Oxygen Transport

Vertebrates are aerobic organisms

They have a closed circulatory system and a mechanism for extraction of O₂ from air (or water), and release of carbon dioxide (CO₂) in waste products. Inspired O₂ leads to an efficient utilization of metabolic fuels, such as glucose and fatty acids; expired CO₂ is a major product of cellular metabolism. This utilization of O₂ as a metabolic substrate is accompanied by the generation of reactive oxygen species (ROS) that are capable of damaging virtually all biological macromolecules (Chapter 37). Organisms protect themselves from radical damage in several ways: sequestering O₂, limiting production of ROS, and detoxifying them. Heme proteins, interestingly, participate in these protective mechanisms by sequestering and transporting oxygen. The major heme proteins in mammals are myoglobin (Mb) and hemoglobin (Hb). Mb is found primarily in skeletal and striated muscle, and serves to store O₂ in the cytoplasm and deliver it on demand to the mitochondrion. Hb is restricted to erythrocytes where it facilitates the transport of O₂ and CO₂ between the lungs and peripheral tissues. This chapter presents the molecular features of Mb and Hb, the biochemical and physiologic relationships between the structures of Mb and Hb and their interaction with O₂ and other small molecules, and the pathologic aspects of selected Hb mutations.

Most oxygen in the body is bound to a carrier protein containing heme

Photosynthetic organisms release diatomic oxygen into the earth's atmosphere during energy production, contributing to the current level of 21% oxygen in air. In mixtures of gases. Each component makes a specific contribution, known as its partial pressure (Dalton's Law), that is directly proportional to its concentration. It is also customary to use the partial pressure of a gas as a measure of its concentration in physiologic fluids. For atmospheric O₂ at a barometric pressure (sea level) of 760 mmHg or torr (101.3 kP or kPa; 1 atmosphere absolute or ATA), the partial pressure of oxygen, pO₂, is 150–160 mmHg. The amount of O₂ in solution is, in turn, directly proportional to its partial pressure. Thus, in the arterial blood (37°C, pH 7.4) the pO₂ is 100 mmHg, which produces a concentration of dissolved O₂ of 0.13 mmol/L. This level of dissolved O₂, however, is inadequate to support efficient aerobic metabolism.

Rather, the major fraction of O₂ transported in blood and stored in muscle is complexed with the iron (ferrous, Fe²⁺) proteins Hb and Mb, respectively. Hb is a tetrameric protein with four O₂-binding sites (heme groups). In arterial blood with a Hb concentration of 150 g/L (2.3 mmol/L) and O₂ saturation of 97.4%, the contribution of protein-bound O₂ is about 8.7 mmol/L. This concentration represents a dramatic 67-fold increase over physically dissolved O₂. The total oxygen-carrying capacity of the arterial blood, in dissolved and protein-bound forms, is 8.8 mmol/L – almost 200 mL of dissolved oxygen per liter of blood.

Globins constitute an ancient family of soluble metalloproteins

Globins are ubiquitous family of proteins, found in microorganisms, plants, invertebrates, and vertebrates. Present-day globins, with their spectacular diversity of function, are most likely derived from a single ancestral globin. While the extent of amino acid identity among invertebrate and vertebrate globins varies widely and can often appear random, two features are noteworthy: the invariant residues PheCD1 and HisF8 and the characteristic patterns of hydrophobic residues in helical segments (Fig. 5.1). Human Mb consists of a single globin polypeptide (153 amino acid residues, 17,053 Da). Human Hb is a tetrameric assembly of two α-globin polypeptides (141 residues, 15,868 Da) and two β-globin polypeptides (146 residues, 15,126 Da). A single heme prosthetic group is noncovalently associated with each globin apoprotein.

