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Biochemistry_Lippincott_83	Biochemistry_Lippinco	For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW The previous chapter described the types of secondary and tertiary structures that are the bricks and mortar of protein architecture. By arranging these fundamental structural elements in different combinations, widely diverse proteins can be constructed that are capable of various specialized functions. This chapter examines the relationship between structure and function for the clinically important globular hemeproteins. Fibrous structural proteins are discussed in Chapter 4. II. GLOBULAR HEMEPROTEINS	Biochemistry_Lippinco. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW The previous chapter described the types of secondary and tertiary structures that are the bricks and mortar of protein architecture. By arranging these fundamental structural elements in different combinations, widely diverse proteins can be constructed that are capable of various specialized functions. This chapter examines the relationship between structure and function for the clinically important globular hemeproteins. Fibrous structural proteins are discussed in Chapter 4. II. GLOBULAR HEMEPROTEINS
Biochemistry_Lippincott_84	Biochemistry_Lippinco	II. GLOBULAR HEMEPROTEINS Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group. (See p. 54 for a discussion of prosthetic groups.) The role of the heme group is dictated by the environment created by the three-dimensional structure of the protein. For example, the heme group of a cytochrome functions as an electron carrier that is alternately oxidized and reduced (see p. 75). In contrast, the heme group of the enzyme catalase is part of the active site of the enzyme that catalyzes the breakdown of hydrogen peroxide (see p. 148). In hemoglobin and myoglobin, the two most abundant hemeproteins in humans, the heme group serves to reversibly bind oxygen (O2). A. Heme structure	Biochemistry_Lippinco. II. GLOBULAR HEMEPROTEINS Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group. (See p. 54 for a discussion of prosthetic groups.) The role of the heme group is dictated by the environment created by the three-dimensional structure of the protein. For example, the heme group of a cytochrome functions as an electron carrier that is alternately oxidized and reduced (see p. 75). In contrast, the heme group of the enzyme catalase is part of the active site of the enzyme that catalyzes the breakdown of hydrogen peroxide (see p. 148). In hemoglobin and myoglobin, the two most abundant hemeproteins in humans, the heme group serves to reversibly bind oxygen (O2). A. Heme structure
Biochemistry_Lippincott_85	Biochemistry_Lippinco	A. Heme structure Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+), as shown in Figure 3.1. The iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring. The heme Fe2+ can form two additional bonds, one on each side of the planar porphyrin ring. In myoglobin and hemoglobin, one of these positions is coordinated to the side chain of a histidine residue of the globin molecule, whereas the other position is available to bind O2 (Fig. 3.2). (See pp. 278 and 282, respectively, for a discussion of heme synthesis and degradation.) B. Myoglobin structure and function	Biochemistry_Lippinco. A. Heme structure Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+), as shown in Figure 3.1. The iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring. The heme Fe2+ can form two additional bonds, one on each side of the planar porphyrin ring. In myoglobin and hemoglobin, one of these positions is coordinated to the side chain of a histidine residue of the globin molecule, whereas the other position is available to bind O2 (Fig. 3.2). (See pp. 278 and 282, respectively, for a discussion of heme synthesis and degradation.) B. Myoglobin structure and function
Biochemistry_Lippincott_86	Biochemistry_Lippinco	B. Myoglobin structure and function Myoglobin, a hemeprotein present in heart and skeletal muscle, functions both as an oxygen reservoir and as an oxygen carrier that increases the rate of oxygen transport within the muscle cell. [Note: Surprisingly, mouse myoglobin double knockouts (see p. 502) have an apparently normal phenotype.] Myoglobin consists of a single polypeptide chain that is structurally similar to the individual polypeptide chains of the tetrameric hemoglobin molecule. This homology makes myoglobin a useful model for interpreting some of the more complex properties of hemoglobin. 1.	Biochemistry_Lippinco. B. Myoglobin structure and function Myoglobin, a hemeprotein present in heart and skeletal muscle, functions both as an oxygen reservoir and as an oxygen carrier that increases the rate of oxygen transport within the muscle cell. [Note: Surprisingly, mouse myoglobin double knockouts (see p. 502) have an apparently normal phenotype.] Myoglobin consists of a single polypeptide chain that is structurally similar to the individual polypeptide chains of the tetrameric hemoglobin molecule. This homology makes myoglobin a useful model for interpreting some of the more complex properties of hemoglobin. 1.
Biochemistry_Lippincott_87	Biochemistry_Lippinco	1. α-Helical content: Myoglobin is a compact molecule, with ~80% of its polypeptide chain folded into eight stretches of α-helix. These α-helical regions, labeled A to H in Figure 3.2A, are terminated either by the presence of proline, whose five-membered ring cannot be accommodated in an α-helix (see p. 16) or by β-bends and loops stabilized by hydrogen bonds and ionic bonds (see p. 19). [Note: Ionic bonds are also termed electrostatic interactions or salt bridges.] 2. Location of polar and nonpolar amino acid residues: The interior of the globular myoglobin molecule is composed almost entirely of nonpolar amino acids. They are packed closely together, forming a structure stabilized by hydrophobic interactions between these clustered residues (see p. 19). In contrast, polar amino acids are located almost exclusively on the surface, where they can form hydrogen bonds, both with each other and with water. 3.	Biochemistry_Lippinco. 1. α-Helical content: Myoglobin is a compact molecule, with ~80% of its polypeptide chain folded into eight stretches of α-helix. These α-helical regions, labeled A to H in Figure 3.2A, are terminated either by the presence of proline, whose five-membered ring cannot be accommodated in an α-helix (see p. 16) or by β-bends and loops stabilized by hydrogen bonds and ionic bonds (see p. 19). [Note: Ionic bonds are also termed electrostatic interactions or salt bridges.] 2. Location of polar and nonpolar amino acid residues: The interior of the globular myoglobin molecule is composed almost entirely of nonpolar amino acids. They are packed closely together, forming a structure stabilized by hydrophobic interactions between these clustered residues (see p. 19). In contrast, polar amino acids are located almost exclusively on the surface, where they can form hydrogen bonds, both with each other and with water. 3.
