Lippincotts Biochemistry 6th Edition PDF

UNIT I: Protein Structure and Function Amino Acids 1 I. OVERVIEW Proteins are the most abundant and functionally diverse molecules in living systems. Virtually every life process depends on this class of macromolecules. For example, enzymes and polypeptide hormones direct and regulate metabolism in the body, whereas contractile proteins in muscle permit movement. In bone, the protein collagen forms a framework for the deposition of calcium phosphate crystals, acting like the steel cables in reinforced concrete. In the bloodstream, proteins, such as hemoglobin and plasma albumin, shuttle molecules essential to life, whereas immunoglobulins fight infectious bacteria and viruses. In short, proteins display an incredible diversity of functions, yet all share the common structural feature of being linear polymers of amino acids. This chapter describes the properties of amino acids. Chapter 2 explores how these simple building blocks are joined to form proteins that have unique three-dimensional structures, making them capable of performing specific biologic functions. Figure 1.1 Structural features of amino acids (shown in their fully protonated form). II. STRUCTURE Although more than 300 different amino acids have been described in nature, only 20 are commonly found as constituents of mammalian proteins. [Note: These are the only amino acids that are coded for by DNA, the genetic material in the cell (see p. 395).] Each amino acid has a carboxyl group, a primary amino group (except for proline, which has a secondary amino group), and a distinctive side chain (“R group”) bonded to the α-carbon atom (Figure 1.1A). At physiologic pH (approximately 7.4), the carboxyl group is dissociated, forming the negatively charged carboxylate ion (–COO–), and the amino group is protonated (–NH3+). In proteins, almost all of these carboxyl and amino groups are combined through peptide linkage and, in general, are not available for chemical reaction except for hydrogen bond formation (Figure 1.1B). Thus, it is the nature of the side chains that ultimately dictates the role an amino acid plays in a protein. It is, therefore, useful to classify the amino acids according to the properties of their side chains, that is, whether they are nonpolar (have an even distribution of electrons) or polar (have an uneven distribution of electrons, such as acids and bases) as shown in Figures 1.2 and 1.3. A. Amino acids with nonpolar side chains Each of these amino acids has a nonpolar side chain that does not gain or lose protons or participate in hydrogen or ionic bonds (see Figure 1.2). The side chains of these amino acids can be thought of as “oily” or lipid-like, a property that promotes hydrophobic inter-actions (see Figure 2.10, p. 19). 1. Location of nonpolar amino acids in proteins: In proteins found in aqueous solutions (a polar environment) the side chains of the nonpolar amino acids tend to cluster together in the interior of the protein (Figure 1.4). This phenomenon, known as the hydrophobic effect, is the result of the hydrophobicity of the nonpolar R groups, which act much like droplets of oil that coalesce in an aqueous environment. The nonpolar R groups, thus, fill up the interior of the folded protein and help give it its three-dimensional shape. However, for proteins that are located in a hydrophobic environment, such as a membrane, the nonpolar R groups are found on the outside surface of the protein, interacting with the lipid environment see Figure 1.4. The importance of these hydrophobic interactions in stabilizing protein structure is discussed on p. 19. Figure 1.2 Classification of the 20 amino acids commonly found in proteins, according to the charge and polarity of their side chains at acidic pH is shown here and continues in Figure 1.3. Each amino acid is shown in its fully protonated form, with dissociable hydrogen ions represented in red print. The pK values for the α-carboxyl and α-amino groups of the nonpolar amino acids are similar to those shown for glycine. Figure 1.3 Classification of the 20 amino acids commonly found in proteins, according to the charge and polarity of their side chains at acidic pH (continued from Figure 1.2). Figure 1.4 Location of nonpolar amino acids in soluble and membrane proteins. Sickle cell anemia, a sickling disease of red blood cells, results from the replacement of polar glutamate with nonpolar valine at the sixth position in the β subunit of hemoglobin (see p. 36). Figure 1.5 Comparison of the secondary amino group found in proline with the primary amino group found in other amino acids such as alanine. 2. Proline: Proline differs from other amino acids in that its side chain and α-amino N form a rigid, five-membered ring structure (Figure 1.5). Proline, then, has a secondary (rather than a primary) amino group. It is frequently referred to as an “imino acid.” The unique geometry of proline contributes to the formation of the fibrous structure of collagen (see p. 45) and often interrupts the α-helices found in globular proteins (see p. 26). B. Amino acids with uncharged polar side chains These amino acids have zero net charge at physiologic pH, although the side chains of cysteine and tyrosine can lose a proton at an alkaline pH (see Figure 1.3). Serine, threonine, and tyrosine each contain a polar hydroxyl group that can participate in hydrogen bond formation (Figure 1.6). The side chains of asparagine and glutamine each contain a carbonyl group and an amide group, both of which can also participate in hydrogen bonds. 1. Disulfide bond: The side chain of cysteine contains a sulfhydryl (thiol) group (– SH), which is an important component of the active site of many enzymes. In proteins, the –SH groups of two cysteines can be oxidized to form a covalent cross- link called a disulfide bond (–S–S–). Two disulfide-linked cysteines are referred to as “cystine.” (See p. 19 for a further discussion of disulfide bond formation.) Many extracellular proteins are stabilized by disulfide bonds. Albumin, a blood protein that functions as a transporter for a variety of molecules, is an example. Figure 1.6 Hydrogen bond between the phenolic hydroxyl group of tyrosine and another molecule containing a carbonyl group. 2. Side chains as sites of attachment for other compounds: The polar hydroxyl group of serine; threonine; and, rarely, tyrosine, can serve as a site of attachment for structures such as a phosphate group. In addition, the amide group of asparagine, as well as the hydroxyl group of serine or threonine, can serve as a site of attachment for oligosaccharide chains in glycoproteins (see p. 165). C. Amino acids with acidic side chains The amino acids aspartic and glutamic acid are proton donors. At physiologic pH, the side chains of these amino acids are fully ionized, containing a negatively charged carboxylate group (–COO–). They are, therefore, called aspartate or glutamate to emphasize that these amino acids are negatively charged at physiologic pH (see Figure 1.3). D. Amino acids with basic side chains The side chains of the basic amino acids accept protons (see Figure 1.3). At physiologic pH, the R groups of lysine and arginine are fully ionized and positively charged. In contrast, histidine is weakly basic, and the free amino acid is largely uncharged at physiologic pH. However, when histidine is incorporated into a protein, its R group can be either positively charged (protonated) or neutral, depending on the ionic environment provided by the protein. This is an important property of histidine that contributes to the buffering role it plays in the functioning of proteins such as hemoglobin (see p. 31). [Note: Histidine is the only amino acid with a side chain that can ionize within the physiologic pH range.] Figure 1.7 Abbreviations and symbols for the commonly occurring amino acids. E. Abbreviations and symbols for commonly occurring amino acids Each amino acid name has an associated three-letter abbreviation and a one-letter symbol (Figure 1.7). The one-letter codes are determined by the following rules. 1. Unique first letter: If only one amino acid begins with a given letter, then that letter is used as its symbol. For example, V = valine. 2. Most commonly occurring amino acids have priority: If more than one amino acid begins with a particular letter, the most common of these amino acids receives this letter as its symbol. For example, glycine is more common than glutamate, so G = glycine. 3. Similar sounding names: Some one-letter symbols sound like the amino acid they represent. For example, F = phenylalanine, or W = tryptophan (“twyptophan” as Elmer Fudd would say). 