The secondary structure of mammalian globins is dominated by a high proportion of α-helix, with over 75% of the amino acids associated with eight helical segments. These α-helices are organized into a tightly packed, nearly spherical, tertiary structure, designated the globin fold (Fig. 5.2). So universal is this overall tertiary structure among all globins that the conventional nomenclature for globin residues follows that defined initially for sperm whale Mb, namely helices A, B, C, etc., starting at the N-terminus, separated by corners AB, BC, etc., with residues numbered within each helix and corner. For example, residue A14, an amino acid that participates in electrostatic stabilization between helix A and the GH corner, corresponds to Lys¹⁵ in insect Hb, Lys¹⁶ in Mb and α-globin, and Lys¹⁷ in β-globin (arrow, lower center, Fig. 5.2).

Polar amino acids are located almost exclusively on the exterior surface of globin polypeptides and contribute to the remarkably high solubility of these proteins (e.g. 370 g/L (37% protein solution; 5.7 mmol/L) Hb in the erythrocyte). Amino acids that are both polar and hydrophobic, such as threonine, tyrosine and tryptophan, are oriented with their polar functions toward the protein's exterior. Hydrophobic residues are buried within the interior, where they stabilize the folding of the polypeptide and form a pocket that accommodates the heme prosthetic group. Notable exceptions to this general distribution of amino acid residues in globins are the two histidines that play indispensable roles deep within the heme pocket (Fig. 5.3). Their side chains are oriented perpendicular to and lay on either side of the heme prosthetic group. One of the side chain imidazole nitrogens of the invariant proximal histidine (HisF8) is close enough to bond directly to the pentacoordinate Fe²⁺ atom. On the opposite side of the heme plane the distal histidine (HisE7), which is too far from the heme iron for direct bonding, functions to stabilize bound O₂ by hydrogen bonding.

Heme, the O₂-binding moiety common to Mb and Hb, is a porphyrin molecule to which an iron atom (Fe²⁺) is coordinated

The Fe-porphyrin prosthetic group of heme is planar and hydrophobic, with the exception of two propionate groups which are exposed to solvent. Heme becomes an integral component of the holoprotein during polypeptide synthesis; it is heme that gives globins, their characteristic purple-red color – purple in the deoxygenated state in venous blood, red in the oxygenated state in arterial blood.

Globins increase the aqueous solubility of the otherwise poorly soluble, hydrophobic heme prosthetic group. Once sequestered inside a hydrophobic pocket created by the folded globin polypeptide, heme is in a protective environment that minimizes the spontaneous oxidation of Fe²⁺ (ferrous) to Fe³⁺ (ferric: rusting) in the presence of O₂. Such an environment is also essential for globins to bind and release O₂. Should the iron atom become oxidized to the ferric state, heme can no longer interact reversibly with O₂, compromising its function in O₂ storage and transport.

Mb binds O2 that has been released from Hb in tissue capillaries and subsequently diffused into tissues

Located in the cytosol of skeletal, cardiac and some smooth muscle cells, Mb stores a supply of O₂ that is readily available to cellular organelles, particularly the mitochondrion, that carry out oxidative metabolism. With its single ligand-binding site, the reversible reaction of Mb with O₂:

may be described by the following equations:

where K_a is an affinity or equilibrium constant, and Y is the fractional O₂ saturation. Combining these two equations, expressing the concentration of O₂ in terms of its partial pressure (pO₂), and substituting the term P₅₀ for 1/K_a yields the equation for the O₂ saturation curve of Mb:

By definition, the constant P₅₀ is the value of pO₂ at which Y = 0.5 or half the ligand sites are occupied by O₂. In a plot of Y versus pO₂, the equation for ligand binding by Mb describes a hyperbola (Fig. 5.4) with a P₅₀ of 4 mmHg. The low value of P₅₀ reflects a high affinity for O₂. In the capillary beds of muscle tissues, pO₂ values are in the range of 20–40 mmHg. Predictably, working muscles exhibit lower pO₂ values than muscles at rest. With its high affinity for O₂, myocyte Mb readily becomes saturated with O₂ that has entered from the blood. As O₂ is consumed during aerobic metabolism, it dissociates through mass action from Mb and diffuses into mitochondria, the power plants of the muscle cell. Whales and other diving mammals have unusually high concentrations of Mb in their muscle tissue; the Mb is thought to function as a large oxygen reservoir for prolonged periods under water.

