobin have played important roles in the history of biochemistry. They were the first proteins for which three-dimensional structures were determined by X-ray crystallography.

1962 Chemistry Furthermore, the possibility that variations in protein sequence could lead to disease was first proposed and demonstrated for sickle-cell anemia, a blood disease caused by a change in a single amino acid in one hemoglobin chain. Hemoglobin has been and continues to be a valuable source of knowledge and insight, both in itself and as a prototype for many other proteins that we will encounter throughout our study of biochemistry.

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7.1 Myoglobin and Hemoglobin Bind Oxygen at Iron Atoms in Heme

Sperm whale myoglobin was the first protein for which the three-dimensional structure was determined. X-ray crystallographic studies pioneered by John Kendrew revealed the structure of this protein in the 1950s (Figure 7.1). Myoglobin consists largely of α helices that are linked to one another by turns to form a globular structure. Myoglobin can exist in an oxygen-free form called deoxymyoglobin or in a form with an oxygen molecule bound called oxymyoglobin. The ability of myoglobin, and hemoglobin as well, to bind oxygen depends on the presence of a bound prosthetic group called heme. The heme group gives muscle and blood their distinctive red color.

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Although the heme-bound iron can be in either the ferrous (Fe2+) or ferric (Fe3+) oxidation states, only the Fe2+ state is capable of binding oxygen. The iron ion can form two additional bonds, one on each side of the heme plane. These binding sites are called the fifth and sixth coordination sites. Fifth coordination site is occupied by a histidine residue from the protein. This histidine is referred to as the proximal histidine.

Heme consists of an organic component and a central iron atom. The organic component, called protoporphyrin, is made up of four pyrrole rings linked by methine bridges to form a tetrapyrrole ring. Four methyl groups, two vinyl groups, and two propionate side chains are attached. The iron atom lies in the center of the protoporphyrin, bonded to the four pyrrole nitrogen atoms.

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Oxygen binding occurs at the sixth coordination site. In deoxymyoglobin, the sixth coordination site remains unoccupied. The iron ion is slightly too large to fit into the well-defined hole within the porphyrin ring; it lies approximately 0.4 Å outside the porphyrin plane (Figure 7.2, left). Binding of the oxygen molecule at the sixth coordination site of the iron ion substantially rearranges the electrons within the iron so that the ion becomes effectively smaller, allowing it to move into the plane of the porphyrin (Figure 7.2, right).

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proximal histidine

The change in electronic structure that occurs when the iron ion moves into the plane of the porphyrin is paralleled by alterations in the magnetic properties of hemoglobin; these changes are the basis for functional magnetic resonance imaging (fMRI), one of the most powerful methods for examining brain function. MRI detects signals that originate primarily from the protons in water molecules and are altered by the magnetic properties of hemoglobin. With the use of appropriate techniques, images can be generated that reveal differences in the relative amount of deoxy- and oxyhemoglobin and thus the relative activity of various part of the brain.

Changes in heme electronic structure upon oxygen binding are the basis for functional imaging studies

When a specific part of the brain is active, blood vessels relax to allow more blood flow to that region. Thus more-active region of the brain will be richer in oxyhemoglobin. These noninvasive methods identify area of the brain that process sensory information.

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The Structure of Myoglobin Prevents the Release of Reactive Oxygen Species

Oxygen binding to iron in heme is accompanied by the partial transfer of an electron from the ferrous ion to oxygen. In many ways, the structure is best described as a complex between ferric ion (Fe3+) and superoxide anion (O2-), as illustrated in Figure 7.4.

It is crucial that oxygen, when it is released, leaves as dioxygen rather than superoxide, for two important reasons First, superoxide itself another species that can be generated from it are reactive oxygen species that can be damaging to many biological materials. Second, the release of superoxide leaves the iron ion in the ferric state. This species, termed metmyoglobin, does not bind oxygen. Thus, potential oxygen-storage capacity is lost. 9

Features of myoglobin stabilize the oxygen complex such that superoxide is less likely to be released. In particular, the binding pocket of myoglobin includes an additional histidine residue (termed the distal histidine) that donates a hydrogen bond to the bound oxygen molecule (Figure 7.5). The superoxide character of the bound oxygen species strengthens this interaction. Thus, the protein component of myoglobin controls the intrinsic reactivity of heme, making it more suitable for reversible oxygen binding.

