BIOCHEMISTRY: A TEXTBOOK FOR MEDICAL UNIVERSITIES - E. S. Severin - 2004

SECTION 1. STRUCTURE, PROPERTIES, AND FUNCTIONS OF PROTEINS

V. Functional features of oligomeric proteins using hemoglobin as an example

Oligomeric Proteins exhibit properties that are absent in monomeric proteins. METABOLISM/18.html">The Influence of quaternary Structure on the Functional Properties of a protein can be examined by comparing the Structure and function of two related heme-containing Proteins: Myoglobin and Hemoglobin. Both proteins share a common evolutionary origin, a similar conformation of individual polypeptide chains, and a similar function (they participate in Oxygen transport), but myoglobin is a monomeric protein, whereas hemoglobin is a tetramer. The presence of a quaternary structure in hemoglobin imparts properties to this protein that are absent in myoglobin.

A. STRUCTURE AND Functions of myoglobin

Myoglobin belongs to the Class of heme-containing proteins, meaning it contains a prosthetic group, heme, which is bound rather tightly to the protein moiety. Myoglobin is classified as a globular protein and consists of a single polypeptide chain.

1. Cellular localization and function

Myoglobin is found in red Muscles and is involved in oxygen storage. Under conditions of intensive muscular work, when the partial pressure of oxygen in the tissue drops, O₂ is released from the complex with myoglobin and is utilized in the Cell/35.html">Mitochondria of Cells to generate the energy necessary for Muscle Function.

2. Structure of myoglobin

Myoglobin contains a non-protein part (heme) and a protein part (apomyoglobin).

Heme — a molecule with a cyclic tetrapyrrole structure, in which 4 pyrrole rings are connected by methylene bridges and contain 4 methyl, 2 vinyl, and 2 propionate side chains. This organic portion of heme is called protoporphyrin. There are 15 possible arrangements of the side chains, but only one isomer, designated protoporphyrin IX, is present in Hemoproteins. In heme, the 4 nitrogen atoms of the protoporphyrin IX pyrrole rings are linked by four coordination bonds to Fе²⁺ located at the center of the molecule (Fig. 1-29).

Fig. 1-29. STRUCTURE OF THE heme moiety found in myoglobin and hemoglobin.

Apomyoglobin — the protein moiety of myoglobin; its Primary Structure is represented by a sequence of 153 Amino Acids folded into 8 α-helices in the Secondary structure. The α-helices are designated by Latin letters from A to H, starting from the N-terminus of the polypeptide chain, and contain from 7 to 23 amino acids each. To designate individual amino acids in the Introduction/19.html">Primary structure of apomyoglobin, either their ordinal number from the N-terminus is used (e.g., His₆₄, Phe₁₃₈), or the letter of the α-Helix along with the ordinal number of the given amino acid within that helix, starting from the N-terminus (e.g., His F₈).

The tertiary structure has the appearance of a compact globule (with practically no free space inside), formed by loops and turns in the regions of non-helical protein segments. The interior of the molecule consists almost entirely of hydrophobic radicals, with the exception of two His residues located in the active center.

3. Binding of heme to apomyoglobin

Heme is a specific Ligand of apomyoglobin that attaches to the protein moiety in a cleft between two α-helices, F and E. The heme-binding site is formed predominantly by hydrophobic amino acid residues surrounding the hydrophobic pyrrole rings of heme. Two propionic acid side chains, ionized at physiological pH values, project onto The surface of the molecule.

In addition to hydrophobic amino acids, the active center of apomyoglobin also includes 2 His residues (His₆₄ and His₉₃, or His E₇ and His F₈) that play an important role in protein function. They are located on opposite sides of the heme plane and are part of the F and E helices, between which the heme is situated. The iron atom in heme can form 6 coordination bonds, 4 of which hold Fе²⁺ at the center of protoporphyrin IX (connecting it to the nitrogen atoms of the pyrrole rings), while the 5th bond arises between Fе²⁺ and the nitrogen atom of the imidazole ring of His F₈ (Fig. 1-30).