Hb is the principal O₂-transporting protein in human blood; it is localized exclusively in erythrocytes

Adult Hb (HbA) is a tetrahedral array of two identical α-globin, and two identical β-globin subunits, a geometry that predicts several types of subunit–subunit interactions in the quaternary structure (Fig. 5.5). Importantly, within the Hb tetrahedron each subunit is in contact with the other three. Experimental analysis of the quaternary structure indicates multiple noncovalent interactions (hydrogen bonds and electrostatic bonds) between each pair of dissimilar subunits, i.e. at the α–β interfaces. In contrast, there are fewer and predominantly hydrophobic interactions between identical subunits, at the α₁–α₂ or β₁–β₂ interfaces. The actual number and nature of contacts differ in the presence or absence of O₂. Strong associations within each αβ heterodimer and at the interface between the two heterodimers (see Fig. 5.5) are now recognized as major factors determining O₂ binding and release. Thus, Hb is more appropriately considered a dimer of heterodimers, (αβ)₂, rather than an α₂β₂ tetramer. Although a solution of HbA is theoretically a dynamic mixture of heterodimers and tetramers, under physiologic conditions (high Hb and neutral pH) the equilibria greatly favor the tetramer: 99% for oxygenated Hb, and 99.9% for deoxygenated Hb.

Hb binds oxygen cooperatively, with a Hill coefficient of ~2.7

As a gas delivery vehicle, Hb must be able to bind O₂ efficiently as it enters the lung alveoli during respiration and to release O₂ to the extracellular environment with similar efficiency as erythrocytes circulate through tissue capillaries. This remarkable duality of function is achieved by cooperative interactions among globin subunits. When deoxygenated Hb becomes oxygenated, significant structural changes extend throughout the protein molecule. In the heme pocket, as a consequence of O₂ coordination to iron and a new orientation of atoms in the heme structure, the proximal histidine and helix F to which it belongs, shift their positions (see Fig. 5.3). This subtle conformational change triggers major structural realignments elsewhere within that globin subunit. In turn, these tertiary structural changes are transmitted, even amplified, in the overall quaternary structure, such that a 12–15° rotation and a 0.10 nm displacement of the α₁β₁ dimer relative to the α₂β₂ dimer take place. Because of the inherent asymmetry of the α₂β₂ tetramer, these combined motions result in quite dramatic changes within and, more importantly, between the αβ heterodimers. Because of structural changes in hemoglobin as a result of binding of oxygen and other effectors, the binding affinity for subsequent molecules of oxygen may be increased (positive cooperativity) or decreased (negative cooperativity).

Hb can bind up to four molecules of O₂ in a cooperative manner

With its multiple ligand-binding sites and structural changes in response to binding, the oxygen affinity and the fractional saturation of Hb are more complex functions than those of Mb. Consequently, the equation for the fractional O₂ saturation curve must be modified to:

where n is the Hill coefficient. In a plot of Y versus pO₂ when n > 1, the equation for ligand binding describes a sigmoid (S-shaped) curve (see Fig. 5.4). The Hill coefficient, determined experimentally, is a measure of cooperativity among ligand-binding sites, i.e. the extent to which the binding of O₂ to one subunit influences the affinity of O₂ to other subunits. For fully cooperative binding, n is equal to the number of sites (four in Hb), an indication that binding at one site maximally enhances binding at other sites in the same molecule. The normal Hill coefficient for adult Hb (n = 2.7) reflects strongly cooperative ligand binding. Hb has a considerably lower affinity for O₂, reflected in a P₅₀ of 27 ± 2 mmHg, compared to myoglobin (P₅₀ = 4 mmHg). In the absence of cooperativity, even with multiple sites, the Hill coefficient would be 1, i.e. binding of one molecule of O₂ would not influence the binding of other molecules. Decreased or absent cooperativity is observed for Hb mutants that have lost functional subunit–subunit contacts (Table 5.1). The steepest slope of the saturation curve for Hb lies in a range of pO₂ that is found in most tissues (see Fig. 5.4). Thus, relatively small changes in pO₂ will result in considerably larger changes in the interaction of Hb with O₂. Accordingly, slight shifts of the curve in either direction will also dramatically influence O₂ affinity.