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Human Hemoglobin Is an Assembly of Four Myoglobin-like Subunits

The three-dimensional structure of hemoglobin from horse heart was solved by Max Perutz shortly after the determination of the myoglobin structure. Since then, the structures of hemoglobins from other sources including human beings have been determined. Hemoglobin consist of four polypeptide chains, two identical α chains and two identical β chains (Figure 7.6). The recurring structure is called a globin fold. Consistent with this structural similarity, the amino acid sequences of the α and β chains of human hemoglobin are readily aligned with the amino acid sequence of sperm whale myoglobin with 25% and 24% identity, respectively, and good conservation of key residues such as the proximal and distal histidine residues. Thus, the α and β chains are related to each other and to myoglobin by divergent evolution. 11

Hemoglobin A (HbA) α1 β1 α2 β2

7.2 Hemoglobin Binds Oxygen Cooperatively

For myoglobin, a binding curve indicating a simple chemical equilibrium is observed (Figure 7.7). Notice that the fraction of myoglobin with bound oxygen rises sharply as pO2 increases and then levels off. Half-saturation (P50 for 50% saturated) is at the relatively low value of 2 torr (mmHg), indicating high affinity

Oxygen-binding curve: a plot of the fractional saturation versus the concentration of oxygen. The fractional saturation, Y, is defined as the fraction of possible binding sites that contain bound oxygen. The concentration of oxygen is measured by its partial pressure, pO2

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)( 502

2

PpOpOY+

=

Mb+O2 MbO2

Mb+O2 MbO2

In contrast, the oxygen-binding curve for hemoglobin in red blood cells shows some remarkable features (Figure 7.8). It does not look like a simple binding curve such as that for myoglobin; instead, it resembles an "S." Such curves are referred to as sigmoid because of their S-like shape.

Notice that oxygen binding is significantly weaker than that for myoglobin. Half-saturation is at the higher value of P50 = 26 torr. A sigmoid binding curve indicates that a protein shows a special binding behavior. The shape of the curve reveals that the binding of oxygen at one site within the hemoglobin molecule increases the likelihood that oxygen binds at the remaining unoccupied sites. Conversely, the unloading of oxygen at one heme facilitates the unloading of oxygen at the others. This sort of binding behavior is cooperative, because the binding reactions at individual sites in each hemoglobin molecule are not independent of one another.

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)( 502

2nn

n

PpOpOY+

=

Hb+nO2 Hb(O2)n

What is the physiological significance of the cooperative binding of oxygen by hemoglobin? Oxygen must be transported in the blood from the lungs, where the partial pressure of oxygen is relatively high (approximately 100 torr), to the actively metabolizing tissues, where the partial pressure of oxygen is much lower (typically, 20 torr). Let us consider how the cooperative behavior indicated by the sigmoid curve leads to efficient oxygen transport (Figure 7.9).

In the lungs, hemoglobin becomes nearly saturated with oxygen such that 98% of the oxygen-binding sites are occupied. When hemoglobin moves to the tissues and releases O2, the saturation level drops to 32%. Thus, a total of 98 - 32 = 66% of the potential oxygen-binding sites contribute to oxygen transport. 14

If myoglobin were employed for oxygen transport, it would be 98% saturated in the lungs, but would remain 91% saturated in the tissues, and so only 98 - 91 = 7% of the sites would contribute to oxygen transport; Myoglobin binds oxygen too tightly to be useful in oxygen transport. The situation might have been improved without cooperativity by the evolution of a noncooperative oxygen carrier with an optimized affinity for oxygen.

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For such a protein, the most oxygen that could be transported from a region in which pO2 is 100 torr to one in which it is 20 torr is 63 - 25 = 38%. Thus, the cooperative binding and release of oxygen by hemoglobin enables it to deliver nearly 10 times as much oxygen as could be delivered by myoglobin and more than 1.7 times as much as could be delivered by any noncooperative protein.

Closer examination of oxygen concentrations in tissues at rest and during exercise underscore the effectiveness of hemoglobin as an oxygen carrier (Figure 7.10). Under resting conditions, the oxygen in muscle is approximately 40 torr but, during exercise, the concentration is reduced to 20 torr. In the decrease from 100 torr in the lungs to 40 torr in resting muscle, the oxygen saturation of hemoglobin is reduced from 98% to 77%, and so 98- 77= 21% of the oxygen is released over a drop of 60 torr.