Biochemistry_Lippincott_88	Biochemistry_Lippinco	3. Binding of the heme group: The heme group of the myoglobin molecule sits in a crevice, which is lined with nonpolar amino acids. Notable exceptions are two histidine residues (see Fig. 3.2B). One, the proximal histidine (F8), binds directly to the Fe2+ of heme. The second, or distal histidine (E7), does not directly interact with the heme group but helps stabilize the binding of O2 to Fe2+ . Thus, the protein, or globin, portion of myoglobin creates a special microenvironment for the heme that permits the reversible binding of one oxygen molecule (oxygenation). The simultaneous loss of electrons by Fe2+ (oxidation to the ferric [Fe3+] form) occurs only rarely. C. Hemoglobin structure and function	Biochemistry_Lippinco. 3. Binding of the heme group: The heme group of the myoglobin molecule sits in a crevice, which is lined with nonpolar amino acids. Notable exceptions are two histidine residues (see Fig. 3.2B). One, the proximal histidine (F8), binds directly to the Fe2+ of heme. The second, or distal histidine (E7), does not directly interact with the heme group but helps stabilize the binding of O2 to Fe2+ . Thus, the protein, or globin, portion of myoglobin creates a special microenvironment for the heme that permits the reversible binding of one oxygen molecule (oxygenation). The simultaneous loss of electrons by Fe2+ (oxidation to the ferric [Fe3+] form) occurs only rarely. C. Hemoglobin structure and function
Biochemistry_Lippincott_89	Biochemistry_Lippinco	C. Hemoglobin structure and function Hemoglobin is found exclusively in red blood cells (RBC), where its main function is to transport O2 from the lungs to the capillaries of the tissues. Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains (two α chains and two β chains) held together by noncovalent interactions (Fig. 3.3). Each chain (subunit) has stretches of αhelical structure and a hydrophobic heme-binding pocket similar to that described for myoglobin. However, the tetrameric hemoglobin molecule is structurally and functionally more complex than myoglobin. For example, hemoglobin can transport protons (H+) and carbon dioxide (CO2) from the tissues to the lungs and can carry four molecules of O2 from the lungs to the cells of the body. Furthermore, the oxygen-binding properties of hemoglobin are regulated by interaction with allosteric effectors (see p. 29).	Biochemistry_Lippinco. C. Hemoglobin structure and function Hemoglobin is found exclusively in red blood cells (RBC), where its main function is to transport O2 from the lungs to the capillaries of the tissues. Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains (two α chains and two β chains) held together by noncovalent interactions (Fig. 3.3). Each chain (subunit) has stretches of αhelical structure and a hydrophobic heme-binding pocket similar to that described for myoglobin. However, the tetrameric hemoglobin molecule is structurally and functionally more complex than myoglobin. For example, hemoglobin can transport protons (H+) and carbon dioxide (CO2) from the tissues to the lungs and can carry four molecules of O2 from the lungs to the cells of the body. Furthermore, the oxygen-binding properties of hemoglobin are regulated by interaction with allosteric effectors (see p. 29).
Biochemistry_Lippincott_90	Biochemistry_Lippinco	Obtaining O2 from the atmosphere solely by diffusion greatly limits the size of organisms. Circulatory systems overcome this, but transport molecules such as hemoglobin are also required because O2 is only slightly soluble in aqueous solutions such as blood.	Biochemistry_Lippinco. Obtaining O2 from the atmosphere solely by diffusion greatly limits the size of organisms. Circulatory systems overcome this, but transport molecules such as hemoglobin are also required because O2 is only slightly soluble in aqueous solutions such as blood.
Biochemistry_Lippincott_91	Biochemistry_Lippinco	1. Quaternary structure: The hemoglobin tetramer can be envisioned as composed of two identical dimers, (αβ)1 and (αβ)2. The two polypeptide chains within each dimer are held tightly together primarily by hydrophobic interactions (Fig. 3.4). [Note: In this instance, hydrophobic amino acid residues are localized not only in the interior of the molecule but also in a region on the surface of each subunit. Multiple interchain hydrophobic interactions form strong associations between α-subunits and β-subunits in the dimers.] In contrast, the two dimers are held together primarily by polar bonds. The weaker interactions between the dimers allow them to move with respect to one other. This movement results in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin (see Fig. 3.4). a.	Biochemistry_Lippinco. 1. Quaternary structure: The hemoglobin tetramer can be envisioned as composed of two identical dimers, (αβ)1 and (αβ)2. The two polypeptide chains within each dimer are held tightly together primarily by hydrophobic interactions (Fig. 3.4). [Note: In this instance, hydrophobic amino acid residues are localized not only in the interior of the molecule but also in a region on the surface of each subunit. Multiple interchain hydrophobic interactions form strong associations between α-subunits and β-subunits in the dimers.] In contrast, the two dimers are held together primarily by polar bonds. The weaker interactions between the dimers allow them to move with respect to one other. This movement results in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin (see Fig. 3.4). a.
Biochemistry_Lippincott_92	Biochemistry_Lippinco	a. T form: The deoxy form of hemoglobin is called the “T,” or taut (tense) form. In the T form, the two αβ dimers interact through a network of ionic bonds and hydrogen bonds that constrain the movement of the polypeptide chains. The T conformation is the low-oxygen-affinity form of hemoglobin. b. R form: The binding of O2 to hemoglobin causes the rupture of some of the polar bonds between the two αβ dimers, allowing movement. Specifically, the binding of O2 to the heme Fe2+ pulls the iron into the plane of the heme (Fig. 3.5). Because the iron is also linked to the proximal histidine (F8), the resulting movement of the globin chains alters the interface between the αβ dimers. This leads to a structure called the “R,” or relaxed form (see Fig. 3.4). The R conformation is the high-oxygen-affinity form of hemoglobin. oxygen (O2) is not bound. B. Into the plane of the heme upon O2 binding. D. Oxygen binding to myoglobin and hemoglobin	Biochemistry_Lippinco. a. T form: The deoxy form of hemoglobin is called the “T,” or taut (tense) form. In the T form, the two αβ dimers interact through a network of ionic bonds and hydrogen bonds that constrain the movement of the polypeptide chains. The T conformation is the low-oxygen-affinity form of hemoglobin. b. R form: The binding of O2 to hemoglobin causes the rupture of some of the polar bonds between the two αβ dimers, allowing movement. Specifically, the binding of O2 to the heme Fe2+ pulls the iron into the plane of the heme (Fig. 3.5). Because the iron is also linked to the proximal histidine (F8), the resulting movement of the globin chains alters the interface between the αβ dimers. This leads to a structure called the “R,” or relaxed form (see Fig. 3.4). The R conformation is the high-oxygen-affinity form of hemoglobin. oxygen (O2) is not bound. B. Into the plane of the heme upon O2 binding. D. Oxygen binding to myoglobin and hemoglobin
Biochemistry_Lippincott_93	Biochemistry_Lippinco	Myoglobin can bind only one molecule of O2, because it contains only one heme group. In contrast, hemoglobin can bind four molecules of O2, one at each of its four heme groups. The degree of saturation (Y) of these oxygen-binding sites on all myoglobin or hemoglobin molecules can vary between zero (all sites are empty) and 100% (all sites are full), as shown in Figure 3.6. [Note: Pulse oximetry is a noninvasive, indirect method of measuring the oxygen saturation of arterial blood based on differences in light absorption by oxyhemoglobin and deoxyhemoglobin.] 1. Oxygen-dissociation curve: A plot of Y measured at different partial pressures of oxygen (pO2) is called the oxygen-dissociation curve. [Note: pO2 may also be represented as PO2.] The curves for myoglobin and hemoglobin show important differences (see Fig. 3.6). This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half	Biochemistry_Lippinco. Myoglobin can bind only one molecule of O2, because it contains only one heme group. In contrast, hemoglobin can bind four molecules of O2, one at each of its four heme groups. The degree of saturation (Y) of these oxygen-binding sites on all myoglobin or hemoglobin molecules can vary between zero (all sites are empty) and 100% (all sites are full), as shown in Figure 3.6. [Note: Pulse oximetry is a noninvasive, indirect method of measuring the oxygen saturation of arterial blood based on differences in light absorption by oxyhemoglobin and deoxyhemoglobin.] 1. Oxygen-dissociation curve: A plot of Y measured at different partial pressures of oxygen (pO2) is called the oxygen-dissociation curve. [Note: pO2 may also be represented as PO2.] The curves for myoglobin and hemoglobin show important differences (see Fig. 3.6). This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half
Biochemistry_Lippincott_94	Biochemistry_Lippinco	important differences (see Fig. 3.6). This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half saturation of the binding sites (P50) is ~1 mm Hg for myoglobin and 26 mm Hg for hemoglobin. The higher the oxygen affinity (that is, the more tightly O2 binds), the lower the P50.	Biochemistry_Lippinco. important differences (see Fig. 3.6). This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half saturation of the binding sites (P50) is ~1 mm Hg for myoglobin and 26 mm Hg for hemoglobin. The higher the oxygen affinity (that is, the more tightly O2 binds), the lower the P50.