Fig. 1-30. Arrangement of heme in the active center of apomyoglobin and apohemoglobin protomers.

His E₇, although not bound to heme, is essential for the correct orientation and attachment of another ligand—O₂—to myoglobin.

The amino acid environment of heme creates conditions for a fairly strong yet Reversible Binding of O₂ to the Fе²⁺ of myoglobin. The hydrophobic amino acid residues surrounding the heme prevent Water from penetrating the myoglobin binding center and inhibit The oxidation of Fе²⁺ to Fе³⁺. Ferric iron within heme is incapable of binding O₂.

B. Structure and functions of hemoglobin

Hemoglobins are related proteins found in the erythrocytes of humans and vertebrates. These proteins perform 2 important functions:

✵ transport of O₂ from the Lungs to peripheral Tissues;

✵ Participation in the transport of CO₂ and protons from peripheral tissues to the lungs for subsequent elimination from the body.

Every day, the Blood must transport about 600 L of O₂ from the lungs to the tissues. Since O₂ is poorly soluble in water, virtually all oxygen in the blood is bound to hemoglobin in erythrocytes.

The amount of O₂ delivered to tissues and the metabolic rate depend on hemoglobin's ability to bind O₂ in the lungs and release it relatively easily in tissue capillaries. On the other hand, O₂ is a strong oxidizing agent; an excess of O₂ entering tissues can lead to molecular damage and disrupt cellular structure and function. Therefore, a critical characteristic of hemoglobin is its ability to regulate its affinity for O₂ depending on tissue conditions.

Like myoglobin, hemoglobins are classified as hemoproteins, but they possess a quaternary structure (consisting of 4 polypeptide chains), which enables The regulation of their functions.

1. Human Hemoglobins

Adult Hemoglobins

In human adult erythrocytes, hemoglobin accounts for 90% of all proteins in The Cell.

Hemoglobin A — the primary hemoglobin of the adult Organism, accounting for about 98% of total hemoglobin; it is a tetramer consisting of 2 α and 2 β polypeptide chains (2α2β).

Hemoglobin A₂ is present in the adult body in a lower concentration, accounting for about 2% of total hemoglobin. It consists of 2 α and 2 δ chains.

Hemoglobin A_1c — hemoglobin A modified by the covalent attachment of glucose (so-called glycated hemoglobin).

Hemoglobins synthesized during embryonic development:

Embryonic hemoglobin is synthesized in the embryonic yolk sac a few weeks after Fertilization. It is a tetramer of 22ε. Two weeks after The formation of the fetal Liver, hemoglobin F synthesis begins there, replacing embryonic hemoglobin by 6 months.

Hemoglobin F — fetal hemoglobin, synthesized in the fetal liver and Bone Marrow until birth. It has a tetrameric structure consisting of 2 α and 2 γ chains. After birth, it is gradually replaced by hemoglobin A, which begins to be synthesized in the bone marrow cells as early as the 8th month of fetal development.

2. Structure of Hemoglobin A

Structure of Hemoglobin Protomers

The conformation of individual hemoglobin protomers is strikingly similar to that of myoglobin, despite the fact that only 24 amino acid residues are identical in the primary structure of their polypeptide chains. Like apomyoglobin, hemoglobin protomers consist of 8 helices folded into a compact globular structure containing a Hydrophobic core and a heme-binding "pocket." The binding of heme to globin (the protein moiety) is analogous to that in myoglobin — featuring a hydrophobic heme environment, except for 2 residues, His E₇ and His F₈ (Fig. 1-31). However, The quaternary structure of hemoglobin represents a more complex Structural and functional entity than myoglobin.