Hemoglobin subunits may assume two different conformations that differ in O₂ affinity

The mechanism underlying the cooperativity in oxygen binding by hemoglobin involves a shift between two conformational states of the hemoglobin molecule which differ in oxygen affinity. These two quaternary conformations are known as the T (tense) and R (relaxed) states, respectively. In the T state, interactions between the heterodimers are stronger; in the R state, these noncovalent bonds are, in summation, weaker. O₂ affinity is lower in the T state and higher in the R state. Transition between these states is accompanied by the breaking of existing noncovalent bonds and formation of new ones at the heterodimer interfaces (Fig. 5.6). Contact between the two αβ heterodimers (see Fig. 5.5) is stabilized by a mixture of hydrogen and electrostatic bonds. Approximately 30 amino acids participate in the noncovalent interactions that characterize the deoxygenated and oxygenated Hb conformations.

Several models have been developed to describe the transition between the T and R states of Hb. At one extreme is a model in which each Hb subunit sequentially responds to O₂ binding with a conformational change, thereby permitting hybrid intermediates of the T and R states. At the opposite extreme is a model in which all four subunits switch concertedly; hybrid states are forbidden, and binding of O₂ to one subunit shifts the equilibrium of all subunits from the T to the R state simultaneously. The molecular structures of deoxygenated and partially and fully ligated Hb have been studied extensively by a broad range of thermodynamic and kinetic techniques. Yet, progress toward reconciling inconsistencies among classic and more recent models has been slow. These different viewpoints on conformational changes in multi-subunit proteins are discussed further in the section on allosteric enzymes in Chapter 6.

Hb is an allosteric protein; its affinity for O2 is regulated by small molecules

Hb is one of the best studied examples of an allosteric protein, These small bind to proteins at sites that are spatially distinct from the ligand-binding sites, thus their designation as allosteric (other site) effectors. Through long-range conformational effects, they alter the ligand or substrate binding affinity of the protein. Allosteric proteins are typically multi-subunit proteins. The O₂ binding affinity of Hb is affected positively by O₂, as well as by a number of chemically diverse allosteric effectors, including H⁺, CO₂, and 2,3-bisphosphoglycerate (2,3-BPG) (Fig. 5.7). When an allosteric effector affects its own binding to the protein (at other sites), the process is termed homotropic, e.g. the effect of binding of O₂ at one site on Hb enhances the affinity for binding of O₂ to other sites on Hb. When the allosteric effector is different from the ligand whose binding is altered, the process is termed heterotropic, e.g. the effect of H⁺ (pH) on the P₅₀ for oxygen binding to Hb. These interactions lead to horizontal shifts in the O₂ binding curves (see Fig. 5.7).

Acidic pH (protons) decreases the O₂ affinity of Hb

The O₂ affinity of Hb is exquisitely sensitive to pH, a phenomenon known as the Bohr effect. The Bohr effect is most readily described as a right shift in the O₂ saturation curve with decreasing pH. Thus, an increased concentration of H⁺ (decreased pH) favors an increased P₅₀ (lower affinity) for O₂ binding to Hb, equivalent to an H⁺-dependent shift of Hb from the R to the T state.

To understand the Bohr effect at the level of protein structure and to appreciate the role of H⁺ as a heterotropic allosteric effector, it is important to recall that Hb is a highly charged molecule. The residues that participate in the Bohr effect include the N-terminal Val amino group of α-globin and the C-terminal His side chain of β-globin. The pK_a values of these weak acids differ sufficiently between the deoxygenated and oxygenated forms of Hb to cause the uptake of 1.2–2.4 protons by the deoxygenated, compared to oxygenated, tetramer.