4. Letter close to initial letter: For the remaining amino acids, a one-letter symbol is assigned that is as close in the alphabet as possible to the initial letter of the amino acid, for example, K = lysine. Furthermore, B is assigned to Asx, signifying either aspartic acid or asparagine, Z is assigned to Glx, signifying either glutamic acid or glutamine, and X is assigned to an unidentified amino acid. Figure 1.8 D and L forms of alanine are mirror images. F. Optical properties of amino acids The α-carbon of an amino acid is attached to four different chemical groups (asymmetric) and is, therefore, a chiral, or optically active carbon atom. Glycine is the exception because its α-carbon has two hydrogen substituents. Amino acids with an asymmetric center at the α-carbon can exist in two forms, designated D and L, that are mirror images of each other (Figure 1.8). The two forms in each pair are termed stereoisomers, optical isomers, or enantiomers. All amino acids found in proteins are of the L configuration. However, D-amino acids are found in some antibiotics and in bacterial cell walls. (See p. 252 for a discussion of D-amino acids.) III. ACIDIC AND BASIC PROPERTIES OF AMINO ACIDS Amino acids in aqueous solution contain weakly acidic α-carboxyl groups and weakly basic α-amino groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain. Thus, both free amino acids and some amino acids combined in peptide linkages can act as buffers. Recall that acids may be defined as proton donors and bases as proton acceptors. Acids (or bases) described as “weak” ionize to only a limited extent. The concentration of protons in aqueous solution is expressed as pH, where pH = log 1/[H+] or –log [H+]. The quantitative relationship between the pH of the solution and concentration of a weak acid (HA) and its conjugate base (A–) is described by the Henderson-Hasselbalch equation. Figure 1.9 Titration curve of acetic acid. A. Derivation of the equation Consider the release of a proton by a weak acid represented by HA: The “salt” or conjugate base, A–, is the ionized form of a weak acid. By definition, the dissociation constant of the acid, Ka, is [Note: The larger the Ka, the stronger the acid, because most of the HA has dissociated into H+ and A–. Conversely, the smaller the K a, the less acid has dissociated and, therefore, the weaker the acid.] By solving for the [H +] in the above equation, taking the logarithm of both sides of the equation, multiplying both sides of the equation by –1, and substituting pH = –log [H+] and pKa = –log Ka, we obtain the Henderson-Hasselbalch equation: B. Buffers A buffer is a solution that resists change in pH following the addition of an acid or base. A buffer can be created by mixing a weak acid (HA) with its conjugate base (A–). If an acid such as HCl is added to a buffer, A – can neutralize it, being converted to HA in the process. If a base is added, HA can neutralize it, being converted to A– in the process. Maximum buffering capacity occurs at a pH equal to the pKa, but a conjugate acid–base pair can still serve as an effective buffer when the pH of a solution is within approximately ±1 pH unit of the pKa. If the amounts of HA and A– are equal, the pH is equal to the pKa. As shown in Figure 1.9, a solution containing acetic acid (HA = CH3 – COOH) and acetate (A– = CH3 –COO–) with a pKa of 4.8 resists a change in pH from pH 3.8 to 5.8, with maximum buffering at pH 4.8. At pH values less than the pKa, the protonated acid form (CH3 – COOH) is the predominant species in solution. At pH values greater than the pKa, the deprotonated base form (CH3 – COO–) is the predominant species. Figure 1.10 Ionic forms of alanine in acidic, neutral, and basic solutions. C. Titration of an amino acid 1. Dissociation of the carboxyl group: The titration curve of an amino acid can be analyzed in the same way as described for acetic acid. Consider alanine, for example, which contains an ionizable α-carboxyl and α-amino group. [Note: Its –CH3 R group is nonionizable.] At a low (acidic) pH, both of these groups are protonated (shown in Figure 1.10). As the pH of the solution is raised, the – COOH group of form I can dissociate by donating a proton to the medium. The release of a proton results in the formation of the carboxylate group, – COO–. This structure is shown as form II, which is the dipolar form of the molecule (see Figure 1.10). This form, also called a zwitterion, is the isoelectric form of alanine, that is, it has an overall (net) charge of zero. 2. Application of the Henderson-Hasselbalch equation: The dissociation constant of the carboxyl group of an amino acid is called K1, rather than Ka, because the molecule contains a second titratable group. The Henderson-Hasselbalch equation can be used to analyze the dissociation of the carboxyl group of alanine in the same way as described for acetic acid: where I is the fully protonated form of alanine, and II is the isoelectric form of alanine (see Figure 1.10). This equation can be rearranged and converted to its logarithmic form to yield: 3. Dissociation of the amino group: The second titratable group of alanine is the amino (– NH3+) group shown in Figure 1.10. This is a much weaker acid than the – COOH group and, therefore, has a much smaller dissociation constant, K2. [Note: Its pKa is, therefore, larger.] Release of a proton from the protonated amino group of form II results in the fully deprotonated form of alanine, form III (see Figure 1.10). Figure 1.11 The titration curve of alanine. 4. pKs of alanine: The sequential dissociation of protons from the carboxyl and amino groups of alanine is summarized in Figure 1.10. Each titratable group has a pKa that is numerically equal to the pH at which exactly one half of the protons have been removed from that group. The pKa for the most acidic group (–COOH) is pK1, whereas the pKa for the next most acidic group (– NH3+) is pK2. [Note: The pKa of the α-carboxyl group of amino acids is approximately 2, whereas that of the α-amino is approximately 9.] 5. Titration curve of alanine: By applying the Henderson-Hasselbalch equation to each dissociable acidic group, it is possible to calculate the complete titration curve of a weak acid. Figure 1.11 shows the change in pH that occurs during the addition of base to the fully protonated form of alanine (I) to produce the completely deprotonated form (III). Note the following: a. Buffer pairs: The – COOH/– COO– pair can serve as a buffer in the pH region around pK1, and the – NH3+/– NH2 pair can buffer in the region around pK2. b. When pH = pK: When the pH is equal to pK1 (2.3), equal amounts of forms I and II of alanine exist in solution. When the pH is equal to pK2 (9.1), equal amounts of forms II and III are present in solution. c. Isoelectric point: At neutral pH, alanine exists predominantly as the dipolar form II in which the amino and carboxyl groups are ionized, but the net charge is zero. The isoelectric point (pI) is the pH at which an amino acid is electrically neutral, that is, in which the sum of the positive charges equals the sum of the negative charges. For an amino acid, such as alanine, that has only two dissociable hydrogens (one from the α-carboxyl and one from the α-amino group), the pI is the average of pK1 and pK2 (pI = [2.3 + 9.1]/2 = 5.7) as shown i n Figure 1.11. The pI is, thus, midway between pK1 (2.3) and pK2 (9.1). pI corresponds to the pH at which the form II (with a net charge of zero) predominates and at which there are also equal amounts of forms I (net charge of +1) and III (net charge of –1). Separation of plasma proteins by charge typically is done at a pH above the pI of the major proteins. Thus, the charge on the proteins is negative. In an electric field, the proteins will move toward the positive electrode at a rate determined by their net negative charge. Variations in the mobility pattern are suggestive of certain diseases. 6. Net charge of amino acids at neutral pH: At physiologic pH, amino acids have a negatively charged group (– COO–) and a positively charged group (– NH3+), both attached to the α-carbon. [Note: Glutamate, aspartate, histidine, arginine, and lysine have additional potentially charged groups in their side chains.] Substances such as amino acids that can act either as an acid or a base are defined as amphoteric and are referred to as ampholytes (amphoteric electrolytes). Figure 1.12 The Henderson-Hasselbalch equation is used to predict: A, changes in pH as the concentrations of HCO3– or CO2 are altered, or B, the ionic forms of drugs. D. Other applications of the Henderson-Hasselbalch equation The Henderson-Hasselbalch equation can be used to calculate how the pH of a physiologic solution responds to changes in the concentration of a weak acid and/or its corresponding “salt” form. For example, in the bicarbonate buffer system, the Henderson-Hasselbalch equation predicts how shifts in the bicarbonate ion concentration, [HCO3–], and CO2 influence pH (Figure 1.12A). The equation is also useful for calculating the abundance of ionic forms of acidic and basic drugs. For example, most drugs are either weak acids or weak bases (Figure 1.12B). Acidic drugs (HA) release a proton (H+), causing a charged anion (A–) to form. HA H+ + A- Weak bases (BH+) can also release a H+. However, the protonated form of basic drugs is usually charged, and the loss of a proton produces the uncharged base (B). BH+ B + H+ A drug passes through membranes more readily if it is uncharged. Thus, for a weak acid, such as aspirin, the uncharged HA can permeate through membranes, but A– cannot. For a weak base, such as morphine, the uncharged form, B, penetrates through the cell membrane, but BH+ does not. Therefore, the effective concentration of the permeable form of each drug at its absorption site is determined by the relative concentrations of the charged (impermeant) and uncharged (permeant) forms. The ratio between the two forms is determined by the pH at the site of absorption, and by the strength of the weak acid or base, which is represented by the pKa of the ionizable group. The Henderson-Hasselbalch equation is useful in determining how much drug is found on either side of a membrane that separates two compartments that differ in pH, for example, the stomach (pH 1.0–1.5) and blood plasma (pH 7.4). IV. CONCEPT MAPS Students sometimes view biochemistry as a list of facts or equations to be memorized, rather than a body of concepts to be understood. Details provided to enrich understanding of these concepts inadvertently turn into distractions. What seems to be missing is a road map—a guide that provides the student with an understanding of how various topics fit together to make sense. Therefore, a series of biochemical concept maps have been created to graphically illustrate relationships between ideas presented in a chapter and to show how the information can be grouped or organized. A concept map is, thus, a tool for visualizing the connections between concepts. Material is represented in a hierarchic fashion, with the most inclusive, most general concepts at the top of the map and the more specific, less general concepts arranged beneath. The concept maps ideally function as templates or guides for organizing information, so the student can readily find the best ways to integrate new information into knowledge they already possess. Figure 1.13 Symbols used in concept maps. A. How is a concept map constructed? 