The Role of Histidine E₇ in the Functioning of Myoglobin and Hemoglobin

Heme has a high affinity for carbon monoxide (CO). In an aqueous medium, heme free of the protein moiety binds to CO 25,000 times stronger than to O₂. The high affinity of heme for CO compared to O₂ is due to the different spatial arrangement of the Fe²⁺-heme complexes with CO and O₂ (Fig. 1-31, A).

Fig. 1-31. Spatial arrangement of CO and O₂ bound to free heme (A) and to heme within hemoglobin or myoglobin (B).

In the Fe²⁺-heme complex with CO, the Fe²⁺, carbon, and oxygen atoms lie in a straight line, whereas in the Fe²⁺-heme complex with O₂, the iron and oxygen atoms are at an angle, reflecting their optimal spatial arrangement.

In myoglobin and hemoglobin, His E₇ is located above Fe²⁺ in the O₂-binding region, which disrupts the optimal arrangement of CO in the protein binding center and weakens its interaction with heme. Conversely, this same His E₇ creates optimal conditions for O₂ binding (Fig. 1-31, B). As a result, the affinity of heme for CO in proteins is only 200 times greater than its affinity for O₂.

The reduction in the affinity of heme-containing proteins for CO is of great biological significance. CO is produced in small quantities during the Catabolism of certain substances, notably heme. This endogenously produced CO blocks about 1% of heme-containing proteins. If heme affinity for CO were not reduced by the protein environment, endogenous carbon monoxide could cause severe poisoning.

Quaternary Structure of Hemoglobin

Four polypeptide chains joined together form an almost regular spherical shape, where each α-chain contacts two β-chains (Fig. 1-32).

Fig. 1-32. Structure of hemoglobin.

Due to the Abundance of hydrophobic radicals in the contact regions between the α₁ and β₁ subunits, as well as the α₂ and β₂ subunits, strong interactions are formed between these polypeptide chains, primarily driven by hydrophobic forces, alongside ionic and Hydrogen Bonds. As a result, α₁β₁ and α₂β₂ dimers are formed. These dimers are held together within the tetrameric hemoglobin molecule mainly by polar (ionic and hydrogen) bonds; therefore, shifting the pH of the environment toward acidic or alkaline values disrupts the bonds between the dimers first. Moreover, the dimers can easily move relative to each other.

Since the surface of the protomers is irregular, The polypeptide chains in the central region cannot pack tightly against one another, resulting in the formation of a "central cavity" running through the entire hemoglobin molecule.

3. Binding of hemoglobin to O₂ in the lungs and its dissociation from the complex in tissues

The primary function of hemoglobin is to deliver O₂ from the lungs to the tissues. The oligomeric structure of hemoglobin ensures its rapid oxygenation in the lungs (formation of oxyhemoglobin — Hb(O₂)₄), The ability to release oxygen from hemoglobin in tissue capillaries at a relatively high O₂ partial pressure, and the capacity to regulate hemoglobin's affinity for O₂ depending on the metabolic needs of the tissues.

Cooperative conformational changes of protomers

O₂ binds to hemoglobin protomers via Fe²⁺, which is coordinated with four nitrogen atoms of the heme pyrrole rings and the nitrogen atom of His F₈ in the protein moiety of the protomer. The binding of O₂ to the remaining free coordination site of Fe²⁺ occurs on the opposite side of the heme plane in the vicinity of His E₇ (similarly to myoglobin). His E₇ does not interact directly with O₂, but it provides optimal conditions for its binding (Fig. 1-33).

Fig. 1-33. Changes in THE POSITION OF Fe²⁺ and the protein moiety of hemoglobin upon O₂ binding.

In deoxyhemoglobin, due to the covalent bond with the protein moiety, the Fe²⁺ ion projects out of the heme plane toward His F₈. The attachment of O₂ to the Fe²⁺ atom of one protomer pulls it into the plane of the heme, which in turn shifts the His F₈ residue and the polypeptide chain to which it belongs. Because the protomer is linked to the other protomers and proteins possess conformational flexibility, a conformational change occurs throughout the entire protein. The conformational changes taking place in the other protomers facilitate the binding of the next O₂ molecule, which induces further conformational shifts in the protein and accelerates the binding of subsequent O₂ molecules. The fourth O₂ molecule binds to hemoglobin 300 times more easily than the first one (Fig. 1-34).