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In a decrease from 40 torr to 20 torr, the oxygen saturation is reduced from 77% to 32%, corresponding to an oxygen release of 45% over a drop of 20 torr. Thus, because the change in oxygen concentration from rest to exercise corresponds to the steepest part of the oxygen-binding curve, oxygen is effectively delivered to tissues where it is most needed.

Oxygen Binding Markedly Changes the Quaternary Structure of Hemoglobin

The cooperative binding of oxygen by hemoglobin requires that the binding of oxygen at one site in the hemoglobin tetramer influence the oxygen- binding properties at the other sites. Hemoglobin undergoes substantial changes in quaternary structure on oxygen binding: the α1β1 and α2β2 dimers rotate approximately 15 degrees with respect to one another (Figure 7.11). Deoxyhemoglobin is often referred to as the T (for tense) state because it is quite constrained by subunit- subunit interactions. Oxyhemoglobin is referred to as the R (for relaxed) state.

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Importantly, in the R state, the oxygen-binding sites are free of strain and are capable of binding oxygen with higher affinity than are the sites in the T state. By triggering the shift of the hemoglobin tetramer from the T state to the R state, the binding of oxygen to one site increases the binding affinity of other sites.

Hemoglobin Cooperativity Can Be Potentially Explained by Several Models

Two limiting models have been developed to explain the cooperative binding of ligands to a multisubunit assembly such as hemoglobin. In the concerted model, also known as the MWC model after Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux who first proposed it, the overall assembly can exist only in two forms: the T state and the R state. The binding of ligands simply shifts the equilibrium between these two states (Figure 7.12).

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Thus, as a hemoglobin tetramer binds each oxygen molecule, the probability that the tetramer is in the R state increases. Deoxyhemoglobin tetramers are almost exclusively in the T state. However, the binding of oxygen to one site in the molecule shifts the equilibrium toward the R state. If a molecule assumes the R quaternary structure, the oxygen affinity of its sites increases. Additional oxygen molecules are now more likely to bind to the three unoccupied sites.

nn

nn

cLcLcY)1()1()1()1( 11

αααααα

+++

+++=

−−

)( 502

2nn

n

PpOpOY+

=

[ ] [ ]00 / RTL =

TR KKc /=

RR KpOKS //][ 2==α

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The binding curve is shallow at low oxygen concentrations when all of the molecules are in the T state, becomes steeper as the fraction of molecules in the R state increases, flattens out again when all of the sites within the R-state molecules become filled (Figure 7.13). These events produce the sigmoid binding curve so important for efficient oxygen transport. 20

nn

nn

cLcLcY)1()1()1()1( 11

αααααα

+++

+++=

−−

In the concerted model, each tetramer can exist in only two states, the T state and the R state. In an alternative model, the sequential model, the binding of a ligand to one site in an assembly increases the binding affinity of neighboring sites without inducing a full conversion from the T into the R state (Figure 7. 14).

Is the cooperative binding of oxygen by hemoglobin best described by the concerted or the sequential model?

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Neither model in its pure form fully accounts for the behavior of hemoglobin. Instead, a combined model is required. Hemoglobin behavior is concerted in that hemoglobin with three sites occupied by oxygen is in the quaternary structure associated with the R state. The remaining open binding site has an affinity for oxygen more than 20-fold greater than that of fully deoxygenated hemoglobin binding its first oxygen. However, the behavior is not fully concerted, because hemoglobin with oxygen bound to only one of four sites remains primarily in the T-state quaternary structure. Yet, this molecule binds oxygen three times as strongly as does fully deoxygenated hemoglobin, an observation consistent only with a sequential model. These results highlight the fact that the concerted and sequential models represent idealized limiting cases, which real systems may approach but rarely attain.

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Structural Changes at the heme groups transmitted to the αlβ1-α2β2 Interface

We now examine how oxygen binding at one site is able to shift the equilibrium between the T and R states of the entire hemoglobin tetramer. As in myoglobin, oxygen binding causes each iron atom in hemoglobin to move from outside the plane of the porphyrin into the plane. This histidine residue is part of an α helix, which also moves (Figure 7.15). The carboxyl terminal end of this α helix lies in the interface between the two αβ dimers.

the structural transition at the iron ion in one subunit is directly transmitted to the other subunits. The rearrangement of the dimer interface provides a pathway for communication between subunits, enabling the cooperative binding of oxygen.