1. Concept boxes and links: Educators define concepts as “perceived regularities in events or objects.” In the biochemical maps, concepts include abstractions (for example, free energy), processes (for example, oxidative phosphorylation), and compounds (for example, glucose 6-phosphate). These broadly defined concepts are prioritized with the central idea positioned at the top of the page. The concepts that follow from this central idea are then drawn in boxes (Figure 1.13A). The size of the type indicates the relative importance of each idea. Lines are drawn between concept boxes to show which are related. The label on the line defines the relationship between two concepts, so that it reads as a valid statement, that is, the connection creates meaning. The lines with arrowheads indicate in which direction the connection should be read (Figure 1.14). 2. Cross-links: Unlike linear flow charts or outlines, concept maps may contain cross- links that allow the reader to visualize complex relationships between ideas represented in different parts of the map (Figure 1.13B), or between the map and other chapters in this book (Figure 1.13C). Cross-links can, thus, identify concepts that are central to more than one topic in biochemistry, empowering students to be effective in clinical situations and on the United States Medical Licensure Examination (USMLE) or other examinations that require integration of material. Students learn to visually perceive nonlinear relationships between facts, in contrast to cross-referencing within linear text. V. CHAPTER SUMMARY Each amino acid has an α-carboxyl group and a primary α-amino group (except for proline, which has a secondary amino group). At physiologic pH, the α- carboxyl group is dissociated, forming the negatively charged carboxylate ion (– COO–), and the α-amino group is protonated (– NH3+). Each amino acid also contains one of 20 distinctive side chains attached to the α-carbon atom. The chemical nature of this R group determines the function of an amino acid in a protein and provides the basis for classification of the amino acids as nonpolar, uncharged polar, acidic (polar negative), or basic (polar positive). All free amino acids, plus charged amino acids in peptide chains, can serve as buffers. The quantitative relationship between the pH of a solution and the concentration of a weak acid (HA) and its conjugate base (A–) is described by the Henderson- Hasselbalch equation. Buffering occurs within ±1 pH unit of the pKa and is maximal when pH = pKa, at which [A–] = [HA]. The α-carbon of each amino acid (except glycine) is attached to four different chemical groups and is, therefore, a chiral, or optically active carbon atom. The L-form of amino acids is found in proteins synthesized by the human body. Figure 1.14 Key concept map for amino acids. Study Questions Choose the ONE best answer. 1.1 Which one of the following statements concerning the titration curve for a nonpolar amino acid is correct? The letters A through D designate certain regions on the curve below. A. Point A represents the region where the amino acid is deprotonated. B. Point B represents a region of minimal buffering. C. Point C represents the region where the net charge on the amino acid is zero. D. Point D represents the pK of the amino acid’s carboxyl group. E. The amino acid could be lysine. Correct answer = C. C represents the isoelectric point, or pI, and as such is midway between pK1 and pK2 for a nonpolar amino acid. The amino acid is fully protonated at Point A. Point B represents a region of maximum buffering, as does Point D. Lysine is a basic amino acid, and has an ionizable side chain. 1.2 Which one of the following statements concerning the peptide shown below is correct? Val-Cys-Glu-Ser-Asp-Arg-Cys A. The peptide contains asparagine. B. The peptide contains a side chain with a secondary amino group. C. The peptide contains a side chain that can be phosphorylated. D. The peptide cannot form an internal disulfide bond. E. The peptide would move to the cathode (negative electrode) during electrophoresis at pH 5. Correct answer = C. The hydroxyl group of serine can accept a phosphate group. Asp is aspartate. Proline contains a secondary amino group. The two cysteine residues can, under oxidizing conditions, form a disulfide (covalent) bond. The net charge on the peptide at pH 5 is negative, and it would move to the anode. 1.3 A 2-year-old child presents with metabolic acidosis after ingesting an unknown number of flavored aspirin tablets. At presentation, her blood pH was 7.0. Given that the pKa of aspirin (salicylic acid) is 3, calculate the ratio of its ionized to un-ionized forms at pH 7.0. Correct answer = 10,000 to 1. pH = pKa + log [A–]/[HA]. Therefore, 7 = 3 + × and × = 4. The ratio of A– (ionized) to HA (un-ionized), then, is 10,000 to 1 because the log of 10,000 is 4. Structure of Proteins 2 I. OVERVIEW The 20 amino acids commonly found in proteins are joined together by peptide bonds. The linear sequence of the linked amino acids contains the information necessary to generate a protein molecule with a unique three-dimensional shape. The complexity of protein structure is best analyzed by considering the molecule in terms of four organizational levels: primary, secondary, tertiary, and quaternary ( Figure 2.1). An examination of these hierarchies of increasing complexity has revealed that certain structural elements are repeated in a wide variety of proteins, suggesting that there are general “rules” regarding the ways in which proteins achieve their native, functional form. These repeated structural elements range from simple combinations of α-helices and β- sheets forming small motifs, to the complex folding of polypeptide domains of multifunctional proteins (see p. 19). II. PRIMARY STRUCTURE OF PROTEINS The sequence of amino acids in a protein is called the primary structure of the protein. Understanding the primary structure of proteins is important because many genetic diseases result in proteins with abnormal amino acid sequences, which cause improper folding and loss or impairment of normal function. If the primary structures of the normal and the mutated proteins are known, this information may be used to diagnose or study the disease. A. Peptide bond In proteins, amino acids are joined covalently by peptide bonds, which are amide linkages between the α-carboxyl group of one amino acid and the α-amino group of another. For example, valine and alanine can form the dipeptide valylalanine through the formation of a peptide bond (Figure 2.2). Peptide bonds are resistant to conditions that denature proteins, such as heating and high concentrations of urea (see p. 20). Prolonged exposure to a strong acid or base at elevated temperatures is required to break these bonds nonenzymically. Figure 2.1 Four hierarchies of protein structure. 1. Naming the peptide: By convention, the free amino end (N-terminal) of the peptide chain is written to the left and the free carboxyl end (C-terminal) to the right. Therefore, all amino acid sequences are read from the N- to the C-terminal end of the peptide. For example, in Figure 2.2A, the order of the amino acids is “valine, alanine.” Linkage of many amino acids through peptide bonds results in an unbranched chain called a polypeptide. Each component amino acid in a polypeptide is called a “residue” because it is the portion of the amino acid remaining after the atoms of water are lost in the formation of the peptide bond. When a polypeptide is named, all amino acid residues have their suffixes (-ine, -an, -ic, or -ate) changed to -yl, with the exception of the C-terminal amino acid. For example, a tripeptide composed of an N-terminal valine, a glycine, and a C-terminal leucine is called valylglycylleucine. 2. Characteristics of the peptide bond: The peptide bond has a partial double- bond character, that is, it is shorter than a single bond and is rigid and planar (Figure 2.2B). This prevents free rotation around the bond between the carbonyl carbon and the nitrogen of the peptide bond. However, the bonds between the α-carbons and the α-amino or α-carboxyl groups can be freely rotated (although they are limited by the size and character of the R groups). This allows the polypeptide chain to assume a variety of possible configurations. The peptide bond is almost always a trans bond (instead of cis, see Figure 2.2B), in large part because of steric interference of the R groups when in the cis position. 