High-Yield Terms
Heme: is formed when iron is inserted into the chemical compound protoporphyrin
Hemin: normal heme contains iron in the ferrous oxidation state (Fe2+), whereas hemin contains iron in the ferric oxidation state (Fe3+)
Methemoglobin: the form of the hemoglobin protein that contains ferric iron (Fe3+) in the heme prosthetic groups due to oxidation
Hemoglobinopathy: any disease resulting from either (or both) quantitative or qualitative defects in α-globin or β-globin proteins
Thalassemia: specifically refers to quantitative hemoglobinopathies due to either α-globin or β-globin protein defects
Sickle cell anemia: most commonly occurring qualitative hemoglobinopathy, results from a single amino acid substitution in the adult β-globin gene
Cooley anemia: is thalassemia major, which is either β0– and β+-thalassemia
Myoglobin and hemoglobin are hemeproteins whose physiological importance is principally related to their ability to bind molecular oxygen. Hemoglobin is a heterotetrameric oxygen transport protein found in red blood cells (erythrocytes), whereas myoglobin is a monomeric protein found mainly in muscle tissue where it serves as an intracellular storage site for oxygen. The oxygen carried by hemeproteins such as hemoglobin and myoglobin is bound directly to the ferrous iron (Fe2+) atom of the heme prosthetic group. Oxidation of the iron to the ferric (Fe3+) state renders the molecule incapable of normal oxygen binding. When the iron in heme is in the ferric state, the molecule is referred to as hemin.
Myoglobin
The tertiary structure of myoglobin is that of a typical water-soluble globular protein. Its secondary structure is unusual in which it contains a very high proportion (75%) of α-helical secondary structure. Each myoglobin molecule contains a single heme group inserted into a hydrophobic cleft in the protein. Hydrophobic interactions between the tetrapyrrole ring and hydrophobic amino acid R groups on the interior of the cleft in the protein strongly stabilize the heme–protein conjugate. In addition, a nitrogen atom from a histidine R group located above the plane of the heme ring is coordinated with the iron atom further stabilizing the interaction between the heme and the protein. In oxymyoglobin the remaining bonding site on the iron atom (the 6th coordinate position) is occupied by the oxygen, whose binding is stabilized by a second histidine residue.
Hemoglobin
Adult hemoglobin is a heterotetrameric [α(2):β(2)] hemeprotein (Figure 6-1) found in erythrocytes where it is responsible for binding oxygen in the lung and transporting the bound oxygen throughout the body, where it is used in aerobic metabolic pathways. Each subunit of a hemoglobin tetramer has a heme prosthetic group identical to that described for myoglobin. The quaternary structure of hemoglobin leads to physiologically important allosteric interactions between the subunits, a property lacking in monomeric myoglobin, which is otherwise very similar to the α-subunit of hemoglobin.
FIGURE 6-1: Hemoglobin. Shown is the three-dimensional structure of deoxyhemoglobin with a molecule of 2,3-bisphosphoglycerate (dark blue) bound. The two α subunits are colored in the darker shades of green and blue, the two β subunits in the lighter shades of green and blue, and the heme prosthetic groups in red. (Adapted from Protein Data Bank ID no. 1b86.) Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
Oxygen-Binding Characteristics
Comparison of the oxygen-binding properties of myoglobin and hemoglobin illustrates the allosteric properties of hemoglobin that result from its quaternary structure and differentiate hemoglobin’s oxygen-binding properties from that of myoglobin (Figure 6-2). The curve of oxygen binding to hemoglobin is sigmoidal typical of allosteric proteins in which the substrate, in this case oxygen, is a positive homotropic effector. When oxygen binds to the first subunit of deoxyhemoglobin, it increases the affinity of the remaining subunits for oxygen. As additional oxygen is bound to the second and third subunits, oxygen binding is further, incrementally, strengthened, so that at the oxygen tension in lung alveoli, hemoglobin is fully saturated with oxygen. As oxyhemoglobin circulates to deoxygenated tissue, oxygen is incrementally unloaded and the affinity of hemoglobin for oxygen is reduced. Thus at the lowest oxygen tensions found in very active tissues, the binding affinity of hemoglobin for oxygen is very low allowing maximal delivery of oxygen to the tissue. In contrast, the oxygen-binding curve for myoglobin is hyperbolic in character indicating the absence of allosteric interactions in this process.