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2,3-Bisphosphoglycerate in Red Cells Is Crucial in Determining the Oxygen Affinity of Hemoglobin

For hemoglobin to function efficiently, the T state must remain stable until the binding of sufficient oxygen has converted it into the R state. The T state of hemoglobin is highly unstable, pushing the equilibrium so far toward the R state that little oxygen would be released in physiological conditions.

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Thus, an additional mechanism is needed to properly stabilize the T state. This mechanism was discovered by comparing the oxygen-binding properties of hemoglobin in red blood cells with fully purified hemoglobin (Figure 7.16). Pure hemoglobin binds oxygen much more tightly than does hemoglobin in red blood cells.

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This dramatic difference is due to the presence within these cells of 2,3-bisphosphoglycerate (2,3 –BPG). This highly anionic compound is present in red blood cells at approximately the same concentration as that of hemoglobin (~2 mM). Without 2,3-BPG, hemoglobin would be an extremely inefficient oxygen transporter, releasing only 8% of its cargo in the tissues. How does 2,3-BPG lower the oxygen affinity of hemoglobin so significantly?

On T-to-R transition, this pocket collapses and 2,3-BPG is released. Thus, in order for the structural transition from T to R to take place, the bonds between hemoglobin and 2,3-BPG must be broken. In the presence of 2,3-BPG, hemoglobin remains in the lower-affinity T state until higher oxygen concentrations are reached. The regulation of hemoglobin by 2,3-BPG is remarkable because 2,3 -BPG does not in any way resemble oxygen, the molecule on which hemoglobin carries out its primary function. 2,3-BPG is referred to as an allosteric effect (from allos, "other," and stereos, "structure"). 26

Examination of the crystal structure of deoxyhemoglobin in the presence of 2,3-BPG reveals that a single molecule of 2,3-BPG binds in the center of the tetramer, in a pocket present only in the T form (Figure 7.17).

The binding of 2,3-BPG to hemoglobin has other crucial physiological consequences. The globin gene expressed by human fetuses differs from that expressed by adults; fetal hemoglobin tetramers include two α chains and two γ chains. The γ chain, a result of another gene duplication, is 72% identical in amino acid sequence with the β chain.

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One noteworthy change is the substitution of a serine residue for His 143 in the β chain, part of the 2,3- BPG-binding site. This change removes two positive charges from the 2,3- BPG-binding site (one from each chain) and reduces the affinity of 2,3-BPG for fetal hemoglobin . Consequently, the oxygen-binding affinity of fetal hemoglobin is higher than that of maternal (adult) hemoglobin (Figure 7.18). This difference in oxygen affinity allows oxygen to be effectively transferred from maternal to fetal red blood cells.

pKa=6.5

7.3 Hydrogen Ions and Carbon Dioxide Promote the Release of Oxygen: The Bohr Effect

We have seen how cooperative release of oxygen from hemoglobin helps deliver oxygen to tissues where it is most needed, as revealed by their low oxygen partial pressures. This ability is enhanced by the ability of hemoglobin to respond to other cues in its physiological environment signaling the need for oxygen. Rapidly metabolizing tissues, such as contracting muscle, generate large amounts of hydrogen ions and carbon dioxide. So that oxygen is released where the need is greatest, hemoglobin has evolved to release oxygen more readily in response to higher levels of these substances. Like 2,3-BPG, hydrogen ions and carbon dioxide are allosteric effectors of hemoglobin that bind to sites on the molecule that are distinct from the oxygen-binding sites. The regulation of oxygen binding by hydrogen ions and carbon dioxide is called the Bohr effect after Christian Bohr, who described this phenomenon in 1904. 28

The oxygen affinity of hemoglobin decreases as pH decreases from a value of 7.4 (Figure 7.19). Consequently, as hemoglobin moves into a region of lower pH, its tendency to release oxygen increases. For example, transport from the lungs, with pH 7.4 and an oxygen partial pressure of 100 torr, to active muscle, with a pH of 7.2 and an oxygen partial pressure of 20 torr, a release of oxygen amounting to 77% of total carrying capacity. Only 66% of the oxygen would be released in the absence of any change in pH.

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In deoxyhemoglobin, the terminal carboxylate group of β146 forms a salt bridge with a lysine residue in the a subunit of the other αβ dimer. This interaction locks the side chain of histidine β146 in a position from which it can participate in a salt bridge with negatively charged aspartate 94 in the same chain, provided that the imidazole group of the histidine residue is protonated (Figure 7.20).