3. Polarity of the peptide bond: Like all amide linkages, the – C =O and – NH groups of the peptide bond are uncharged and neither accept nor release protons over the pH range of 2–12. Thus, the charged groups present in polypeptides consist solely of the N-terminal (α-amino) group, the C-terminal (α-carboxyl) group, and any ionized groups present in the side chains of the constituent amino acids. The – C=O and – NH groups of the peptide bond are polar, however, and are involved in hydrogen bonds (for example, in α-helices and β-sheets), as described on pp. 16–17. Figure 2.2 A. Formation of a peptide bond, showing the structure of the dipeptide valylalanine. B. Characteristics of the peptide bond. B. Determination of the amino acid composition of a polypeptide The first step in determining the primary structure of a polypeptide is to identify and quantitate its constituent amino acids. A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid at 110°C for 24 hours. This treatment cleaves the peptide bonds and releases the individual amino acids, which can be separated by cation-exchange chromatography. In this technique, a mixture of amino acids is applied to a column that contains a resin to which a negatively charged group is tightly attached. [Note: If the attached group is positively charged, the column becomes an anion-exchange column.] The amino acids bind to the column with different affinities, depending on their charges, hydrophobicity, and other characteristics. Each amino acid is sequentially released from the chromatography column by eluting with solutions of increasing ionic strength and pH (Figure 2.3). The separated amino acids contained in the eluate from the column are quantitated by heating them with ninhydrin (a reagent that forms a purple compound with most amino acids, ammonia, and amines). The amount of each amino acid is determined spectrophotometrically by measuring the amount of light absorbed by the ninhydrin derivative. The analysis described above is performed using an amino acid analyzer, an automated machine whose components are depicted in Figure 2.3. C. Sequencing of the peptide from its N-terminal end Sequencing is a stepwise process of identifying the specific amino acid at each position in the peptide chain, beginning at the N-terminal end. Phenylisothiocyanate, known as Edman reagent, is used to label the amino-terminal residue under mildly alkaline conditions (Figure 2.4). The resulting phenylthiohydantoin (PTH) derivative introduces an instability in the N-terminal peptide bond such that it can be hydrolyzed without cleaving the other peptide bonds. The identity of the amino acid derivative can then be determined. Edman reagent can be applied repeatedly to the shortened peptide obtained in each previous cycle. The process is now automated. D. Cleavage of the polypeptide into smaller fragments Many polypeptides have a primary structure composed of more than 100 amino acids. Such molecules cannot be sequenced directly from end to end. However, these large molecules can be cleaved at specific sites and the resulting fragments sequenced. By using more than one cleaving agent (enzymes and/or chemicals) on separate samples of the purified polypeptide, overlapping fragments can be generated that permit the proper ordering of the sequenced fragments, thereby providing a complete amino acid sequence of the large polypeptide (Figure 2.5). Enzymes that hydrolyze peptide bonds are termed peptidases (proteases). [Note: Exopeptidases cut at the ends of proteins and are divided into aminopeptidases and carboxypeptidases. Carboxypeptidases are used in determining the C-terminal amino acid. Endopeptidases cleave within a protein.] Figure 2.3 Determination of the amino acid composition of a polypeptide using an amino acid analyzer. E. Determination of a protein’s primary structure by DNA sequencing The sequence of nucleotides in a protein-coding region of the DNA specifies the amino acid sequence of a polypeptide. Therefore, if the nucleotide sequence can be determined, it is possible, from knowledge of the genetic code (see p. 432), to translate the sequence of nucleotides into the corresponding amino acid sequence of that polypeptide. This indirect process, although routinely used to obtain the amino acid sequences of proteins, has the limitations of not being able to predict the positions of disulfide bonds in the folded chain and of not identifying any amino acids that are modified after their incorporation into the polypeptide (posttranslational modification, see p. 443). Therefore, direct protein sequencing is an extremely important tool for determining the true character of the primary sequence of many polypeptides. Figure 2.4 Determination of the amino (N)-terminal residue of a polypeptide by Edman degradation. PTH = phenylthiohydantoin. III. SECONDARY STRUCTURE OF PROTEINS The polypeptide backbone does not assume a random three-dimensional structure but, instead, generally forms regular arrangements of amino acids that are located near each other in the linear sequence. These arrangements are termed the secondary structure of the polypeptide. The α-helix, β-sheet, and β-bend (β-turn) are examples of secondary structures commonly encountered in proteins. [Note: The collagen α-chain helix, another example of secondary structure, is discussed on p. 45.] Figure 2.5 Overlapping of peptides produced by the action of trypsin and cyanogen bromide. A. α-Helix Several different polypeptide helices are found in nature, but the α-helix is the most common. It is a spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side chains of the component amino acids extending outward from the central axis to avoid interfering sterically with each other (Figure 2.6). A very diverse group of proteins contains α-helices. For example, the keratins are a family of closely related, fibrous proteins whose structure is nearly entirely α-helical. They are a major component of tissues such as hair and skin, and their rigidity is determined by the number of disulfide bonds between the constituent polypeptide chains. In contrast to keratin, myoglobin, whose structure is also highly α-helical, is a globular, flexible molecule (see p. 26). Figure 2.6 α-Helix showing peptide backbone. 1. Hydrogen bonds: An α-helix is stabilized by extensive hydrogen bonding between the peptide-bond carbonyl oxygens and amide hydrogens that are part of the polypeptide backbone (see Figure 2.6). The hydrogen bonds extend up and are parallel to the spiral from the carbonyl oxygen of one peptide bond to the – NH – group of a peptide linkage four residues ahead in the polypeptide. This insures that all but the first and last peptide bond components are linked to each other through intrachain hydrogen bonds. Hydrogen bonds are individually weak, but they collectively serve to stabilize the helix. 2. Amino acids per turn: Each turn of an α-helix contains 3.6 amino acids. Thus, amino acid residues spaced three or four residues apart in the primary sequence are spatially close together when folded in the α-helix. 3. Amino acids that disrupt an α-helix: Proline disrupts an α-helix because its secondary amino group is not geometrically compatible with the right-handed spiral of the α-helix. Instead, it inserts a kink in the chain, which interferes with the smooth, helical structure. Large numbers of charged amino acids (for example, glutamate, aspartate, histidine, lysine, and arginine) also disrupt the helix by forming ionic bonds or by electrostatically repelling each other. Finally, amino acids with bulky side chains, such as tryptophan, or amino acids, such as valine or isoleucine, that branch at the β-carbon (the first carbon in the R group, next to the α-carbon) can interfere with formation of the α-helix if they are present in large numbers. Figure 2.7 A. Structure of a β-sheet. B. An antiparallel β-sheet with the β-strands represented as broad arrows. C. A parallel β-sheet formed from a single polypeptide chain folding back on itself. B. β-Sheet The β-sheet is another form of secondary structure in which all of the peptide bond components are involved in hydrogen bonding (Figure 2.7A). The surfaces of β-sheets appear “pleated,” and these structures are, therefore, often called β-pleated sheets. When illustrations are made of protein structure, β-strands are often visualized as broad arrows (Figure 2.7B). 1. Comparison of a β-sheet and an α-helix: Unlike the α-helix, β-sheets are composed of two or more peptide chains (β-strands), or segments of polypeptide chains, which are almost fully extended. Note also that the hydrogen bonds are perpendicular to the polypeptide backbone in β-sheets (see Figure 2.7A). 