Fig. 1-34. Cooperative Conformational Changes in hemoglobin protomers upon O₂ binding.

The alteration in the conformation (and, consequently, the functional properties) of all protomers of an oligomeric protein upon ligand binding to only one of them is termed cooperative conformational changes of protomers.

Similarly, in tissues, the dissociation of each O₂ molecule alters the conformation of all protomers and facilitates the release of subsequent O₂ molecules.

O₂ dissociation curves for myoglobin and hemoglobin

The cooperativity in the functioning of hemoglobin protomers can also be observed in the O₂ dissociation curves for myoglobin and hemoglobin (Fig. 1-35).

Fig. 1-35. Oxygen dissociation curves for myoglobin and hemoglobin as a function of oxygen partial pressure.

The ratio of O₂-occupied binding sites of a protein to the total number of such sites capable of binding is called the oxygen saturation fraction of these proteins. The dissociation curves show the degree to which these proteins are saturated with O₂ at various oxygen partial pressures.

The O₂ dissociation curve for myoglobin has the shape of a simple hyperbola. This indicates that myoglobin binds reversibly to its ligand without being influenced by any external factors (see scheme below).

The processes of oxymyoglobin formation and breakdown are in equilibrium, and this equilibrium shifts to the left or right depending on whether oxygen is added to or removed from the system. Myoglobin binds the oxygen released by hemoglobin in tissue capillaries, and can itself release O₂ in response to increased oxygen demand by Muscle tissue and during intensive O₂ consumption resulting from physical exertion.

Myoglobin has a very high affinity for

O₂. Even at an O₂ partial pressure of 1 — 2 mm Hg, myoglobin remains 50% saturated with O₂.

O₂ dissociation curve for hemoglobin. The graph in Fig. 1-35 shows that hemoglobin has a significantly lower affinity for O₂; half-saturation of hemoglobin with O₂ occurs at a higher O₂ pressure (about 26 mm Hg).

The hemoglobin dissociation curve is sigmoidal (S-shaped). This indicates that the hemoglobin protomers function cooperatively: the more O₂ the protomers release, the easier the subsequent O₂ molecules are dissociated.

In the capillaries of resting muscles, where the O₂ pressure is about 40 mm Hg, most of the oxygen returns to the lungs in the form of oxyhemoglobin. During physical exertion, the O₂ pressure in muscle capillaries drops to 10 — 20 mm Hg. It is in this range (from 10 to 40 mm Hg) that the "steep region" of the S-shaped curve is located, where the cooperative behavior of the protomers is most pronounced.

Consequently, thanks to their unique structure, each of the proteins considered is adapted to perform its specific function: myoglobin binds the O₂ released by hemoglobin, stores it within the cell, and releases it in case of extreme necessity; hemoglobin binds O₂ in the lungs, where its saturation reaches 100%, and releases O₂ in the tissue capillaries in response to changes in local O₂ pressure.

4. Transport of H⁺ and CO₂ from tissues to the lungs via hemoglobin. The Bohr effect

The oxidation of organic substances to generate energy takes place in cellular mitochondria using O₂ delivered by hemoglobin from the lungs. This oxidation process yields final breakdown products—CO₂ and H₂O—the amounts of which are proportional to the intensity of oxidative metabolism.

The CO₂ produced in tissues is transported into erythrocytes. There, catalyzed by the enzyme Carbonic anhydrase, The rate of H₂CO₃ formation increases. This weak carbonic acid can dissociate into H⁺ and HCO₃^-.

CO₂ + H₂O <-> H₂CO₃ <-> H⁺ + HCO₃^-.