Biochemistry_Lippincott_95	Biochemistry_Lippinco	a. Myoglobin: The oxygen-dissociation curve for myoglobin has a hyperbolic shape (see Fig. 3.6). This reflects the fact that myoglobin reversibly binds a single molecule of O2. Thus, oxygenated (MbO2) and deoxygenated (Mb) myoglobin exist in a simple equilibrium:	Biochemistry_Lippinco. a. Myoglobin: The oxygen-dissociation curve for myoglobin has a hyperbolic shape (see Fig. 3.6). This reflects the fact that myoglobin reversibly binds a single molecule of O2. Thus, oxygenated (MbO2) and deoxygenated (Mb) myoglobin exist in a simple equilibrium:
Biochemistry_Lippincott_96	Biochemistry_Lippinco	The equilibrium is shifted to the right or to the left as O2 is added to or removed from the system. [Note: Myoglobin is designed to bind O2 released by hemoglobin at the low pO2 found in muscle. Myoglobin, in turn, releases O2 within the muscle cell in response to oxygen demand.] b. Hemoglobin: The oxygen-dissociation curve for hemoglobin is sigmoidal in shape (see Fig. 3.6), indicating that the subunits cooperate in binding O2. Cooperative binding of O2 by the four subunits of hemoglobin means that the binding of an oxygen molecule at one subunit increases the oxygen affinity of the remaining subunits in the same hemoglobin tetramer (Fig. 3.7). Although it is more difficult for the first oxygen molecule to bind to hemoglobin, the subsequent binding of oxygen molecules occurs with high affinity, as shown by the steep upward curve in the region near 20–30 mm Hg (see Fig. 3.6). E. Allosteric effectors	Biochemistry_Lippinco. The equilibrium is shifted to the right or to the left as O2 is added to or removed from the system. [Note: Myoglobin is designed to bind O2 released by hemoglobin at the low pO2 found in muscle. Myoglobin, in turn, releases O2 within the muscle cell in response to oxygen demand.] b. Hemoglobin: The oxygen-dissociation curve for hemoglobin is sigmoidal in shape (see Fig. 3.6), indicating that the subunits cooperate in binding O2. Cooperative binding of O2 by the four subunits of hemoglobin means that the binding of an oxygen molecule at one subunit increases the oxygen affinity of the remaining subunits in the same hemoglobin tetramer (Fig. 3.7). Although it is more difficult for the first oxygen molecule to bind to hemoglobin, the subsequent binding of oxygen molecules occurs with high affinity, as shown by the steep upward curve in the region near 20–30 mm Hg (see Fig. 3.6). E. Allosteric effectors
Biochemistry_Lippincott_97	Biochemistry_Lippinco	The ability of hemoglobin to reversibly bind O2 is affected by the pO2, the pH of the environment, the partial pressure of carbon dioxide (pCO2), and the availability of 2,3-bisphosphoglycerate (2,3-BPG). These are collectively called allosteric (“other site”) effectors, because their interaction at one site on the tetrameric hemoglobin molecule causes structural changes that affect the binding of O2 to the heme iron at other sites on the molecule. [Note: The binding of O2 to monomeric myoglobin is not influenced by allosteric effectors.] 1. Oxygen: The sigmoidal oxygen-dissociation curve reflects specific structural changes that are initiated at one subunit and transmitted to other subunits in the hemoglobin tetramer. The net effect of this cooperativity is that the affinity of hemoglobin for the last oxygen molecule bound is ~300 times greater than its affinity for the first oxygen molecule bound. Oxygen, then, is an allosteric effector of hemoglobin. It stabilizes the R form.	Biochemistry_Lippinco. The ability of hemoglobin to reversibly bind O2 is affected by the pO2, the pH of the environment, the partial pressure of carbon dioxide (pCO2), and the availability of 2,3-bisphosphoglycerate (2,3-BPG). These are collectively called allosteric (“other site”) effectors, because their interaction at one site on the tetrameric hemoglobin molecule causes structural changes that affect the binding of O2 to the heme iron at other sites on the molecule. [Note: The binding of O2 to monomeric myoglobin is not influenced by allosteric effectors.] 1. Oxygen: The sigmoidal oxygen-dissociation curve reflects specific structural changes that are initiated at one subunit and transmitted to other subunits in the hemoglobin tetramer. The net effect of this cooperativity is that the affinity of hemoglobin for the last oxygen molecule bound is ~300 times greater than its affinity for the first oxygen molecule bound. Oxygen, then, is an allosteric effector of hemoglobin. It stabilizes the R form.
Biochemistry_Lippincott_98	Biochemistry_Lippinco	a. Loading and unloading oxygen: The cooperative binding of O2 allows hemoglobin to deliver more O2 to the tissues in response to relatively small changes in the pO2. This can be seen in Figure 3.6, which indicates pO2 in the alveoli of the lung and the capillaries of the tissues. For example, in the lung, oxygen concentration is high, and hemoglobin becomes virtually saturated (or “loaded”) with O2. In contrast, in the peripheral tissues, oxyhemoglobin releases (or “unloads”) much of its O2 for use in the oxidative metabolism of the tissues (Fig. 3.8).	Biochemistry_Lippinco. a. Loading and unloading oxygen: The cooperative binding of O2 allows hemoglobin to deliver more O2 to the tissues in response to relatively small changes in the pO2. This can be seen in Figure 3.6, which indicates pO2 in the alveoli of the lung and the capillaries of the tissues. For example, in the lung, oxygen concentration is high, and hemoglobin becomes virtually saturated (or “loaded”) with O2. In contrast, in the peripheral tissues, oxyhemoglobin releases (or “unloads”) much of its O2 for use in the oxidative metabolism of the tissues (Fig. 3.8).