2. Parallel and antiparallel sheets: A β-sheet can be formed from two or more separate polypeptide chains or segments of polypeptide chains that are arranged either antiparallel to each other (with the N-terminal and C-terminal ends of the β- strands alternating as shown in Figure 2.7B) or parallel to each other (with all the N- termini of the β-strands together as shown in Figure 2.7C). When the hydrogen bonds are formed between the polypeptide backbones of separate polypeptide chains, they are termed interchain bonds. A β-sheet can also be formed by a single polypeptide chain folding back on itself (see Figure 2.7C). In this case, the hydrogen bonds are intrachain bonds. In globular proteins, β-sheets always have a right- handed curl, or twist, when viewed along the polypeptide backbone. [Note: Twisted β-sheets often form the core of globular proteins.] The α-helix and β-sheet structures provide maximal hydrogen bonding for peptide bond components within the interior of polypeptides. C. β-Bends (reverse turns, β-turns) β-Bends reverse the direction of a polypeptide chain, helping it form a compact, globular shape. They are usually found on the surface of protein molecules and often include charged residues. [Note: β-Bends were given this name because they often connect successive strands of antiparallel β-sheets.] β-Bends are generally composed of four amino acids, one of which may be proline, the amino acid that causes a kink in the polypeptide chain. Glycine, the amino acid with the smallest R group, is also frequently found in β-bends. β-Bends are stabilized by the formation of hydrogen and ionic bonds. Figure 2.8 Some common structural motifs involving β-helices and β-sheets. The names describe their schematic appearance. D. Nonrepetitive secondary structure Approximately one half of an average globular protein is organized into repetitive structures, such as the α-helix and β-sheet. The remainder of the polypeptide chain is described as having a loop or coil conformation. These nonrepetitive secondary structures are not random, but rather simply have a less regular structure than those described above. [Note: The term “random coil” refers to the disordered structure obtained when proteins are denatured (see p. 20).] E. Supersecondary structures (motifs) Globular proteins are constructed by combining secondary structural elements (that is, α-helices, β-sheets, and coils), producing specific geometric patterns or motifs. These form primarily the core (interior) region of the molecule. They are connected by loop regions (for example, β-bends) at the surface of the protein. Supersecondary structures are usually produced by the close packing of side chains from adjacent secondary structural elements. Thus, for example, α-helices and β-sheets that are adjacent in the amino acid sequence are also usually (but not always) adjacent in the final, folded protein. Some of the more common motifs are illustrated in Figure 2.8. Motifs may be associated with particular functions. Proteins that bind to DNA contain a limited number of motifs. The helix-loop-helix motif is an example found in a number of proteins that function as transcription factors (see p. 450). Figure 2.9 Formation of a disulfide bond by the oxidation of two cysteine residues, producing one cystine residue. IV. TERTIARY STRUCTURE OF GLOBULAR PROTEINS The primary structure of a polypeptide chain determines its tertiary structure. “Tertiary” refers both to the folding of domains (the basic units of structure and function, see discussion below), and to the final arrangement of domains in the polypeptide. The structure of globular proteins in aqueous solution is compact, with a high density (close packing) of the atoms in the core of the molecule. Hydrophobic side chains are buried in the interior, whereas hydrophilic groups are generally found on the surface of the molecule. A. Domains Domains are the fundamental functional and three-dimensional structural units of polypeptides. Polypeptide chains that are greater than 200 amino acids in length generally consist of two or more domains. The core of a domain is built from combinations of supersecondary structural elements (motifs). Folding of the peptide chain within a domain usually occurs independently of folding in other domains. Therefore, each domain has the characteristics of a small, compact globular protein that is structurally independent of the other domains in the polypeptide chain. Figure 2.10 Hydrophobic interactions between amino acids with nonpolar side chains. B. Interactions stabilizing tertiary structure The unique three-dimensional structure of each polypeptide is determined by its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide to form a compact structure. The following four types of interactions cooperate in stabilizing the tertiary structures of globular proteins. 1. Disulfide bonds: A disulfide bond is a covalent linkage formed from the sulfhydryl group (–SH) of each of two cysteine residues to produce a cystine residue (Figure 2.9). The two cysteines may be separated from each other by many amino acids in the primary sequence of a polypeptide or may even be located on two different polypeptide chains. The folding of the polypeptide chain(s) brings the cysteine residues into proximity and permits covalent bonding of their side chains. A disulfide bond contributes to the stability of the three-dimensional shape of the protein molecule and prevents it from becoming denatured in the extracellular environment. For example, many disulfide bonds are found in proteins such as immunoglobulins that are secreted by cells. 2. Hydrophobic interactions: Amino acids with nonpolar side chains tend to be located in the interior of the polypeptide molecule, where they associate with other hydrophobic amino acids (Figure 2.10). In contrast, amino acids with polar or charged side chains tend to be located on the surface of the molecule in contact with the polar solvent. [Note: Recall that proteins located in nonpolar (lipid) environments, such as a membrane, exhibit the reverse arrangement (see Figure 1.4, p. 4).] In each case, a segregation of R groups occurs that is energetically most favorable. 3. Hydrogen bonds: Amino acid side chains containing oxygen- or nitrogen-bound hydrogen, such as in the alcohol groups of serine and threonine, can form hydrogen bonds with electron-rich atoms, such as the oxygen of a carboxyl group or carbonyl group of a peptide bond (Figure 2.11; see also Figure 1.6, p. 4). Formation of hydrogen bonds between polar groups on the surface of proteins and the aqueous solvent enhances the solubility of the protein. 4. Ionic interactions: Negatively charged groups, such as the carboxylate group (– COO–) in the side chain of aspartate or glutamate, can interact with positively charged groups such as the amino group (– NH3+) in the side chain of lysine (see Figure 2.11). Figure 2.11 Interactions of side chains of amino acids through hydrogen bonds and ionic bonds (salt bridges). C. Protein folding Interactions between the side chains of amino acids determine how a long polypeptide chain folds into the intricate three-dimensional shape of the functional protein. Protein folding, which occurs within the cell in seconds to minutes, involves nonrandom, ordered pathways. As a peptide folds, secondary structures form driven by the hydrophobic effect (that is, hydrophobic groups come together as water is released). These small structures combine to form larger structures. Additional events stabilize secondary structure and initiate formation of tertiary structure. In the last stage, the peptide achieves its fully folded, native (functional) form characterized by a low- energy state (Figure 2.12). [Note: Some biologically active proteins or segments thereof lack a stable tertiary structure. They are referred to as “intrinsically disordered” proteins.] D. Denaturation of proteins Protein denaturation results in the unfolding and disorganization of a protein’s secondary and tertiary structures without the hydrolysis of peptide bonds. Denaturing agents include heat, organic solvents, strong acids or bases, detergents, and ions of heavy metals such as lead. Denaturation may, under ideal conditions, be reversible, such that the protein refolds into its original native structure when the denaturing agent is removed. However, most proteins, once denatured, remain permanently disordered. Denatured proteins are often insoluble and precipitate from solution. E. Role of chaperones in protein folding The information needed for correct protein folding is contained in the primary structure of the polypeptide. However, most proteins when denatured do not resume their native conformations even under favorable environmental conditions. This is because, for many proteins, folding is a facilitated process that requires a specialized group of proteins, referred to as “molecular chaperones,” and adenosine triphosphate hydrolysis. The chaperones, also known as “heat shock proteins” (Hsp), interact with a polypeptide at various stages during the folding process. Some chaperones bind hydrophobic regions of an extended polypeptide and are important in keeping the protein unfolded until its synthesis is completed (for example, Hsp70). Others form cage-like macromolecular structures composed of two stacked rings. The partially folded protein enters the cage, binds the central cavity through hydrophobic interactions, folds, and is released (for example, mitochondrial Hsp60). [Note: Cage- like chaperones are sometimes referred to as “chaperonins.”] Chaperones, then, facilitate correct protein folding by binding to and stabilizing exposed, aggregation- prone hydrophobic regions in nascent (and denatured) polypeptides, preventing premature folding. Figure 2.12 Steps in protein folding (simplified). V. QUATERNARY STRUCTURE OF PROTEINS Many proteins consist of a single polypeptide chain and are defined as monomeric proteins. However, others may consist of two or more polypeptide chains that may be structurally identical or totally unrelated. The arrangement of these polypeptide subunits is called the quaternary structure of the protein. Subunits are held together primarily by noncovalent interactions (for example, hydrogen bonds, ionic bonds, and hydrophobic interactions). Subunits may either function independently of each other or may work cooperatively, as in hemoglobin, in which the binding of oxygen to one subunit of the tetramer increases the affinity of the other subunits for oxygen (see p. 29). Isoforms are proteins that perform