Identification of specific amino acid residues of the α- and β-globins that participate in the Bohr effect is complicated by differential interactions of other charged solutes with deoxy- and oxy-Hb. Thus, a preferential binding of a given anion, i.e. Cl⁻ and/or organic phosphates, to deoxygenated Hb involves the alteration of the pKs of some cationic groups, thereby contributing to the overall observed Bohr effect. For example, there is compelling evidence showing that Val^1(α) is relevant to the Bohr effect only in the presence of Cl⁻. The pK_a of this group shifts from 8.0 in deoxygenated Hb to 7.25 in oxygenated Hb in the presence of physiologic concentration of Cl⁻ (≈100 mmol/L). Further, the participation of the Val^1(α) groups in the chloride-dependent Bohr effect is strongly modulated by CO₂ because of the formation of CO₂ (carbamino) adducts of Hb (described below).

As Hb binds O₂, protons dissociate from selected weak acid functions. Conversely, in acidic media, protonation of the conjugate bases inhibits O₂ binding

During their circulation between pulmonary alveoli and peripheral tissue capillaries, erythrocytes encounter markedly different conditions of pO₂ and pH. The high pO₂ in the lungs promotes ligand saturation and forces protons from the Hb molecule to stabilize the R state. In the capillary bed, particularly in metabolically active tissues, the pH is slightly lower, due to the production of acidic metabolites, such as lactate. Oxygenated Hb, upon entering this environment, will acquire some ‘excess’ protons and shift toward the T state, promoting release of O₂ for uptake by tissues for aerobic metabolism.

Like H⁺, CO₂ is is increased in venous capillaries and is a negative allosteric effector of the O₂ affinity of Hb

Closely related to the Bohr effect is the ability of CO₂ to alter the O₂ affinity of Hb. The increase in pCO₂ in venous capillaries decreases the affinity of Hb for O₂. Accordingly, a right shift in the ligand saturation curve occurs as pCO₂ increases. It should be emphasized that the allosteric effector is, in fact, CO₂, not HCO₃⁻: CO₂ reacts reversibly with the unprotonated N-terminal amino groups of the globin polypeptides to form carbamino adducts:

This transient covalent chemical modification of Hb is not only a specialized example of allosteric control, resulting in a stabilization of deoxygenated Hb; it also represents one form of transport of CO₂ to the lungs for clearance from the body. Between 5% and 10% of the total CO₂ content of blood exists as carbamino adducts.

There is a strong physiologic correlation between pCO₂ and the O₂ affinity of Hb. CO₂ is a major product of mitochondrial oxidation and, like H⁺, is particularly abundant in metabolically active tissues. Upon diffusing into blood, CO₂ can react with oxygenated Hb, shift the equilibrium toward the T state, and thereby promote the dissociation of bound O₂ (see Fig. 5.7). The vast majority of peripheral tissue CO₂, however, is hydrated by erythrocyte carbonic anhydrase to carbonic acid (H₂CO₃), a weak acid that dissociates partially to H⁺ and HCO₃⁻:

Interestingly, from both carbamino adduct formation and hydration/dissociation reactions involving CO₂, an additional pool of protons is generated. These are protons that become available to participate in the Bohr effect and facilitate O₂–CO₂ exchange. During its return to the lungs, blood transports two forms of CO₂: carbamino-Hb and the H₂CO₃/HCO₃⁻ acid–conjugate base pair. Blood and Hb are now exposed to a low pCO₂, and through mass action the carbamino adduct formation is reversed and binding of O₂ is again favored. Similarly, in the pulmonary capillaries, erythrocyte carbonic anhydrase converts H₂CO₃ to CO₂ and H₂O, which are expired into the atmosphere (see Chapter 25).

Working muscles not only produce the allosteric effectors H⁺ and CO₂ as byproducts of aerobic metabolism but also liberate heat. Because the binding of O₂ to heme is an exothermic process, the O₂ affinity of Hb decreases with increasing temperature. Thus, the microenvironment of an exercising muscle profoundly favors a more efficient release of Hb-bound O₂ to the surrounding tissue.

2-3-Bisphoglycerate (2,3-BPG), an intermediate in carbohydrate metabolism, is an important allosteric effector of Hb

2,3-BPG is synthesized in human erythrocytes in a one-step shunt off the glycolytic pathway (Chapter 12). Like H⁺ and CO₂, 2,3-BPG is an indispensable negative allosteric effector that, when bound to Hb, causes a marked increase in P₅₀ (see Fig. 5.7). Were it not for the high erythrocyte concentration of 2,3-BPG (4.1 mmol/L, nearly equal to that of Hb), the O₂ saturation curve of Hb would approach that of Mb!