FIGURE 6-2: Oxygen saturation curves for myoglobin and hemoglobin. The saturation curve for myoglobin shows the typical rapid oxygen concentration–dependent saturation of this monomeric oxygen-binding protein. The other 2 curves show the typical sigmoidal saturation curves for cooperative oxygen binding exhibited by fetal hemoglobin (HbF) and adult hemoglobin (HbA). Also indicated in the diagram are the typical oxygen concentrations in peripheral tissues and the lungs. Note that whereas myoglobin can be fully oxygen saturated in the tissues, hemoglobin requires much higher oxygen tension to become fully saturated, which only occurs in the lungs. The position of HbF saturation to the left of HbA (ie, at lower oxygen tension) reflects the fact that fetal hemoglobin binds oxygen with higher affinity than adult hemoglobin and this is so that the fetus can acquire oxygen from the maternal circulation. Reproduced with permission of themedicalbiochemistrypage, LLC.
High-Yield Concept
In hemoglobin, when the iron is oxidized to the ferric state the resulting form of the protein is called methemoglobin. Methemoglobin, which is incapable of binding oxygen, generally represents less than 2% of the hemoglobin in the blood.
In addition to oxygen, heme proteins can also bind carbon monoxide (CO). The binding of CO to heme is much stronger than that of oxygen and it is this preferential binding of CO that is largely responsible for the asphyxiation due to CO poisoning.
The tertiary configuration of low-affinity, deoxygenated hemoglobin (Hb) is known as the taut (T) state. Conversely, the quaternary structure of the fully oxygenated high-affinity form of hemoglobin (HbO2) is known as the relaxed (R) state.
In the context of the affinity of hemoglobin for oxygen there are 4 primary regulators, each of which exerts a negative effect. These regulators are CO2, hydrogen ion (H+), chloride ion (Cl−), and 2,3-bisphosphoglycerate (2,3BPG, or also just BPG). Although they can influence O2 binding independent of each other, CO2, H+, and Cl− primarily function as a consequence of each other on the affinity of hemoglobin for O2.
In the high O2 environment (high PO2) of the lungs, there is sufficient O2 to overcome the inhibitory nature of the T state of hemoglobin to oxygen binding. During the O2 binding–induced alteration from the T form to the R form, several amino acid side groups on the surface of hemoglobin subunits will dissociate protons as depicted in the following equation. This proton dissociation plays an important role in the expiration of the CO2 that arrives from the tissues. However, because of the high PO2, the pH of the blood in the lungs (≈7.4-7.5) is not sufficiently low enough to exert a negative influence on hemoglobin-binding O2. When the oxyhemoglobin reaches the tissues, the PO2 is sufficiently low, as well as the pH (≈7.2), that the T state is favored and the O2 released.
Within the tissues, metabolizing cells produce CO2, which diffuses into the blood and enters the circulating erythrocytes. Within erythrocytes the CO2 is rapidly converted to carbonic acid through the action of carbonic anhydrase. The carbonic acid then rapidly ionizes leading to increased production of H+ and thus a reduction in the pH.
The bicarbonate ion produced in this dissociation reaction diffuses out of the erythrocyte and is carried in the blood to the lungs. This effective CO2 transport process is referred to as isohydric transport. Approximately 80% of the CO2 produced in metabolizing cells is transported to the lungs in this way. A small percentage of CO2 is transported in the blood as a dissolved gas.
In the tissues, the H+ dissociated from carbonic acid is buffered by hemoglobin, which in turn exerts a negative influence on O2 binding, forcing release of the O2 which then diffuses into the tissues. As indicated earlier, within the lungs the high PO2 allows for effective O2 binding by hemoglobin leading to the T-to-R-state transition and the release of protons. The protons combine with the bicarbonate that arrived from the tissues forming carbonic acid, which then enters the erythrocytes. Through a reversal of the carbonic anhydrase reaction, CO2 and H2O are produced. The CO2 diffuses out of the blood, into the lung alveoli, and is released on expiration. The effects of hydrogen ion concentration on the O2-binding affinity of hemoglobin are referred to as the Bohr effect (Figure 6-3).