the same function but have different primary structures. They can arise from different genes or from tissue-specific processing of the product of a single gene. If the proteins function as enzymes, they are referred to as isozymes (see p. 65). VI. PROTEIN MISFOLDING Protein folding is a complex process that can sometimes result in improperly folded molecules. These misfolded proteins are usually tagged and degraded within the cell (see p. 444). However, this quality control system is not perfect, and intracellular or extracellular aggregates of misfolded proteins can accumulate, particularly as individuals age. Deposits of misfolded proteins are associated with a number of diseases. A. Amyloid diseases Misfolding of proteins may occur spontaneously or be caused by a mutation in a particular gene, which then produces an altered protein. In addition, some apparently normal proteins can, after abnormal proteolytic cleavage, take on a unique conformational state that leads to the formation of long, fibrillar protein assemblies consisting of β-pleated sheets. Accumulation of these insoluble, spontaneously aggregating proteins, called amyloids, has been implicated in degenerative diseases such as Parkinson and Huntington and particularly in the age-related neurodegenerative disorder, Alzheimer disease. The dominant component of the amyloid plaque that accumulates in Alzheimer disease is amyloid β (Aβ), an extracellular peptide containing 40–42 amino acid residues. X-ray crystallography and infrared spectroscopy demonstrate a characteristic β-pleated sheet conformation in nonbranching fibrils. This peptide, when aggregated in a β-pleated sheet configuration, is neurotoxic and is the central pathogenic event leading to the cognitive impairment characteristic of the disease. The Aβ that is deposited in the brain in Alzheimer disease is derived by enzymic cleavages (by secretases) from the larger amyloid precursor protein, a single transmembrane protein expressed on the cell surface in the brain and other tissues (Figure 2.13). The Aβ peptides aggregate, generating the amyloid that is found in the brain parenchyma and around blood vessels. Most cases of Alzheimer disease are not genetically based, although at least 5% of cases are familial. A second biologic factor involved in the development of Alzheimer disease is the accumulation of neurofibrillary tangles inside neurons. A key component of these tangled fibers is an abnormal form (hyperphosphorylated and insoluble) of the tau (τ) protein, which, in its healthy version, helps in the assembly of the microtubular structure. The defective τ appears to block the actions of its normal counterpart. Figure 2.13 Formation of amyloid plaques found in Alzheimer disease (AD). [Note: Mutations to presenilin, the catalytic subunit of γ-secretase, are the most common cause of familial AD.] B. Prion diseases The prion protein (PrP) has been strongly implicated as the causative agent of transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle (popularly called “mad cow” disease). After an extensive series of purification procedures, scientists were surprised to find that the infectivity of the agent causing scrapie in sheep was associated with a single protein species that was not complexed with detectable nucleic acid. This infectious protein is designated PrPSc (Sc = scrapie). It is highly resistant to proteolytic degradation and tends to form insoluble aggregates of fibrils, similar to the amyloid found in some other diseases of the brain. A noninfectious form of PrPC (C = cellular), encoded by the same gene as the infectious agent, is present in normal mammalian brains on the surface of neurons and glial cells. Thus, PrPC is a host protein. No primary structure differences or alternate posttranslational modifications have been found between the normal and the infectious forms of the protein. The key to becoming infectious apparently lies in changes in the three-dimensional conformation of PrPC. It has been observed that a number of α-helices present in noninfectious PrPC are replaced by β-sheets in the infectious form (Figure 2.14). It is presumably this conformational difference that confers relative resistance to proteolytic degradation of infectious prions and permits them to be distinguished from the normal PrPC in infected tissue. The infective agent is, thus, an altered version of a normal protein, which acts as a “template” for converting the normal protein to the pathogenic conformation. The TSEs are invariably fatal, and no treatment is currently available that can alter this outcome. Figure 2.14 One proposed mechanism for multiplication of infectious prion agents. PrP = prion protein; PrPc = prion protein cellular; PrPSc = prion protein scrapie. VII. CHAPTER SUMMARY Central to understanding protein structure is the concept of the native conformation (Figure 2.15), which is the functional, fully folded protein structure (for example, an active enzyme or structural protein). The unique three-dimensional structure of the native conformation is determined by its primary structure, that is, its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide chain to form secondary, tertiary, and (sometimes) quaternary structures, which cooperate in stabilizing the native conformation of the protein. In addition, a specialized group of proteins named chaperones is required for the proper folding of many species of proteins. Protein denaturation results in the unfolding and disorganization of the protein’s structure, which are not accompanied by hydrolysis of peptide bonds. Denaturation may be reversible or, more commonly, irreversible. Disease can occur when an apparently normal protein assumes a conformation that is cytotoxic, as in the case of Alzheimer disease and t h e transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease. In Alzheimer disease, normal proteins, after abnormal chemical processing, take on a unique conformational state that leads to the formation of neurotoxic amyloid β peptide (Aβ) assemblies consisting of β- pleated sheets. In TSEs, the infective agent is an altered version of a normal prion protein that acts as a “template” for converting normal protein to the pathogenic conformation. Figure 2.15 Key concept map for protein structure. Study Questions Choose the ONE best answer. 2.1 Which one of the following statements concerning protein structure is correct? A. Proteins consisting of one polypeptide have quaternary structure that is stabilized by covalent bonds. B. The peptide bonds that link amino acids in a protein most commonly occur in the cis configuration. C. The formation of a disulfide bond in a protein requires the participating cysteine residues to be adjacent in the primary structure. D. The denaturation of proteins leads to irreversible loss of secondary structural elements such as the α-helix. E. The primary driving force for protein folding is the hydrophobic effect. Correct answer = E. The hydrophobic effect, or the tendency of nonpolar entities to associate in a polar environment, is the driving force of protein folding. Quaternary structure requires more than one polypeptide, and, when present, it is stabilized primarily by noncovalent bonds. The peptide bond is almost always trans. The two cysteine residues participating in disulfide bond formation may be a great distance apart in the amino acid sequence of a polypeptide (or on two separate polypeptides) but are brought into close proximity by the three-dimensional folding of the polypeptide. Denaturation may be reversible or irreversible. 2.2 A particular point mutation results in disruption of the α-helical structure in a segment of the mutant protein. The most likely change in the primary structure of the mutant protein is: A. glutamate to aspartate. B. lysine to arginine. C. methionine to proline. D. valine to alanine. Correct answer = C. Proline, because of its secondary amino group, is incompatible with an α-helix. Glutamate, aspartate, lysine, and arginine are charged amino acids, and valine is a branched amino acid. Charged and branched (bulky) amino acids may disrupt an α-helix. 2.3 In comparing the α-helix to the β-sheet, which statement is correct only for the β- sheet? A. Extensive hydrogen bonds between the carbonyl oxygen (C=O) and the amide hydrogen (N-H) of the peptide bond are formed. B. It may be found in typical globular proteins. C. It is stabilized by interchain hydrogen bonds. D. it is an example of secondary structure. E. It may be found in supersecondary structures. Correct answer = C. The β-sheet is stabilized by interchain hydrogen bonds formed between separate polypeptide chains and by intrachain hydrogen bonds formed between regions of a single polypeptide. The α-helix, however, is stabilized only by intrachain hydrogen bonds. Statements A, B, D, and E are true for both of these secondary structural elements. 