Biochemistry_Lippincott_99	Biochemistry_Lippinco	b. Significance of the sigmoidal oxygen-dissociation curve: The steep slope of the oxygen-dissociation curve over the range of oxygen concentrations that occur between the lungs and the tissues permits hemoglobin to carry and deliver O2 efficiently from sites of high to sites of low pO2. A molecule with a hyperbolic oxygen-dissociation curve, such as myoglobin, could not achieve the same degree of O2 release within this range of pO2. Instead, it would have maximum affinity for O2 throughout this oxygen pressure range and, therefore, would deliver no O2 to the tissues.	Biochemistry_Lippinco. b. Significance of the sigmoidal oxygen-dissociation curve: The steep slope of the oxygen-dissociation curve over the range of oxygen concentrations that occur between the lungs and the tissues permits hemoglobin to carry and deliver O2 efficiently from sites of high to sites of low pO2. A molecule with a hyperbolic oxygen-dissociation curve, such as myoglobin, could not achieve the same degree of O2 release within this range of pO2. Instead, it would have maximum affinity for O2 throughout this oxygen pressure range and, therefore, would deliver no O2 to the tissues.
Biochemistry_Lippincott_100	Biochemistry_Lippinco	2. Bohr effect: The release of O2 from hemoglobin is enhanced when the pH is lowered (proton concentration [H+] is increased) or when the hemoglobin is in the presence of an increased pCO2. Both result in decreased oxygen affinity of hemoglobin and, therefore, a shift to the right in the oxygen-dissociation curve (Fig. 3.9). Both, then, stabilize the T (deoxy) form. This change in oxygen binding is called the Bohr effect. Conversely, raising the pH or lowering the concentration of CO2 results in a greater oxygen affinity, a shift to the left in the oxygen-dissociation curve, and stabilization of the R (oxy) form. a. Source of the protons that lower pH: The concentration of both H+ and	Biochemistry_Lippinco. 2. Bohr effect: The release of O2 from hemoglobin is enhanced when the pH is lowered (proton concentration [H+] is increased) or when the hemoglobin is in the presence of an increased pCO2. Both result in decreased oxygen affinity of hemoglobin and, therefore, a shift to the right in the oxygen-dissociation curve (Fig. 3.9). Both, then, stabilize the T (deoxy) form. This change in oxygen binding is called the Bohr effect. Conversely, raising the pH or lowering the concentration of CO2 results in a greater oxygen affinity, a shift to the left in the oxygen-dissociation curve, and stabilization of the R (oxy) form. a. Source of the protons that lower pH: The concentration of both H+ and
Biochemistry_Lippincott_101	Biochemistry_Lippinco	a. Source of the protons that lower pH: The concentration of both H+ and CO2 in the capillaries of metabolically active tissues is higher than that observed in alveolar capillaries of the lungs, where CO2 is released into the expired air. In the tissues, CO2 is converted by zinc-containing carbonic anhydrase to carbonic acid: which spontaneously loses a H+, becoming bicarbonate (the major blood buffer): The H+ produced by this pair of reactions contributes to the lowering of pH. This differential pH gradient (that is, lungs having a higher pH and tissues a lower pH) favors the unloading of O2 in the peripheral tissues and the loading of O2 in the lung. Thus, the oxygen affinity of the hemoglobin molecule responds to small shifts in pH between the lungs and oxygen-consuming tissues, making hemoglobin a more efficient transporter of O2.	Biochemistry_Lippinco. a. Source of the protons that lower pH: The concentration of both H+ and CO2 in the capillaries of metabolically active tissues is higher than that observed in alveolar capillaries of the lungs, where CO2 is released into the expired air. In the tissues, CO2 is converted by zinc-containing carbonic anhydrase to carbonic acid: which spontaneously loses a H+, becoming bicarbonate (the major blood buffer): The H+ produced by this pair of reactions contributes to the lowering of pH. This differential pH gradient (that is, lungs having a higher pH and tissues a lower pH) favors the unloading of O2 in the peripheral tissues and the loading of O2 in the lung. Thus, the oxygen affinity of the hemoglobin molecule responds to small shifts in pH between the lungs and oxygen-consuming tissues, making hemoglobin a more efficient transporter of O2.
Biochemistry_Lippincott_102	Biochemistry_Lippinco	b. Mechanism of the Bohr effect: The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for H+ than does oxyhemoglobin. This is caused by ionizable groups such as specific histidine side chains that have a higher pKa (see p. 6) in deoxyhemoglobin than in oxyhemoglobin. Therefore, an increase in the concentration of H+ (resulting in a decrease in pH) causes these groups to become protonated (charged) and able to form ionic bonds (salt bridges). These bonds preferentially stabilize the deoxy form of hemoglobin, producing a decrease in oxygen affinity. [Note: Hemoglobin, then, is an important blood buffer.] The Bohr effect can be represented schematically as: where an increase in H+ (or a lower pO2) shifts the equilibrium to the right (favoring deoxyhemoglobin), whereas an increase in pO2 (or a decrease in H+) shifts the equilibrium to the left.	Biochemistry_Lippinco. b. Mechanism of the Bohr effect: The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for H+ than does oxyhemoglobin. This is caused by ionizable groups such as specific histidine side chains that have a higher pKa (see p. 6) in deoxyhemoglobin than in oxyhemoglobin. Therefore, an increase in the concentration of H+ (resulting in a decrease in pH) causes these groups to become protonated (charged) and able to form ionic bonds (salt bridges). These bonds preferentially stabilize the deoxy form of hemoglobin, producing a decrease in oxygen affinity. [Note: Hemoglobin, then, is an important blood buffer.] The Bohr effect can be represented schematically as: where an increase in H+ (or a lower pO2) shifts the equilibrium to the right (favoring deoxyhemoglobin), whereas an increase in pO2 (or a decrease in H+) shifts the equilibrium to the left.
Biochemistry_Lippincott_103	Biochemistry_Lippinco	3. 2,3-BPG effect on oxygen affinity: 2,3-BPG is an important regulator of the binding of O2 to hemoglobin. It is the most abundant organic phosphate in the RBC, where its concentration is approximately that of hemoglobin. 2,3-BPG is synthesized from an intermediate of the glycolytic pathway (Fig. 3.10; see p. 101 for a discussion of 2,3-BPG synthesis in glycolysis). a. 2,3-BPG binding to deoxyhemoglobin: 2,3-BPG decreases the oxygen affinity of hemoglobin by binding to deoxyhemoglobin but not to oxyhemoglobin. This preferential binding stabilizes the T conformation of deoxyhemoglobin. The effect of binding 2,3-BPG can be represented schematically as: b.	Biochemistry_Lippinco. 3. 2,3-BPG effect on oxygen affinity: 2,3-BPG is an important regulator of the binding of O2 to hemoglobin. It is the most abundant organic phosphate in the RBC, where its concentration is approximately that of hemoglobin. 2,3-BPG is synthesized from an intermediate of the glycolytic pathway (Fig. 3.10; see p. 101 for a discussion of 2,3-BPG synthesis in glycolysis). a. 2,3-BPG binding to deoxyhemoglobin: 2,3-BPG decreases the oxygen affinity of hemoglobin by binding to deoxyhemoglobin but not to oxyhemoglobin. This preferential binding stabilizes the T conformation of deoxyhemoglobin. The effect of binding 2,3-BPG can be represented schematically as: b.