FIGURE 6-3: The Bohr effect. Carbon dioxide generated in peripheral tissues combines with water to form carbonic acid, which dissociates into protons and bicarbonate ions. Deoxyhemoglobin acts as a buffer by binding protons and delivering them to the lungs. In the lungs, the uptake of oxygen by hemoglobin releases protons that combine with bicarbonate ion, forming carbonic acid, which when dehydrated by carbonic anhydrase becomes carbon dioxide, which then is exhaled. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
Coupled to the diffusion of bicarbonate out of erythrocytes in the tissues, there must be ion movement into the erythrocytes to maintain electrical neutrality. This is the role of Cl− and is referred to as the chloride shift. In this way, Cl− plays an important role in bicarbonate production and diffusion and thus also negatively influences O2 binding to hemoglobin.
Role of 2,3-Bisphosphoglycerate
The compound 2,3-bisphosphoglycerate (2,3-BPG), derived from the glycolytic intermediate 1,3-bisphosphoglycerate, is a potent allosteric effector on the oxygen-binding properties of hemoglobin (Figure 6-4). In the dexoxygenated T state, a cavity forms in the center of the hemoglobin molecule allowing 2,3-BPG to bind to the β-subunits decreasing their affinity for oxygen. 2,3-BPG can occupy this cavity stabilizing the T state. Conversely, when 2,3-BPG is not available, or not bound, Hb can be converted to HbO2 more readily. Thus, like increased hydrogen ion concentration, increased 2,3-BPG concentration decreases the amount of oxygen bound by Hb at any oxygen concentration. Hemoglobin molecules differing in subunit composition are known to have different 2,3-BPG–binding properties with correspondingly different allosteric responses to 2,3-BPG. For example, HbF (the fetal form of hemoglobin, which contains the γ-subunits) binds 2,3-BPG much less avidly than HbA (the adult form of hemoglobin) with the result that HbF in fetuses of pregnant women binds oxygen with greater affinity than the mothers HbA, thus giving the fetus preferential access to oxygen carried by the mothers circulatory system. Conversely, when individuals acclimate to high altitudes, where oxygen concentrations are lower, the level of 2,3-BPG increases in order to allow for easier release of the oxygen to the tissues.
FIGURE 6-4: Chromosomal structure of the α- and β-globin gene clusters on chromosomes 16 and 11, respectively. The 5′ to 3′ orientation of the genes on each chromosome also reflects the developmental timing of their expression with the 5′-most genes expressed earliest. The ζ (zeta) and ε (epsilon) genes are the embryonic genes in each cluster. Genes with a Ψ (psi) designation represent pseudogenes. Reproduced with permission of themedicalbiochemistrypage, LLC.
High-Yield Concept
In addition to isohydric transport, as much as 15% of CO2 is transported to the lungs bound to N-terminal amino groups of the T form of hemoglobin. This reaction forms what is called carbaminohemoglobin. The formation of H+ via this reaction enhances the release of the bound O2 to the surrounding tissues. Within the lungs, the high O2 content results in O2 binding to hemoglobin with the concomitant release of H+. The released protons then promote the dissociation of the carbamino to form CO2, which is then released with expiration.
The Hemoglobin Genes
The α- and β-globin proteins contained in functional hemoglobin tetramers are derived from gene clusters. The α-globin genes are on chromosome 16 and the β-globin genes are on chromosome 11 (Figure 6-4). Both gene clusters contain not only the major adult genes, α and β, but other expressed sequences that are utilized at different stages of development. In addition to functional genes, both clusters contain nonfunctional pseudogenes.
Hemoglobin synthesis begins in the first few weeks of embryonic development within the yolk sac (Figure 6-5). The major hemoglobin at this stage of development is a tetramer composed of 2 zeta (ζ) chains encoded within the α-cluster and 2 epsilon (ε) chains from the β-cluster. By 6 to 8 weeks of gestation, the expression of this version of hemoglobin declines dramatically coinciding with the change in hemoglobin synthesis from the yolk sac to the liver. Expression from the α-cluster consists of identical proteins from the α1 and α2 genes and this cluster remains on throughout life.
FIGURE 6-5: Developmental patterns of globin gene expression. Reproduced with permission of themedicalbiochemistrypage, LLC.