The reaction equilibrium in erythrocytes located within tissue capillaries shifts to the right, because the protons generated by carbonic acid dissociation can bind to specific sites on the hemoglobin molecule: the His₁₄₆ residues of the two β-chains, the His₁₂₂ residues, and the terminal α-amino groups of the two α-chains. When hemoglobin transitions from the oxy to the deoxy form, all six of these sites acquire a higher affinity for H⁺ as a result of local Changes in the amino acid environment surrounding these sites (specifically, the close approach of negatively charged carboxyl groups of amino acids).

The binding of 3 pairs of protons to hemoglobin decreases its affinity for O₂ and enhances O₂ transport to tissues in need of it (Fig. 1-36, A). This increase in O₂ release by hemoglobin in response to H⁺ concentration is known as the Bohr effect (named after the Danish physiologist Christian Bohr, who first discovered this phenomenon).

In the lung capillaries, the high partial pressure of O₂ leads to the Oxygenation of hemoglobin and the release of 6 protons. The reaction CO₂⁺ + H₂O <-> H₂CO₃ <-> H⁺ + HCO₃^- shifts to the left, and the resulting CO₂ is released into the alveolar space and eliminated via exhaled air (Fig. 1-36, B).

Fig. 1-36. Transport of H⁺ and CO₂ by the blood. The Bohr effect. A — effect of CO₂ and H⁺ concentration on the release of O₂ from the hemoglobin complex in tissues (the Bohr effect); B — oxygenation of deoxyhemoglobin in the lungs, along with the Formation and Elimination of CO₂.

Thus, in the course of evolution, the hemoglobin molecule has acquired the ability to perceive and respond to signals from its environment. An increase in the proton concentration of the medium lowers the affinity of O₂ for hemoglobin and enhances its transport to the tissues (Fig. 1-37).

Fig. 1-37. Effect of pH on the O₂ dissociation curve for hemoglobin.

Most of the CO₂ is transported by the blood in the form of bicarbonate (HCO₃^-). A small amount of CO₂ (about 15 — 20%) can be carried to the lungs by reversibly binding to un-ionized terminal α-amino groups: R-NH₂⁺ + CO₂ = R-NH-COO^- + H⁺, resulting in the formation of carbaminohemoglobin, where R represents the polypeptide chain of hemoglobin. The binding of CO₂ to hemoglobin also decreases its affinity for O₂.

5. 2,3-Bisphosphoglycerate as an allosteric regulator of hemoglobin affinity for O₂

2,3-Bisphosphoglycerate (BPG) is a substance synthesized in erythrocytes from 1,3-bisphosphoglycerate, an intermediate in glucose oxidation.

Regulation of hemoglobin affinity for O₂ by 2,3-bisphosphoglycerate

Under normal conditions, 2,3-bisphosphoglycerate is present in erythrocytes at approximately the same concentration as hemoglobin. By binding to hemoglobin, BGP can also modulate its affinity for O₂.

At the center of the tetrameric hemoglobin molecule lies a cavity formed by the amino acid residues of all four protomers. This central cavity serves as the binding site for BPG.

The dimensions of the central cavity can change: the dissociation of O₂ from oxyhemoglobin induces conformational changes that promote the formation of additional ionic bonds between the α₁β₁ and α₂β₂ dimers. As a result, the Spatial Structure of deoxyhemoglobin becomes more rigid and tense, while the central cavity expands.

The surface of the cavity is lined with amino acid residues, including the positively charged Lys₈₂ and His₁₄₃ radicals of the β-chains and the positively charged α-amino groups of the N-terminal valine residues of the β-chains. Within the expanded cavity of deoxyhemoglobin, highly negatively charged BPG binds via ionic bonds formed with the positively charged Functional groups of the two hemoglobin β-chains. The binding of BPG further stabilizes the rigid structure of deoxyhemoglobin and reduces the protein's affinity for O₂ (Fig. 1-38).