At one end of the twofold symmetry axis within the quaternary structure of Hb there is a shallow cleft defined by cationic amino acids of the juxtaposed β-globin subunits (Fig. 5.8). A single molecule of 2,3-BPG binds to this site. A critical consequence of the conformational differences between the T and R states is that deoxygenated Hb preferentially interacts with the negatively charged 2,3-BPG. Multiple electrostatic interactions stabilize the complex between the polyanionic effector and deoxygenated Hb. The cleft is too narrow in fully oxygenated Hb to accommodate 2,3-BPG.

The importance of 2,3-BPG as an allosteric effector is underscored by observations that its concentration in the erythrocyte changes in response to various physiologic and pathologic conditions. During chronic hypoxia (decreased pO₂) secondary to pulmonary disease, anemia or shock, the level of 2,3-BPG increases. Such compensatory increases have also been described in cigarette smokers and on adaptation to high altitudes. The net result is a greater stabilization of the deoxygenated, low-affinity T state and a further shift of the saturation curve to the right, thereby facilitating release of more O₂ to tissues. Under most circumstances, the rightward shift has an insignificant effect on the O₂ saturation of Hb in the lungs.

Nitric oxide, a potent vasodilator, is stored on Hb as S-nitrosoHb (SNO-Hb)

Nitric oxide (NO) is a gaseous free radical capable of oxidative modification (nitration, nitrosation, nitrosylation) of biological macromolecules. Yet this highly reactive molecule, also known as endothelium-derived relaxing factor (EDRF), is synthesized in endothelial cells and participates in normal vascular physiology, including vasodilation (smooth muscle), hemostasis (platelet), and adhesion molecule expression (endothelial cell). Erythrocytes are the largest intravascular reservoir of bioactive NO, and Hb is indispensable for its formation, storage, and release. SNO-Hb is the product of S-nitrosylation of the Cys^93β side chains of Hb. These Cys thiol groups can accept NO by transfer from intracellular S-nitrosoglutathione or from heme-bound NO (nitrosyl-Hb). NO is released by exchange from SNO-Hb to Cys side chains of anion exchanger 1, an erythrocyte membrane protein that can then deliver NO to the plasma. The formation and breakdown of SNO-Hb are sensitive to pO; NO is released from Hb in response to hypoxia or on conversion to the T state, e.g. in venous capillaries.

Another remarkable process within the erythrocyte is the allosterically regulated conversion of nitrite (NO₂⁻) to NO, a reaction performed by deoxygenated Hb. This intrinsic ‘nitrite reductase’ activity takes advantage of the moderate NO₂⁻ concentration in the erythrocyte (up to 0.3 µmol/L). While the chemistry is complex, the reaction is thought to yield a labile intermediate nitrosyl-metHb(ferric) that can readily transfer NO to Cys^93(β) on oxygenated Hb.

Two other globins have recently been identified in humans

Neuroglobin (Ngb) is expressed primarily in the central nervous system and some endocrine tissues; cytoglobin (Cygb) is ubiquitously expressed, primarily in cells of fibroblast origin. Tissue concentrations of both are <1 mmol/L. The Ngb polypeptide has 151 amino acid residues (16,933 Da), whereas Cygb contains 190 residues (21,405 Da), with ‘extensions’ of 20 amino acids at both the N- and C-termini (see Fig. 5.1). Both human proteins share only about 25% sequence identity with Mb and Hb. Yet all key elements of the globin fold are present: the three-over-three α-helix sandwich; the proximal and distal His residues; and a hydrophobic, heme-containing pocket.