2.4 An 80-year-old man presented with impairment of higher intellectual function and alterations in mood and behavior. His family reported progressive disorientation and memory loss over the last 6 months. There is no family history of dementia. The patient was tentatively diagnosed with Alzheimer disease. Which one of the following best describes Alzheimer disease? A. It is associated with β-amyloid, an abnormal protein with an altered amino acid sequence. B. It results from accumulation of denatured proteins that have random conformations. C. It is associated with the accumulation of amyloid precursor protein. D. It is associated with the deposition of neurotoxic amyloid β peptide aggregates. E. It is an environmentally produced disease not influenced by the genetics of the individual. F. It is caused by the infectious β-sheet form of a host-cell protein. Correct answer = D. Alzheimer disease is associated with long, fibrillar protein assemblies consisting of β-pleated sheets found in the brain and elsewhere. The disease is associated with abnormal processing of a normal protein. The accumulated altered protein occurs in a β- pleated sheet configuration that is neurotoxic. The amyloid β that is deposited in the brain in Alzheimer disease is derived by proteolytic cleavages from the larger amyloid precursor protein, a single transmembrane protein expressed on the cell surface in the brain and other tissues. Most cases of Alzheimer disease are sporadic, although at least 5% of cases are familial. Prion diseases, such as Creutzfeldt- Jakob, are caused by the infectious β-sheet form (PrPSc ) of a host-cell protein (PrPc). Globular Proteins 3 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 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. 76). 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. Figure 3.1 A. Hemeprotein (cytochrome c). B. Structure of heme. A. Structure of heme Heme is a complex of protoporphyrin IX and ferrous iron (Fe 2+) (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 Fe 2+ 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 oxygen (Figure 3.2). (See pp. 278 and 282 for a discussion of the synthesis and degradation of heme.) Figure 3.2 A. Model of myoglobin showing helices A to H. B. Schematic diagram of the oxygen-binding site of myoglobin. B. Structure and function of myoglobin Myoglobin, a hemeprotein present in heart and skeletal muscle, functions both as a reservoir for oxygen and as an oxygen carrier that increases the rate of transport of oxygen within the muscle cell. [Note: Mouse myoglobin double knockouts (see p. 486) have, surprisingly, 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. α-Helical content: Myoglobin is a compact molecule, with approximately 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. 17). [Note: Ionic bonds are also termed electrostatic interactions or salt bridges.] 2. Location of polar and nonpolar amino acid residues: The interior of the 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. 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 (Figure 3.2B). One, the proximal histidine (F8), binds directly to the iron of heme. The second, or distal histidine (E7), does not directly interact with the heme group but helps stabilize the binding of oxygen to the ferrous iron. The protein, or globin, portion of myoglobin thus creates a special microenvironment for the heme that permits the reversible binding of one oxygen molecule (oxygenation). The simultaneous loss of electrons by the ferrous iron (oxidation to the ferric form) occurs only rarely. Figure 3.3 A. Structure of hemoglobin showing the polypeptide backbone. B. Simplified drawing showing the helices. C. Structure and function of hemoglobin Hemoglobin is found exclusively in red blood cells (RBC), where its main function is to transport oxygen (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 (Figure 3.3). Each chain (subunit) has stretches of α-helical 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 H+ and 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). 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. 1. Quaternary structure of hemoglobin: The hemoglobin tetramer can be envisioned as being 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 (Figure 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 allows 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 Figure 3.4). [Note: The binding of O2 to the heme iron pulls the iron into the plane of the heme. Because the iron is also linked to the proximal histidine (F8), there is movement of the globin chains that alters the interface between the αβ dimers.] Figure 3.4 Schematic diagram showing structural changes resulting from oxygenation and deoxygenation of hemoglobin. 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 αβ dimers, allowing movement. This leads to a structure called the “R,” or relaxed form (see Figure 3.4). The R conformation is the high-oxygen-affinity form of hemoglobin. D. Binding of oxygen to myoglobin and hemoglobin Myoglobin can bind only one molecule of O2, because it contains only one heme group. In contrast, hemoglobin can bind four O2 molecules, 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 3.5. [Note: Pulse oximetry is a noninvasive, indirect method of measuring the O2 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 Figure 3.5). 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 (P 50) is approximately 1 mm Hg for myoglobin and 26 mm Hg for hemoglobin. The higher the oxygen affinity (that is, the more tightly oxygen binds), the lower the P50. Figure 3.5 Oxygen-dissociation curves for myoglobin and hemoglobin (Hb). a. Myoglobin: The oxygen-dissociation curve for myoglobin has a hyperbolic shape (see Figure 3.5). This reflects the fact that myoglobin reversibly binds a single molecule of oxygen. Thus, oxygenated (MbO2) and deoxygenated (Mb) myoglobin exist in a simple equilibrium: Mb + O2 MbO2 The equilibrium is shifted to the right or to the left as oxygen is added to or removed from the system. [Note: Myoglobin is designed to bind oxygen released by hemoglobin at the low pO2 found in muscle. Myoglobin, in turn, releases oxygen within the muscle cell in response to oxygen demand.] b. Hemoglobin: The oxygen-dissociation curve for hemoglobin is sigmoidal in shape (see Figure 3.5), indicating that the subunits cooperate in binding oxygen. Cooperative binding of oxygen by the four subunits of hemoglobin means that the binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same hemoglobin tetramer (Figure 3.6). This effect is referred to as heme–heme interaction (see below). Although it is more difficult for the first oxygen molecule to bind to hemoglobin, the subsequent binding of oxygen occurs with high affinity, as shown by the steep upward curve in the region near 20–30 mm Hg (see Figure 3.5). E. Allosteric effects The ability of hemoglobin to reversibly bind oxygen is affected by the pO2 (through heme–heme interactions as described above), the pH of the environment, the partial pressure of carbon dioxide (pCO2) and the availability of 2,3-bisphosphoglycerate. These are collectively called allosteric (“other site”) effectors, because their interaction at one site on the hemoglobin molecule affects the binding of oxygen to heme groups at other sites on the molecule. [Note: The binding of oxygen to monomeric myoglobin is not influenced by allosteric effectors.] 1. Heme–heme interactions: The sigmoidal oxygen-dissociation curve reflects specific structural changes that are initiated at one heme group and transmitted to other heme groups in the hemoglobin tetramer. The net effect is that the affinity of hemoglobin for the last oxygen bound is approximately 300 times greater than its affinity for the first oxygen bound. Figure 3.6 Hemoglobin (Hb) binds successive molecules of oxygen with increasing affinity. a. Loading and unloading oxygen: The cooperative binding of oxygen allows hemoglobin to deliver more oxygen to the tissues in response to relatively small changes in the partial pressure of oxygen. This can be seen in Figure 3.5, which indicates pO2 in the alveoli of the lung and the capillaries of the tissues. For example, in the lung, the concentration of oxygen is high, and hemoglobin becomes virtually saturated (or “loaded”) with oxygen. In contrast, in the peripheral tissues, oxyhemoglobin releases (or “unloads”) much of its oxygen for use in the oxidative metabolism of the tissues (Figure 3.7). 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 oxygen 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 oxygen release within this range of partial pressures of oxygen. Instead, it would have maximum affinity for oxygen throughout this oxygen pressure range and, therefore, would deliver no oxygen to the tissues. Figure 3.7 Transport of oxygen and carbon dioxide by hemoglobin. Fe = iron. 2. Bohr effect: The release of oxygen from hemoglobin is enhanced when the pH is lowered or when the hemoglobin is in the presence of an increased pCO2. Both result in a decreased oxygen affinity of hemoglobin and, therefore, a shift to the right in the oxygen-dissociation curve (Figure 3.8), and 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 affinity for oxygen, a shift to the left in the oxygen-dissociation curve, and stabilization of the R (oxy) form. a. Source of the protons that lower the 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 carbonic anhydrase to carbonic acid: CO2 + H2O H2CO3 which spontaneously loses a proton, becoming bicarbonate (the major blood buffer): H2CO3 HCO3– + H+ 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 oxygen in the peripheral tissues and the loading of oxygen 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 oxygen. Figure 3.8 Effect of pH on the oxygen affinity of hemoglobin. Protons are allosteric effectors of hemoglobin. b. Mechanism of the Bohr effect: The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for protons than does oxyhemoglobin. This effect is caused by ionizable groups such as specific histidine side chains that have a higher pKa in deoxyhemoglobin than in oxyhemoglobin. Therefore, an increase in the concentration of protons (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 protons (or a lower pO2) shifts the equilibrium to the right (favoring deoxyhemoglobin), whereas an increase in pO2 (or a decrease in protons) shifts the equilibrium to the left. Figure 3.9 Synthesis of 2,3-bisphosphoglycerate. [Note: is a phosphoryl group, PO3.] In older literature, 2, 3-bisphosphoglycerate (2,3-BPG) may be referred to as 2,3- 2– diphosphoglycerate (2,3-DPG). 