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Structural and chemical studies have revealed much about the chemical basis of the pH effect.

The formation of these salt bridges stabilizes the T state, leading to a greater tendency for oxygen to be released. For example, at high pH, the side chain of histidine β146 is not protonated and the salt bridge does not form. As the pH drops, however, the side chain of histidine β146 becomes protonated, the salt bridge with aspartate β 94 forms, and the T state is stabilized.

Carbon dioxide, a neutral species, passes through the red-blood-cell membrane into the cell. Carbon dioxide stimulates oxygen release by two mechanisms. First, the presence of high concentrations of carbon dioxide leads to a drop in pH within the red blood cell (Figure 7.21). Carbon dioxide reacts with water to form carbonic acid, H2CO3. accelerated by carbonic anhydrase Carbonic acid is a strong acid with a pKa of 3.5. Thus, once formed, carbonic acid dissociates to form bicarbonate ion, HCO3

-, and H+, resulting in a drop in pH. This drop in pH stabilizes the T state

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In the second mechanism, a direct chemical interaction between carbon dioxide and hemoglobin stimulates oxygen release. The effect of carbon dioxide on oxygen affinity can be seen by comparing oxygen-binding curves in the absence and presence of carbon dioxide at a constant pH (Figure 7.22). the amount of oxygen released approaches 90% Carbon dioxide stabilizes deoxyhemoglobin by reacting with the terminal amino groups to form carbamate groups, negatively charged in contrast with the neutral or positive charges carbamate groups participate in salt-bridge interactions that stabilize the T state 32

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Carbamate formation also provides a mechanism for carbon dioxide transport from tissues to the lungs, but it accounts for only 14% of the total carbon dioxide transport. Most carbon dioxide released from red blood cells is transported to the lungs in the form of HCO3

- (Figure 7.23).

Much of the HCO3- that is formed leaves the cell through a specific membrane-

transport protein that exchanges HCO3- from one side of the membrane for Cl- from

the other side. In the lungs, this process is reversed: HCO3

- is converted back into carbon dioxide and exhaled. Thus, carbon dioxide generated by active tissues contributes to a decrease in red-blood-cell pH and, hence, to oxygen release and is converted into a form that can be transported in the serum and released in the lungs.

7.4 Mutations in Genes Encoding Hemoglobin Subunits Can Result in Disease In modern times, particularly after the sequencing of the human genome, to think of genetically encoded variations in protein sequence as a factor in specific diseases is routine. The notion that diseases might be caused by molecular defects was proposed by Linus Pauling in 1949 (4 years before Watson and Crick's proposal of the DNA double helix) to explain the blood disease sickle-cell anemia.

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The name of the disorder comes from the abnormal sickle shape of red blood cells deprived of oxygen that is seen in people suffering from this disease (Figure 7.24). Pauling proposed that sickle-cell disease might be caused by a specific variation in the amino acid sequence of one hemoglobin chain. Today, we know that this bold hypothesis is correct. In fact, approximately 7% of the world's population are carriers of some disorder of hemoglobin caused by a variation in the amino acid sequence.

Sickle-Cell Anemia Results from the Aggregation of Mutated Deoxyhemoglobin Molecules

The results may be painful swelling of the extremities and a higher risk of stroke or bacterial infection (due to poor circulation). The sickled red cells also do not remain in circulation as long as normal cells do, leading to anemia. What is the molecular defect associated with sickle-cell anemia? Vernon Ingram demonstrated in 1956 that a single amino acid substitution in the β chain of hemoglobin is responsible namely, the substitution of a valine residue for a glutamate residue in position 6. The mutated form is referred to as hemoglobin S (HbS). The HbS substitution substantially decreases the solubility of deoxyhemoglobin, although it does not markedly alter the properties of oxyhemoglobin. 35

The hemoglobin molecules have formed large fibrous aggregates These fibers extend across the red blood cells, distorting them so that they clog small capillaries and impair blood flow.

Examination of the structure of hemoglobin S reveals that the new valine residue lies on the surface of the T-state molecule (Figure7.26). This new hydrophobic patch interacts with another hydrophobic patch formed by Phe 85 and Val 88 of the β chain of a neighboring molecule to initiate the aggregation process.