Fig. 1-38. Interaction of 2,3-bisphosphoglycerate with the amino acid residues in the central cavity of deoxyhemoglobin.

BPG binds to deoxyhemoglobin at a site distinct from the heme group where O₂ binding takes place. Such a ligand is referred to as an "allosteric" ligand, and the site where it binds is called the "allosteric site" (from the Greek "allos" meaning other or different, and "stereos" meaning space or solid).

In the lungs, a high partial pressure of O₂ leads to the oxygenation of hemoglobin. The disruption of ionic bonds between the α₁β₁ and α₂β₂ dimers results in the "relaxation" of the protein molecule, a reduction of the central cavity, and the expulsion of BPG.

Changes in BPG concentration as a mechanism of bodily adaptation to Hypoxia. The concentration of BPG in the erythrocytes of individuals living under specific climatic conditions remains relatively constant. However, during adaptation to high altitudes—when a person ascends to over 4,000 m above sea level—the BPG concentration nearly doubles (from 4.5 to 7.0 mM) within just 2 days. This decreases the affinity of hemoglobin for O₂ and increases the amount of O₂ delivered to the tissues (Fig. 1-39).

Fig. 1-39. Effect of various concentrations of 2,3-bisphosphoglycerate on hemoglobin affinity for O₂.

The exact same adaptation is observed in patients with pulmonary diseases characterized by generalized tissue hypoxia. For instance, in patients suffering from severe obstructive pulmonary emphysema, the partial pressure in the lungs drops from 100 to 50 mm Hg. Yet, under these conditions, erythrocyte production of BPG is upregulated, raising its concentration from 4.5 to 7.0 mM, which substantially enhances O₂ delivery to the tissues.

Clinical significance of BPG concentration in stored blood

In blood stored in certain media, such as citrate-dextrose, the BPG concentration drops from 4.5 to 0.5 mM over a 10-day period. Hemoglobin from such blood exhibits a very high affinity for O₂. Transfusing blood with a depleted BPG concentration into critically ill patients poses a severe risk of inducing tissue hypoxia. Erythrocytes introduced via transfusion take up to 24 hours to restore even half of their normal BPG levels. Adding BPG directly to stored blood fails to restore its normal intracellular concentration because, possessing a high negative charge, BPG cannot cross Erythrocyte membranes. Consequently, modern practice involves adding substances to blood that can readily cross The erythrocyte membrane and maintain normal BPG concentrations.

6. Regulatory Properties of the oligomeric protein hemoglobin

Thus, unlike the monomeric related protein myoglobin, the oligomeric protein hemoglobin is capable of binding 4 different ligands at specific sites: O₂, H⁺, CO₂, and BPG. Although all these ligands bind to spatially distinct regions, conformational changes in the protein induced by the binding of one ligand are transmitted throughout the entire oligomeric structure, altering its affinity for the other ligands. As a result, the amount of O₂ released into tissues depends not only on the partial pressure of O₂ but also on the concentration of allosteric ligands, which significantly expands the regulatory capacity of hemoglobin functions.

As discussed above, in the capillaries of working muscle, an increase in CO₂ and H⁺ concentration decreases the affinity of hemoglobin for O₂, thereby enhancing its release into the tissues. During prolonged hypoxia, the synthesis of 2,3-BPG in erythrocytes is intensified, which similarly reduces hemoglobin affinity for O₂ and, at the same partial pressure of O₂, increases its transport to the tissues.

Consequently, driven by the action of regulatory ligands, oligomeric proteins are able to adapt their conformation and function to changing environmental conditions.

7. Structural and functional features of fetal hemoglobin

Fetal hemoglobin (HbF) replaces embryonic hemoglobin, beginning synthesis in the liver two weeks after its formation in the fetus. From the 6th month of fetal development until birth, it serves as the predominant hemoglobin in erythrocytes. Following birth, it is intensively replaced by adult hemoglobin A (HbA).