Within the β-globin cluster there is an additional set of genes, the fetal β-globin genes identified as the gamma (γ) genes. The 2 fetal genes called Gγ and Aγ, the derivation of which stems from the single amino acid difference between the 2 fetal genes: glycine in Gγ and alanine in Aγ at position 136. These fetal γ genes are expressed as the embryonic genes are turned off. Shortly before birth there is a smooth switch from fetal γ-globin gene expression to adult β-globin gene expression. The switch from fetal γ– to adult β-globin does not directly coincide with the switch from hepatic synthesis to bone marrow synthesis since at birth it can be shown that both γ and β synthesis is occurring in the marrow.
The 2 predominant forms of hemoglobin are: (1) fetal, designated HbF, and (2) adult, designated HbA. Fetal hemoglobin consists of both α2Gγ2 and α2Aγ2 tetramers. Adult hemoglobin (more commonly HbA1) is a tetramer of 2 α- and 2 β-chains. A minor adult hemoglobin, identified as HbA2, is a tetramer of 2 α-chains and 2 δ-chains. The δ gene is expressed with a timing similar to the β gene, but because the promoter has acquired a number of mutations its efficiency of transcription is reduced. The overall hemoglobin composition in a normal adult is approximately 97.5% HbA1, 2% HbA2, and 0.5% HbF (Figure 6-6).
FIGURE 6-6: Major types of hemoglobins found during embryonic, fetal, and adult life. Reproduced with permission of themedicalbiochemistrypage, LLC.
The Hemoglobinopathies
The term hemoglobinopathy refers to any of several types of gene defect in the hemoglobin genes that result in the synthesis of abnormal or reduced levels of one of the globin chains of the hemoglobin molecule (see Clinical Boxes 6-1 and 6-2). Hemoglobinopathies are classified dependent upon whether the defect results in reduced (quantitative) or altered (qualitative) hemoglobin. More commonly, the term hemoglobinopathy is used to imply structural abnormalities in the globin proteins themselves. In contrast, the term thalassemia is used to define hemoglobinopathies that are due to underproduction of normal globin proteins. The 2 conditions may overlap, however, since some conditions which cause abnormalities in globin proteins also affect their production which means that some hemoglobinopathies are also thalassemias.
CLINICAL BOX 6-1: SICKLE CELL ANEMIA
A large number of mutations have been described in the globin genes. These mutations can be divided into 2 distinct types: those that cause qualitative abnormalities (eg, sickle cell anemia) and those that cause quantitative abnormalities (the thalassemias). Taken together these disorders are referred to as the hemoglobinopathies. Of the mutations leading to qualitative alterations in hemoglobin, the missense mutation in the β-globin gene that causes sickle cell anemia is the most common. The mutation causing sickle cell anemia is a single-nucleotide substitution (A-T) in the codon for amino acid 6. The change converts a glutamic acid codon (GAG) to a valine codon (GTG). The form of hemoglobin in persons with sickle cell anemia is referred to as HbS. An additional relatively common mutation at codon 6 is the conversion to a lysine codon (AAG) which results in the generation of a variant hemoglobin called HbC.
The underlying problem in sickle cell anemia is that the valine for glutamic acid substitution results in hemoglobin tetramers that aggregate into arrays upon deoxygenation in the tissues. This aggregation leads to deformation of the erythrocyte into the characteristic sickle shape. The repeated cycles of oxygenation and deoxygenation lead to irreversible sickling of erythrocytes. These distorted erythrocytes are relatively inflexible and unable to traverse the capillary beds. The result is clogging of the fine capillaries such as those in the retina of the eye. In addition to their altered shape and poor capillary migration, sickled erythrocytes are weak and lyse very easily. Because bones are particularly affected by the reduced blood flow, frequent and severe bone pain results. This is the typical symptom during a sickle cell “crisis.” Long term, the recurrent clogging of the capillary beds leads to damage of the internal organs, in particular the kidneys, heart, and lungs. The continual destruction of the sickled erythrocytes leads to chronic anemia and episodes of hyperbilirubinemia. Genetically, sickle cell anemia is an inherited autosomal recessive disorder. However, heterozygous individuals have what is referred to as sickle cell trait. Although these heterozygotic individuals are clinically normal, their erythrocytes can still sickle under conditions of low oxygen pressure. Because of this phenomenon, heterozygotes exhibit phenotypic dominance yet are genetically recessive. Electrophoresis of hemoglobin proteins from individuals suspected of having sickle cell anemia (or several other types of hemoglobin disorders) is an effective diagnostic tool because the variant hemoglobins have different charges.
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