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More-detailed analysis reveals that a single hemoglobin S fiber is formed from 14 chains of multiple interlinked hemoglobin molecules. Why do these aggregates not form when hemoglobin S is oxygenated? Oxygenated hemoglobin S is in the R state, and residues Phe 85 and Val 88 on the β chain are largely berried inside the hemoglobin assembly. Without a partner which to interact, the surface Val residue in position 6 is benign.

Approximately 1 in 100 West Africans suffer from sickle-cell anemia. Why is the HbS mutation so prevalent in Africa and in some other regions? People with one copy of the HbB gene and one copy of the HbS are unaffected. They are said to have sickle-cell trait because they can pass HbS to their offspring. However, people with sickle-cell trait are resistant to malaria, a disease carried by a parasite, Plasmodium falciparum, that lives within red blood cells at one stage in its life cycle. The dire effect of malaria on health and reproductive likelihood in regions where malaria has been historically endemic has favored people with sickle-cell trait, increasing the prevalence of the HbS allele (Figure 7.27).

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Thalassemia Is Caused by an Imbalanced Production of Hemoglobin Chains Thalassemia, the other prevalent inherited disorder of hemoglobin, is caused by the loss or substantial reduction of a single hemoglobin chain. The result is low levels of functional hemoglobin and a decreased production of red blood cells, which may lead to anemia, fatigue, pale skin, and spleen and liver malfunction. Thalassemia is a set of related diseases. In α -thalassemia, the α chain of hemoglobin is not produced in sufficient quantity. Consequently, hemoglobin tetramers form that contain only the β chain. These tetramers, referred to as hemoglobin H (HbH), bind oxygen but with high affinity and no cooperativity. Thus, oxygen release in the tissues is poor. In β-thalassemia, the β chain of hemoglobin is not produced in sufficient quantity. In the absence of β chains, the α chains form insoluble aggregates that precipitate inside immature red blood cells. The loss of red blood cells results in anemia. 38

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Both α- and β-thalassemia are associated with many different genetic variations and display a wide range of clinical severity. The most severe forms of α -thalassemia are usually fatal shortly before or just after birth. However, these forms are relatively rare. An examination of the repertoire of hemoglobin genes in the human genome provides one explanation. Normally, human beings have not two but four alleles for the α chain, arranged such that a pair of genes are located adjacent to each other on one end of each chromosome 16. Thus, the complete loss of α-chain expression requires the disruption of four alleles. β-Thalassemia is more common because we normally have only two alleles for the β chain, one on each copy of chromosome 11.

The Accumulation of Free Alpha-Hemoglobin Chains Is Prevented The presence of four genes expressing the α chain, compared with two for the Β chain, suggests that the α chain would be produced in excess (making the overly simple assumption that production from each gene is comparable). If this is correct, why doesn't the excess α chain precipitate? One mechanism for maintaining α chains in solution was revealed by the discovery of an 11-kd protein in red blood cells called α-hemoglobin stabilizing protein (AHSP). This protein forms a soluble complex with newly synthesized α-chain monomers.

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The crystal structure of a complex between AHSP and α -hemoglobin reveals that AHSP binds to the same face of α-hemoglobin as does β-hemoglobin (Figure 7.28). AHSP binds the α chain in both the deoxygenated and oxygenated forms. In the complex with oxygen bound, the distal histidine, rather than the proximal histidine, binds the iron atom.

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AHSP serves to bind and ensure the proper folding of α -hemoglobin as it is produced. As β-hemoglobin is produced, it displaces AHSP because the α-hemoglobin–β-hemoglobin dimer is more stable than the α-hemoglobin- AHSP complex. Thus, AHSP prevents the accumulation and precipitation of free α-hemoglobin. Studies are underway to determine if mutations in the gene encoding AHSP playa role in modulating the severity of β-thalassemia.

Additional Globins Are Encoded in the Human Genome

In addition to the gene for myoglobin, the two genes for α-hemoglobin, and the one for β-hemoglobin, the human haploid genome contains other globin genes. We have already encountered fetal hemoglobin, which contains the γ chain in place of the β chain. Several other genes encode other hemoglobin subunits that are expressed during development including the δ chain, the ε chain, and the ζ (zeta) chain. Examination of the human genome sequence has revealed two additional globins. Both of these proteins are monomeric proteins, more similar to myoglobin than to hemoglobin. The first, neuroglobin, is expressed primarily in the brain and at especially high levels in the retina. Neuroglobin may playa role in protecting neural tissues from hypoxia (insufficient oxygen). The second, cytoglobin, is expressed more widely throughout the body. 42