Under physiological conditions, HbF exhibits a higher affinity for O₂ than HbA, which creates optimal conditions for O₂ transport from maternal blood to fetal blood. This property of HbF is due to its weaker binding to 2,3-BPG compared to HbA. The PHYSIOLOGICAL CHARACTERISTICS OF HbF stem from its unique structure: instead of the β-globin chains found in HbA, it contains two γ-chains (which are β-like). The binding of 2,3-BPG to HbA involves positively charged amino acid side chains from the two β-chains, some of which are absent in the primary structure of the γ-chains. In a medium devoid of 2,3-BPG, HbA and HbF display identical, high affinities for O₂.

C. Inherited Disorders of the primary structure and function of hemoglobin A — hereditary hemoglobinopathies

The critical importance of the Primary Structure of Proteins for the formation of their conformation and function can be clearly traced through hereditary DISEASES ASSOCIATED WITH alterations in hemoglobin primary structure. Currently, approximately 300 variants of HbA are known, exhibiting only minor changes in the primary structure of their α- or β-chains. Some of these variants have virtually no impact on protein function or human health; others impair protein function and, particularly in extreme situations, diminish human adaptability; while still others cause severe disruptions in HbA function and trigger anemia, leading to grave clinical consequences.

In abnormal hemoglobins, the alterations may affect amino acids that are:

✵ located on the protein surface;

✵ involved in the Formation of the Active Site;

✵ whose substitution disrupts the overall three-dimensional conformation of the molecule;

✵ capable of altering the quaternary structure of the protein and its regulatory properties.

1. Amino acid substitution On the surface of hemoglobin A

As early as 1904, Chicago physician James Herrick described a severe case of anemia in a student, noting A large number of elongated, crescent-shaped erythrocytes in his blood. The condition became known as "Sickle-Cell Anemia," and it was not until 1949 that Linus Pauling and his colleagues proved it was caused by an alteration in the primary structure of HbA.

In the hemoglobin S molecule (as the abnormal hemoglobin is called), two mutant β-chains were found in which glutamate—a highly polar, negatively charged amino acid at position 6—was replaced by valine, which contains a hydrophobic side chain.

Deoxyhemoglobin S contains a region complementary to another region on identical molecules that carries the altered amino acid. As a result, deoxyhemoglobin molecules begin to "stick together," forming elongated fibrillar aggregates that deform the erythrocyte and lead to the formation of abnormal, sickle-shaped red Blood Cells (Fig. 1-40).

Fig. 1-40. Association of deoxyhemoglobin S molecules.

In oxyhemoglobin S, the complementary region is "masked" as a result of a protein conformation change. This inaccessibility prevents oxyhemoglobin S molecules from binding to one another. Consequently, the formation of HbS aggregates is promoted by conditions that increase the concentration of deoxyhemoglobin within the cells (such as physical exertion, hypoxia, lowered pH, high-altitude environments, or air travel).

Because "sickled" erythrocytes have difficulty passing through tissue capillaries, they frequently occlude Blood Vessels, thereby causing localized hypoxia. This further increases the concentration of intracellular deoxyhemoglobin S, accelerates the rate of HbS aggregate formation, and leads to even greater erythrocyte deformation. Impaired O₂ delivery to tissues causes pain and even cellular necrosis in the affected area.

Sickle-cell anemia is a homozygous recessive disorder; it manifests only when mutant genes for the globin β-chains are inherited from both parents. After birth, the disease remains asymptomatic until significant amounts of HbF are replaced by HbS. Affected individuals exhibit clinical symptoms typical of anemia, including dizziness and headaches, shortness of breath, palpitations, pain in the extremities, and increased Susceptibility to infectious diseases.

Heterozygous individuals who carry one normal HbA Gene and one HbS gene have only trace amounts of sickle cells in their blood, enjoy a normal life expectancy, and typically do not exhibit Clinical symptoms of the disease.