Biochemistry_Lippincott_104	Biochemistry_Lippinco	2,3-BPG binding site: One molecule of 2,3-BPG binds to a pocket, formed by the two β-globin chains, in the center of the deoxyhemoglobin tetramer (Fig. 3.11). This pocket contains several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG. [Note: Replacement of one of these amino acids can result in hemoglobin variants with abnormally high oxygen affinity that may be compensated for by increased RBC production (erythrocytosis).] Oxygenation of hemoglobin narrows the pocket and causes 2,3-BPG to be released. c. Oxygen-dissociation curve shift: Hemoglobin from which 2,3-BPG has been removed has high oxygen affinity. However, as seen in the RBC, the presence of 2,3-BPG significantly reduces the oxygen affinity of hemoglobin, shifting the oxygen-dissociation curve to the right (Fig. 3.12). This reduced affinity enables hemoglobin to release O2 efficiently at the partial pressures found in the tissues.	Biochemistry_Lippinco. 2,3-BPG binding site: One molecule of 2,3-BPG binds to a pocket, formed by the two β-globin chains, in the center of the deoxyhemoglobin tetramer (Fig. 3.11). This pocket contains several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG. [Note: Replacement of one of these amino acids can result in hemoglobin variants with abnormally high oxygen affinity that may be compensated for by increased RBC production (erythrocytosis).] Oxygenation of hemoglobin narrows the pocket and causes 2,3-BPG to be released. c. Oxygen-dissociation curve shift: Hemoglobin from which 2,3-BPG has been removed has high oxygen affinity. However, as seen in the RBC, the presence of 2,3-BPG significantly reduces the oxygen affinity of hemoglobin, shifting the oxygen-dissociation curve to the right (Fig. 3.12). This reduced affinity enables hemoglobin to release O2 efficiently at the partial pressures found in the tissues.
Biochemistry_Lippincott_105	Biochemistry_Lippinco	d. 2,3-BPG levels in chronic hypoxia or anemia: The concentration of 2,3-BPG in the RBC increases in response to chronic hypoxia, such as that observed in chronic obstructive pulmonary disease (COPD) like emphysema, or at high altitudes, where circulating hemoglobin may have difficulty receiving sufficient O2. Intracellular levels of 2,3-BPG are also elevated in chronic anemia, in which fewer than normal RBC are available to supply the body’s oxygen needs. Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of O2 in the capillaries of tissues (see Fig. 3.12).	Biochemistry_Lippinco. d. 2,3-BPG levels in chronic hypoxia or anemia: The concentration of 2,3-BPG in the RBC increases in response to chronic hypoxia, such as that observed in chronic obstructive pulmonary disease (COPD) like emphysema, or at high altitudes, where circulating hemoglobin may have difficulty receiving sufficient O2. Intracellular levels of 2,3-BPG are also elevated in chronic anemia, in which fewer than normal RBC are available to supply the body’s oxygen needs. Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of O2 in the capillaries of tissues (see Fig. 3.12).
Biochemistry_Lippincott_106	Biochemistry_Lippinco	e. 2,3-BPG in transfused blood: 2,3-BPG is essential for the normal oxygen transport function of hemoglobin. However, storing blood in the currently available media results in the gradual depletion of 2,3BPG. Consequently, stored blood displays an abnormally high oxygen affinity and fails to unload its bound O2 properly in the tissues. Thus, hemoglobin deficient in 2,3-BPG acts as an oxygen “trap” rather than as an oxygen delivery system. Transfused RBC are able to restore their depleted supplies of 2,3-BPG in 6–24 hours. However, severely ill patients may be compromised if transfused with large quantities of such 2,3-BPG–depleted blood. Stored blood, therefore, is treated with a “rejuvenation” solution that rapidly restores 2,3-BPG. [Note: Rejuvenation also restores ATP lost during storage.] 4. CO2 binding: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see Fig. 1.12 on p. 9). However, some CO2 is carried as carbamate bound to the terminal	Biochemistry_Lippinco. e. 2,3-BPG in transfused blood: 2,3-BPG is essential for the normal oxygen transport function of hemoglobin. However, storing blood in the currently available media results in the gradual depletion of 2,3BPG. Consequently, stored blood displays an abnormally high oxygen affinity and fails to unload its bound O2 properly in the tissues. Thus, hemoglobin deficient in 2,3-BPG acts as an oxygen “trap” rather than as an oxygen delivery system. Transfused RBC are able to restore their depleted supplies of 2,3-BPG in 6–24 hours. However, severely ill patients may be compromised if transfused with large quantities of such 2,3-BPG–depleted blood. Stored blood, therefore, is treated with a “rejuvenation” solution that rapidly restores 2,3-BPG. [Note: Rejuvenation also restores ATP lost during storage.] 4. CO2 binding: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see Fig. 1.12 on p. 9). However, some CO2 is carried as carbamate bound to the terminal
Biochemistry_Lippincott_107	Biochemistry_Lippinco	4. CO2 binding: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see Fig. 1.12 on p. 9). However, some CO2 is carried as carbamate bound to the terminal amino groups of hemoglobin (forming carbaminohemoglobin as shown in Fig. 3.8), which can be represented schematically as follows:	Biochemistry_Lippinco. 4. CO2 binding: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see Fig. 1.12 on p. 9). However, some CO2 is carried as carbamate bound to the terminal amino groups of hemoglobin (forming carbaminohemoglobin as shown in Fig. 3.8), which can be represented schematically as follows:
Biochemistry_Lippincott_108	Biochemistry_Lippinco	The binding of CO2 stabilizes the T, or deoxy, form of hemoglobin, resulting in a decrease in its oxygen affinity (see p. 28) and a right shift in the oxygen-dissociation curve. In the lungs, CO2 dissociates from the hemoglobin and is released in the breath.	Biochemistry_Lippinco. The binding of CO2 stabilizes the T, or deoxy, form of hemoglobin, resulting in a decrease in its oxygen affinity (see p. 28) and a right shift in the oxygen-dissociation curve. In the lungs, CO2 dissociates from the hemoglobin and is released in the breath.