In contrast to Mb and Hb, Ngb and Cygb contain hexacoordinate hemes for both the Fe²⁺ and Fe³⁺ valency states. The distal HisE7, serving as the sixth ligand, must be displaced to permit binding of O₂. Yet the O₂ affinities of Ngb and Cygb are surprisingly high, with P₅₀ values in the range 1–7.5 mmHg and 0.7–1.8 mmHg, respectively, compared to a P₅₀<27 mmHg for Hb. Binding of O₂ to the dimeric Cygb is cooperative (Hill coefficient = 1.2–1.7) but independent of pH. On the other hand, monomeric Ngb exhibits a pH-dependent O₂ affinity. The functions of these minor globins remain elusive. Ngb appears to be comparable to Mb, mediating the delivery of O₂ to retina mitochondria. Cygb is thought to function as an enzyme cofactor, supplying O₂ for the hydroxylation of Pro and Lys side chains in some proteins.

In sickle cell disease (SCD), distortion of erythrocyte structure (sickling) limits capillary blood flow

Clinically, an individual with SCD presents with intermittent episodes of hemolytic anemia, resulting from chronic lysis of red cells, and painful vaso-occlusive crises. Common features also include impaired growth, increased susceptibility to infections, and multiple organ damage. In the African-American population in the US, SCD affects 90,000–100,000 individuals, a frequency of 0.2%. Heterozygous, mostly asymptomatic, carriers, number 8% in this same population.

SCD is caused by an inherited, single point mutation in the gene encoding β-globin, leading to the expression of the Hb variant HbS. Indeed, HbS has been studied biochemically, biophysically, and genetically for over 50 years, making SCD the paradigm of a molecular disease. The mutation is Glu^6(β)→Val: a surface-localized charged amino acid is replaced by a hydrophobic residue. Valine on the mutant β-globin subunit fits into a complementary pocket (sometimes called a ‘sticky patch’) formed on the β-globin subunit of a deoxygenated Hb molecule, a pocket that becomes exposed only upon the release of bound O₂ in tissue capillaries.

HbA remains a true solute at rather high concentrations, largely as a result of a polar exterior surface that is compatible and nonreactive with nearby Hb molecules. In contrast, HbS, when deoxygenated, is less soluble. It forms long, filamentous polymers that readily precipitate, distorting erythrocyte morphology to the characteristic sickle shape. In the homozygous individual with SCD (HbS/HbS), the complex process of nucleation and polymerization occurs rapidly, producing about 10% of circulating erythrocytes that are sickled. In the heterozygous individual (HbA/HbS, sickle cell trait), the kinetics of sickling are decreased by at least a factor of 1000, thereby accounting for the asymptomatic nature of this genotype. In dilute solution, HbS has interactions with O₂ (P₅₀ value, Hill coefficient) that are similar to those for HbA. However, the Bohr effect on concentrated HbS is more pronounced, leading to greater release of O₂ in the capillaries and increased propensity for sickling.

Sickled erythrocytes exhibit less deformability. They no longer move freely through the microvasculature and often block blood flow, especially in the spleen and joints. Moreover, these cells lose water, become fragile, and have a considerably shorter life span, leading to hemolysis and anemia. Except during extreme physical exertion, the heterozygous individual appears normal. For reasons that remain to be elucidated, heterozygosity is associated with an increased resistance to malaria, specifically growth of the infectious agent Plasmodium falciparum in the erythrocyte. This observation represents an example of a selective advantage that the HbA/HbS heterozygote exhibits over either the HbA/HbA normal or the HbS/HbS homozygote and probably offers an explanation for the persistence of HbS in the gene pool.

More than 1000 mutations in the genes encoding the α- and β-globin polypeptides have been documented

As with most mutational events, most lead to few, if any, clinical problems. There are, however, several hundred mutations that give rise to abnormal Hb and pathologic phenotypes. Hb mutants or hemoglobinopathies are usually named after the location (hospital, city or geographical region) in which the abnormal protein was first identified. They are classified according to the type of structural change and altered function and the resulting clinical characteristics (see Tables 5.1, 5.2). While many of these mutants have predictable phenotypes, others are surprisingly pleiotropic in their impact on multiple properties of the Hb molecule. With few exceptions, Hb variants are inherited as autosomal recessive traits. Occasionally, double heterozygotes are identified, e.g. HbSC (Fig. 5.9).