To diagnose the presence of HbS in human erythrocytes, researchers use Electrophoresis, a method based on the movement of charged proteins in an electric field. Because the negatively charged glutamate groups in the β-chains of HbS are replaced by uncharged valine, HbS migrates more slowly than HbA in an alkaline environment.

The high frequency of the HbS gene among populations in Africa (reaching up to 40% in some regions) is due to the fact that heterozygotes are less susceptible to malaria than individuals with normal hemoglobin A. Plasmodium falciparum, the CAUSATIVE AGENT OF malaria, spends an obligatory part of its life cycle within erythrocytes. Because erythrocytes in HbS heterozygotes have a shorter lifespan than normal red blood cells, the malaria parasite fails to complete its required developmental stage. This confers a selective survival advantage to HbS heterozygotes in areas where malaria is traditionally lethal.

Sickle-cell anemia is the first molecular disease ever described.

Almost all Amino Acid Substitutions occurring on the surface of the hemoglobin molecule are harmless. Hemoglobin S is a rare exception.

2. Amino acid alterations in the active site region of hemoglobin

There are approximately 60 interatomic contacts between the heme group and the protein moiety of hemoglobin. Most Mutations that disrupt these contacts to any degree lead to hemoglobinopathies and anemia.

Hemoglobin M is a variant of hemoglobin A in which a mutation in either the α- or β-chain gene results in the substitution of His E₇ or His F₈ by Tyrosine. Consequently, Fe²⁺ is oxidized to Fe³⁺ and stabilized in this state. Hemoglobin containing Fe³⁺ in its heme is termed methemoglobin (hence the name hemoglobin M). Instead of O₂, an H₂O molecule binds to Fe³⁺. Typically, these alterations affect either the α- or the β-chains exclusively, allowing the variant hemoglobin to transport no more than two molecules of O₂. Heterozygous individuals exhibit cyanosis associated with impaired O₂ transport, whereas homozygosity for this gene is lethal.

Hemoglobin Hammersmith is a variant of hemoglobin A in which Serine (a hydrophilic amino acid) replaces phenylalanine (a hydrophobic amino acid) at position D₁. Phe D₁ normally resides in the nonpolar microenvironment of the heme. Its replacement by a hydrophilic amino acid compromises the Stability of the heme-globin linkage, allowing water to penetrate the "hydrophobic pocket" that houses the heme. This water oxidizes Fe²⁺ to Fe³⁺, ultimately resulting in anemia.

3. Amino acid alterations that deform the Tertiary Structure of hemoglobin

In all normal hemoglobins and in myoglobin, the amino acid Glycine is located at the intersection of the two α-helices B and E. Because glycine possesses a hydrogen atom instead of a bulky side chain, it allows these two helices to pack tightly against each other.

In Hemoglobin Riverdale-Bronx (a variant of hemoglobin A), the amino acid Arginine, which features a bulky side chain, replaces glycine at position B₆. As a result, it cannot be accommodated within such a narrow space, causing the protein molecule to alter its conformation and become unstable.

4. Amino acid substitutions in the α₁β₁ and α₂β₂ dimer contact regions that disrupt the allosteric Regulatory Functions of hemoglobin

Almost all hemoglobin A variants involving amino acid substitutions at the α₁β₁ and α₂β₂ dimer contact regions exhibit reduced cooperativity and altered oxygen affinity.

For instance, Hemoglobin Kempsey is a variant of hemoglobin A in which aspartate is replaced by asparagine at position G₁ of the β-chain. Under normal conditions, this aspartic acid residue participates in forming a Hydrogen bond that stabilizes deoxyhemoglobin. Because of this substitution, the hydrogen bond fails to form, disrupting the conformational stability of deoxyhemoglobin and abnormally increasing its oxygen affinity. Patients develop anemia accompanied by pronounced cyanosis.

Thus, the primary structure of a protein dictates the Specific features of its conformation, active site architecture, and biological functions. A single amino acid alteration in even one protein can cause severe functional impairment and lead to hereditary pathology.