Biochemistry_Lippincott_109	Biochemistry_Lippinco	5. CO binding: Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carboxyhemoglobin. When CO binds to one or more of the four heme sites, hemoglobin shifts to the R conformation, causing the remaining heme sites to bind O2 with high affinity. This shifts the oxygen-dissociation curve to the left and changes the normal sigmoidal shape toward a hyperbola. As a result, the affected hemoglobin is unable to release O2 to the tissues (Fig. 3.13). [Note: The affinity of hemoglobin for CO is 220 times greater than for O2. Consequently, even minute concentrations of CO in the environment can produce toxic concentrations of carboxyhemoglobin in the blood. For example, increased levels of CO are found in the blood of tobacco smokers. CO toxicity appears to result from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.] CO poisoning is treated with 100% O2 at high pressure (hyperbaric oxygen therapy), which facilitates the	Biochemistry_Lippinco. 5. CO binding: Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carboxyhemoglobin. When CO binds to one or more of the four heme sites, hemoglobin shifts to the R conformation, causing the remaining heme sites to bind O2 with high affinity. This shifts the oxygen-dissociation curve to the left and changes the normal sigmoidal shape toward a hyperbola. As a result, the affected hemoglobin is unable to release O2 to the tissues (Fig. 3.13). [Note: The affinity of hemoglobin for CO is 220 times greater than for O2. Consequently, even minute concentrations of CO in the environment can produce toxic concentrations of carboxyhemoglobin in the blood. For example, increased levels of CO are found in the blood of tobacco smokers. CO toxicity appears to result from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.] CO poisoning is treated with 100% O2 at high pressure (hyperbaric oxygen therapy), which facilitates the
Biochemistry_Lippincott_110	Biochemistry_Lippinco	from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.] CO poisoning is treated with 100% O2 at high pressure (hyperbaric oxygen therapy), which facilitates the dissociation of CO from the hemoglobin. [Note: CO inhibits Complex IV of the electron transport chain (see p. 76).] In addition to O2, CO2, and CO, nitric oxide gas (NO) also is carried by hemoglobin. NO is a potent vasodilator (see p. 151). It can be taken up (salvaged) or released from RBC, thereby modulating NO availability and influencing vessel diameter.	Biochemistry_Lippinco. from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.] CO poisoning is treated with 100% O2 at high pressure (hyperbaric oxygen therapy), which facilitates the dissociation of CO from the hemoglobin. [Note: CO inhibits Complex IV of the electron transport chain (see p. 76).] In addition to O2, CO2, and CO, nitric oxide gas (NO) also is carried by hemoglobin. NO is a potent vasodilator (see p. 151). It can be taken up (salvaged) or released from RBC, thereby modulating NO availability and influencing vessel diameter.
Biochemistry_Lippincott_111	Biochemistry_Lippinco	F. Minor hemoglobins It is important to remember that human hemoglobin A (HbA) is just one member of a functionally and structurally related family of proteins, the hemoglobins (Fig. 3.14). Each of these oxygen-carrying proteins is a tetramer, composed of two α-globin (or α-like) polypeptides and two βglobin (or β-like) polypeptides. Certain hemoglobins, such as HbF, are normally synthesized only during fetal development, whereas others, such as HbA2, are synthesized in the adult, although at low levels compared with HbA. HbA can also become modified by the covalent addition of a hexose (see 3. below). 1. Fetal hemoglobin: HbF is a tetramer consisting of two α chains identical to those found in HbA, plus two γ chains (α2γ2; see Fig. 3.14). The γ chains are members of the β-globin gene family (see p. 34).	Biochemistry_Lippinco. F. Minor hemoglobins It is important to remember that human hemoglobin A (HbA) is just one member of a functionally and structurally related family of proteins, the hemoglobins (Fig. 3.14). Each of these oxygen-carrying proteins is a tetramer, composed of two α-globin (or α-like) polypeptides and two βglobin (or β-like) polypeptides. Certain hemoglobins, such as HbF, are normally synthesized only during fetal development, whereas others, such as HbA2, are synthesized in the adult, although at low levels compared with HbA. HbA can also become modified by the covalent addition of a hexose (see 3. below). 1. Fetal hemoglobin: HbF is a tetramer consisting of two α chains identical to those found in HbA, plus two γ chains (α2γ2; see Fig. 3.14). The γ chains are members of the β-globin gene family (see p. 34).
Biochemistry_Lippincott_112	Biochemistry_Lippinco	a. HbF synthesis during development: In the first month after conception, embryonic hemoglobins such as Hb Gower 1, composed of two α-like zeta (ζ) chains and two β-like epsilon (ε) chains (ζ2ε2), are synthesized by the embryonic yolk sac. In the fifth week of gestation, the site of globin synthesis shifts, first to the liver and then to the marrow, and the primary product is HbF. HbF is the major hemoglobin found in the fetus and newborn, accounting for ~60% of the total hemoglobin in the RBC during the last months of fetal life (Fig. 3.15). HbA synthesis starts in the bone marrow at about the eighth month of pregnancy and gradually replaces HbF. Figure 3.15 shows the relative production of each type of hemoglobin chain during fetal and postnatal life. [Note: HbF represents <2% of the hemoglobin in most adults and is concentrated in RBC known as F cells.] b. 2,3-BPG binding to HbF: Under physiologic conditions, HbF has a higher oxygen affinity than does HbA as a result of HbF only	Biochemistry_Lippinco. a. HbF synthesis during development: In the first month after conception, embryonic hemoglobins such as Hb Gower 1, composed of two α-like zeta (ζ) chains and two β-like epsilon (ε) chains (ζ2ε2), are synthesized by the embryonic yolk sac. In the fifth week of gestation, the site of globin synthesis shifts, first to the liver and then to the marrow, and the primary product is HbF. HbF is the major hemoglobin found in the fetus and newborn, accounting for ~60% of the total hemoglobin in the RBC during the last months of fetal life (Fig. 3.15). HbA synthesis starts in the bone marrow at about the eighth month of pregnancy and gradually replaces HbF. Figure 3.15 shows the relative production of each type of hemoglobin chain during fetal and postnatal life. [Note: HbF represents <2% of the hemoglobin in most adults and is concentrated in RBC known as F cells.] b. 2,3-BPG binding to HbF: Under physiologic conditions, HbF has a higher oxygen affinity than does HbA as a result of HbF only
Biochemistry_Lippincott_113	Biochemistry_Lippinco	hemoglobin in most adults and is concentrated in RBC known as F cells.] b. 2,3-BPG binding to HbF: Under physiologic conditions, HbF has a higher oxygen affinity than does HbA as a result of HbF only weakly binding 2,3-BPG. [Note: The γ-globin chains of HbF lack some of the positively charged amino acids that are responsible for binding 2,3BPG in the β-globin chains.] Because 2,3-BPG serves to reduce the oxygen affinity of hemoglobin, the weaker interaction between 2,3BPG and HbF results in a higher oxygen affinity for HbF relative to HbA. In contrast, if both HbA and HbF are stripped of their 2,3-BPG, they then have a similar oxygen affinity. The higher oxygen affinity of HbF facilitates the transfer of O2 from the maternal circulation across the placenta to the RBC of the fetus.	Biochemistry_Lippinco. hemoglobin in most adults and is concentrated in RBC known as F cells.] b. 2,3-BPG binding to HbF: Under physiologic conditions, HbF has a higher oxygen affinity than does HbA as a result of HbF only weakly binding 2,3-BPG. [Note: The γ-globin chains of HbF lack some of the positively charged amino acids that are responsible for binding 2,3BPG in the β-globin chains.] Because 2,3-BPG serves to reduce the oxygen affinity of hemoglobin, the weaker interaction between 2,3BPG and HbF results in a higher oxygen affinity for HbF relative to HbA. In contrast, if both HbA and HbF are stripped of their 2,3-BPG, they then have a similar oxygen affinity. The higher oxygen affinity of HbF facilitates the transfer of O2 from the maternal circulation across the placenta to the RBC of the fetus.
Biochemistry_Lippincott_114	Biochemistry_Lippinco	2. Hemoglobin A2: HbA2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and, ultimately, constituting ~2% of the total hemoglobin. It is composed of two α-globin chains and two δ-globin chains (α2δ2; see Fig. 3.14). 3. Hemoglobin A1c: Under physiologic conditions, HbA is slowly glycated (nonenzymically condensed with a hexose), the extent of glycation being dependent on the plasma concentration of the hexose. The most abundant form of glycated hemoglobin is HbA1c. It has glucose residues attached predominantly to the amino groups of the N-terminal valines of the βglobin chains (Fig. 3.16). Increased amounts of HbA1c are found in RBC of patients with diabetes mellitus, because their HbA has contact with higher glucose concentrations during the 120-day lifetime of these cells. (See p. 340 for a discussion of the use of HbA1c levels in assessing average blood glucose levels in patients with diabetes.) III. GLOBIN GENE ORGANIZATION	Biochemistry_Lippinco. 2. Hemoglobin A2: HbA2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and, ultimately, constituting ~2% of the total hemoglobin. It is composed of two α-globin chains and two δ-globin chains (α2δ2; see Fig. 3.14). 3. Hemoglobin A1c: Under physiologic conditions, HbA is slowly glycated (nonenzymically condensed with a hexose), the extent of glycation being dependent on the plasma concentration of the hexose. The most abundant form of glycated hemoglobin is HbA1c. It has glucose residues attached predominantly to the amino groups of the N-terminal valines of the βglobin chains (Fig. 3.16). Increased amounts of HbA1c are found in RBC of patients with diabetes mellitus, because their HbA has contact with higher glucose concentrations during the 120-day lifetime of these cells. (See p. 340 for a discussion of the use of HbA1c levels in assessing average blood glucose levels in patients with diabetes.) III. GLOBIN GENE ORGANIZATION
Biochemistry_Lippincott_115	Biochemistry_Lippinco	III. GLOBIN GENE ORGANIZATION To understand diseases resulting from genetic alterations in the structure or synthesis of hemoglobin, it is necessary to grasp how the hemoglobin genes, which direct the synthesis of the different globin chains, are structurally organized into gene families and also how they are expressed. A. α-Gene family The genes coding for the α-globin and β-globin subunits of the hemoglobin chains occur in two separate gene clusters (or families) located on two different chromosomes (Fig. 3.17). The α-gene cluster on chromosome 16 contains two genes for the α-globin chains. It also contains the ζ gene that is expressed early in development as an α-globin-like component of embryonic hemoglobin. [Note: Globin gene families also contain globinlike genes that are not expressed, that is, their genetic information is not used to produce globin chains. These are called pseudogenes.] B. β-Gene family	Biochemistry_Lippinco. III. GLOBIN GENE ORGANIZATION To understand diseases resulting from genetic alterations in the structure or synthesis of hemoglobin, it is necessary to grasp how the hemoglobin genes, which direct the synthesis of the different globin chains, are structurally organized into gene families and also how they are expressed. A. α-Gene family The genes coding for the α-globin and β-globin subunits of the hemoglobin chains occur in two separate gene clusters (or families) located on two different chromosomes (Fig. 3.17). The α-gene cluster on chromosome 16 contains two genes for the α-globin chains. It also contains the ζ gene that is expressed early in development as an α-globin-like component of embryonic hemoglobin. [Note: Globin gene families also contain globinlike genes that are not expressed, that is, their genetic information is not used to produce globin chains. These are called pseudogenes.] B. β-Gene family
Biochemistry_Lippincott_116	Biochemistry_Lippinco	B. β-Gene family A single gene for the β-globin chain is located on chromosome 11 (see Fig. 3.17). There are an additional four β-globin-like genes: the ε gene (which, like the ζ gene, is expressed early in embryonic development), two γ genes (Gγ and Aγ that are expressed in HbF), and the δ gene that codes for the globin chain found in the minor adult hemoglobin HbA2. C. Steps in globin chain synthesis	Biochemistry_Lippinco. B. β-Gene family A single gene for the β-globin chain is located on chromosome 11 (see Fig. 3.17). There are an additional four β-globin-like genes: the ε gene (which, like the ζ gene, is expressed early in embryonic development), two γ genes (Gγ and Aγ that are expressed in HbF), and the δ gene that codes for the globin chain found in the minor adult hemoglobin HbA2. C. Steps in globin chain synthesis
Biochemistry_Lippincott_117	Biochemistry_Lippinco	C. Steps in globin chain synthesis Expression of a globin gene begins in the nucleus of RBC precursors, where the DNA sequence encoding the gene is transcribed. The ribonucleic acid (RNA) produced by transcription is actually a precursor of the messenger RNA (mRNA) that is used as a template for the synthesis of a globin chain. Before it can serve this function, two noncoding stretches of RNA (introns) must be removed from the mRNA precursor sequence and the remaining three fragments (exons) joined in a linear manner. The resulting mature mRNA enters the cytosol, where its genetic information is translated, producing a globin chain. (A summary of this process is shown in Figure 3.18. A more detailed description of gene expression is presented in Unit VII, Chapters 30–32.) IV. HEMOGLOBINOPATHIES	Biochemistry_Lippinco. C. Steps in globin chain synthesis Expression of a globin gene begins in the nucleus of RBC precursors, where the DNA sequence encoding the gene is transcribed. The ribonucleic acid (RNA) produced by transcription is actually a precursor of the messenger RNA (mRNA) that is used as a template for the synthesis of a globin chain. Before it can serve this function, two noncoding stretches of RNA (introns) must be removed from the mRNA precursor sequence and the remaining three fragments (exons) joined in a linear manner. The resulting mature mRNA enters the cytosol, where its genetic information is translated, producing a globin chain. (A summary of this process is shown in Figure 3.18. A more detailed description of gene expression is presented in Unit VII, Chapters 30–32.) IV. HEMOGLOBINOPATHIES
Biochemistry_Lippincott_118	Biochemistry_Lippinco	IV. HEMOGLOBINOPATHIES Hemoglobinopathies are defined as a group of genetic disorders caused by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin, or, rarely, both. Sickle cell anemia (HbS), hemoglobin C disease (HbC), hemoglobin SC disease (HbS + HbC = HbSC), and the thalassemias are representative hemoglobinopathies that can have severe clinical consequences. The first three conditions result from production of hemoglobin with an altered amino acid sequence (qualitative hemoglobinopathy), whereas the thalassemias are caused by decreased production of normal hemoglobin (quantitative hemoglobinopathy). A. Sickle cell anemia (hemoglobin S disease)	Biochemistry_Lippinco. IV. HEMOGLOBINOPATHIES Hemoglobinopathies are defined as a group of genetic disorders caused by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin, or, rarely, both. Sickle cell anemia (HbS), hemoglobin C disease (HbC), hemoglobin SC disease (HbS + HbC = HbSC), and the thalassemias are representative hemoglobinopathies that can have severe clinical consequences. The first three conditions result from production of hemoglobin with an altered amino acid sequence (qualitative hemoglobinopathy), whereas the thalassemias are caused by decreased production of normal hemoglobin (quantitative hemoglobinopathy). A. Sickle cell anemia (hemoglobin S disease)
Biochemistry_Lippincott_119	Biochemistry_Lippinco	Sickle cell anemia, the most common of the RBC sickling diseases, is a genetic disorder caused by a single nucleotide substitution (a point mutation, see p. 449) in the gene for β-globin. It is the most common inherited blood disorder in the United States, affecting 50,000 Americans. It occurs primarily in the African American population, affecting 1 in 500 newborn African American infants. Sickle cell anemia is an autosomalrecessive disorder. It occurs in individuals who have inherited two mutant genes (one from each parent) that code for synthesis of the β chains of the globin molecules. [Note: The mutant β-globin chain is designated βS, and the resulting hemoglobin, α2βS2, is referred to as HbS.] An infant does not begin showing symptoms of the disease until sufficient HbF has been replaced by HbS so that sickling can occur (see p. 36). Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia (see p.	Biochemistry_Lippinco. Sickle cell anemia, the most common of the RBC sickling diseases, is a genetic disorder caused by a single nucleotide substitution (a point mutation, see p. 449) in the gene for β-globin. It is the most common inherited blood disorder in the United States, affecting 50,000 Americans. It occurs primarily in the African American population, affecting 1 in 500 newborn African American infants. Sickle cell anemia is an autosomalrecessive disorder. It occurs in individuals who have inherited two mutant genes (one from each parent) that code for synthesis of the β chains of the globin molecules. [Note: The mutant β-globin chain is designated βS, and the resulting hemoglobin, α2βS2, is referred to as HbS.] An infant does not begin showing symptoms of the disease until sufficient HbF has been replaced by HbS so that sickling can occur (see p. 36). Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia (see p.
Biochemistry_Lippincott_120	Biochemistry_Lippinco	by HbS so that sickling can occur (see p. 36). Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia (see p. 284), and increased susceptibility to infections, usually beginning in infancy. [Note: The lifetime of a RBC in sickle cell anemia is <20 days, compared with 120 days for normal RBC, hence, the anemia.] Other symptoms include acute chest syndrome, stroke, splenic and renal dysfunction, and bone changes due to marrow hyperplasia. Life expectancy is reduced. Heterozygotes, representing 1 in 12 African Americans, have one normal and one sickle cell gene. The blood cells of such heterozygotes contain both HbS and HbA, and these individuals have sickle cell trait. They usually do not show clinical symptoms (but may under conditions of extreme physical exertion with dehydration) and can have a normal life span.	Biochemistry_Lippinco. by HbS so that sickling can occur (see p. 36). Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia (see p. 284), and increased susceptibility to infections, usually beginning in infancy. [Note: The lifetime of a RBC in sickle cell anemia is <20 days, compared with 120 days for normal RBC, hence, the anemia.] Other symptoms include acute chest syndrome, stroke, splenic and renal dysfunction, and bone changes due to marrow hyperplasia. Life expectancy is reduced. Heterozygotes, representing 1 in 12 African Americans, have one normal and one sickle cell gene. The blood cells of such heterozygotes contain both HbS and HbA, and these individuals have sickle cell trait. They usually do not show clinical symptoms (but may under conditions of extreme physical exertion with dehydration) and can have a normal life span.
Biochemistry_Lippincott_121	Biochemistry_Lippinco	1. Amino acid substitution in HbS β chains:: A molecule of HbS contains two normal α-globin chains and two mutant β-globin chains (βS), in which glutamate at position six has been replaced with valine (Fig. 3.19). Therefore, during electrophoresis at alkaline pH, HbS migrates more slowly toward the anode (positive electrode) than does HbA (Fig. 3.20). This altered mobility of HbS is a result of the absence of the negatively charged glutamate residues in the two β chains, thereby rendering HbS less negative than HbA. [Note: Electrophoresis of hemoglobin obtained from lysed RBC is routinely used in the diagnosis of sickle cell trait and sickle cell anemia (or, sickle cell disease). DNA analysis also is used (see p. 493).] 2.	Biochemistry_Lippinco. 1. Amino acid substitution in HbS β chains:: A molecule of HbS contains two normal α-globin chains and two mutant β-globin chains (βS), in which glutamate at position six has been replaced with valine (Fig. 3.19). Therefore, during electrophoresis at alkaline pH, HbS migrates more slowly toward the anode (positive electrode) than does HbA (Fig. 3.20). This altered mobility of HbS is a result of the absence of the negatively charged glutamate residues in the two β chains, thereby rendering HbS less negative than HbA. [Note: Electrophoresis of hemoglobin obtained from lysed RBC is routinely used in the diagnosis of sickle cell trait and sickle cell anemia (or, sickle cell disease). DNA analysis also is used (see p. 493).] 2.
Biochemistry_Lippincott_122	Biochemistry_Lippinco	Sickling and tissue anoxia: The replacement of the charged glutamate with the nonpolar valine forms a protrusion on the β chain that fits into a complementary site on the β chain of another hemoglobin molecule in the cell (Fig. 3.21). At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a network of insoluble fibrous polymers that stiffen and distort the cell, producing rigid, misshapen RBC. Such sickled cells frequently block the flow of blood in the narrow capillaries. This interruption in the supply of O2 leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually ischemic death (infarction) of cells in the vicinity of the blockage. The anoxia also leads to an increase in deoxygenated HbS. [Note: The mean diameter of RBC is 7.5 µm, whereas that of the microvasculature is 3–4 µm. Compared to normal RBC, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls. This makes moving through	Biochemistry_Lippinco. Sickling and tissue anoxia: The replacement of the charged glutamate with the nonpolar valine forms a protrusion on the β chain that fits into a complementary site on the β chain of another hemoglobin molecule in the cell (Fig. 3.21). At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a network of insoluble fibrous polymers that stiffen and distort the cell, producing rigid, misshapen RBC. Such sickled cells frequently block the flow of blood in the narrow capillaries. This interruption in the supply of O2 leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually ischemic death (infarction) of cells in the vicinity of the blockage. The anoxia also leads to an increase in deoxygenated HbS. [Note: The mean diameter of RBC is 7.5 µm, whereas that of the microvasculature is 3–4 µm. Compared to normal RBC, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls. This makes moving through