3. Effect of 2,3-bisphosphoglycerate on oxygen affinity: 2,3- Bisphosphoglycerate (2,3-BPG) is an important regulator of the binding of oxygen 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 (Figure 3.9; see p. 101 for a discussion of 2,3- BPG synthesis in glycolysis). a. Binding of 2,3-BPG to deoxyhemoglobin: 2,3-BPG decreases the O2 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. Binding site of 2,3-BPG: One molecule of 2,3-BPG binds to a pocket, formed by the two β-globin chains, in the center of the deoxyhemoglobin tetramer (Figure 3.10). 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).] 2,3-BPG is expelled with oxygenation of the hemoglobin. c. Shift of the oxygen-dissociation curve: Hemoglobin from which 2,3-BPG has been removed has a high affinity for oxygen. However, as seen in the RBC, the presence of 2,3-BPG significantly reduces the affinity of hemoglobin for oxygen, shifting the oxygen-dissociation curve to the right (Figure 3.11). This reduced affinity enables hemoglobin to release oxygen efficiently at the partial pressures found in the tissues. Figure 3.10 Binding of 2,3-bisphosphoglycerate (2,3-BPG) by deoxyhemoglobin. d. Response of 2,3-BPG levels to 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 oxygen. Intracellular levels of 2,3-BPG are also elevated in chronic anemia, in which fewer than normal RBCs are available to supply the body’s oxygen needs. Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of oxygen in the capillaries of the tissues (see Figure 3.11). Figure 3.11 Allosteric effect of 2,3-bisphosphoglycerate (2,3-BPG) on the oxygen affinity of hemoglobin. e. Role of 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 a decrease in 2,3-BPG. Stored blood displays an abnormally high oxygen affinity and fails to unload its bound oxygen properly in the tissues. Hemoglobin deficient in 2,3-BPG thus acts as an oxygen “trap” rather than as an oxygen transport 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–“stripped” blood. [Note: The maximum storage time for RBC has been doubled (21 to 42 days, with median time of 15 days) by changes in H+, phosphate, and hexose sugar concentration and by the addition of adenine (see p. 291). Although the content of 2,3-BPG was not greatly improved in the long-term by these changes, adenosine triphosphate production was increased and improved RBC survival.] 4. Binding of CO2: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion (see p. 9). However, some CO 2 is carried as carbamate bound to the N-terminal amino groups of hemoglobin (forming carbaminohemoglobin as shown in Figure 3.7), which can be represented schematically as follows: Hb – NH2 + CO2 Hb – NH – COO- + H+ The binding of CO2 stabilizes the T or deoxy form of hemoglobin, resulting in a decrease in its affinity for oxygen (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. 5. Binding of CO: 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 oxygen 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 oxygen to the tissues (Figure 3.12). [Note: The affinity of hemoglobin for CO is 220 times greater than for oxygen. 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% oxygen 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, thus modulating NO availability and influencing vessel diameter. Figure 3.12 Effect of carbon monoxide (CO) on the oxygen affinity of hemoglobin. CO- Hb = carboxyhemoglobin (carbon monoxyhemoglobin). Figure 3.13 Normal adult human hemoglobins. [Note: The α-chains in these hemoglobins are identical.] Hb = hemoglobin. 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 (Figure 3.13). Each of these oxygen-carrying proteins is a tetramer, composed of two α-globin (or α- like) polypeptides and two β-globin (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 p. 34). 1. Fetal hemoglobin: HbF is a tetramer consisting of two α chains identical to those found in HbA, plus two γ chains (α2γ2; see Figure 3.13). The γ chains are members of the β-globin gene family (see p. 35). 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 about 60% of the total hemoglobin in the RBC during the last months of fetal life (Figure 3.14). HbA synthesis starts in the bone marrow at about the eighth month of pregnancy and gradually replaces HbF. ( Figure 3.14 shows the relative production of each type of hemoglobin chain during fetal and postnatal life.) [Note: HbF represents less than 1% of the hemoglobin in most adults and is concentrated in RBC known as F cells.] b. Binding of 2,3-BPG to HbF: Under physiologic conditions, HbF has a higher affinity for oxygen 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 2,3-BPG in the β-globin chains.] Because 2,3-BPG serves to reduce the affinity of hemoglobin for oxygen, the weaker interaction between 2,3-BPG 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 affinity for oxygen. The higher oxygen affinity of HbF facilitates the transfer of oxygen from the maternal circulation across the placenta to the RBC of the fetus. Figure 3.14 Developmental changes in hemoglobin. 2. Hemoglobin A2: HbA2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and, ultimately, constituting about 2% of the total hemoglobin. It is composed of two α-globin chains and two δ-globin chains (α2δ2; see Figure 3.13). 3. Hemoglobin A1c: Under physiologic conditions, HbA is slowly and nonenzymically glycosylated (glycated), the extent of glycosylation being dependent on the plasma concentration of a particular hexose. The most abundant form of glycosylated hemoglobin is HbA1c. It has glucose residues attached predominantly to the NH2 groups of the N-terminal valines of the β-globin chains (Figure 3.15). 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.) Figure 3.15 Nonenzymic addition of glucose to hemoglobin. The nonenzymic addition of a sugar to a protein is referred to as glycation. III. ORGANIZATION OF THE GLOBIN GENES To understand diseases resulting from genetic alterations in the structure or synthesis of hemoglobins, 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 (Figure 3.16). 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 hemoblobin. [Note: Globin gene famillies also contain globin-like genes that are not expressed, that is, their genetic information is not used to produce globin chains. These are called pseudogenes.] Figure 3.16 Organization of the globin gene families. Hb = hemoglobin. B. β-Gene family A single gene for the β-globin chain is located on chromosome 11 (see Figure 3.16). 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 Expression of a globin gene begins in the nucleus of RBC precursors, where the DNA sequence encoding the gene is transcribed. The 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 3.17. A more detailed description of gene expresion is presented in Unit VI, p. 395.) Figure 3.17 Synthesis of globin chains. mRNA = messenger RNA. 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) Sickle cell anemia, the most common of the RBC sickling diseases, is a genetic disorder of the blood caused by a single nucleotide substitution (a point mutation, see p. 433) 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 one of 500 newborn African American infants in the United States. Sickle cell anemia is an autosomal recessive 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 below). 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 less than 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. 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. 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. Figure 3.18 Amino acid substitutions in hemoglobin S (HbS) and hemoglobin C (HbC). 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 (Figure 3.18). Therefore, during electrophoresis at alkaline pH, HbS migrates more slowly toward the anode (positive electrode) than does HbA (Figure 3.19). 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 disease. DNA analysis also is used (see p. 472).] 2. 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 (Figure 3.20). 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 oxygen leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually 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 and so have difficulty moving through small vessels, thereby causing microvascular occlusion.] 3. Variables that increase sickling: The extent of sickling and, therefore, the severity of disease is enhanced by any variable that increases the proportion of HbS in the deoxy state (that is, reduces the affinity of HbS for O2). These variables include decreased pO2, increased pCO2, decreased pH, dehydration, and an increased concentration of 2,3-BPG in RBC. 4. Treatment: Therapy involves adequate hydration, analgesics, aggressive antibiotic therapy if infection is present, and transfusions in patients at high risk for fatal occlusion of blood vessels. Intermittent transfusions with packed RBC reduce the risk of stroke, but the benefits must be weighed against the complications of transfusion, which include iron overload (hemosiderosis), bloodborne infections, and immunologic complications. Hydroxyurea (hydroxycarbamide), an antitumor drug, is therapeutically useful because it increases circulating levels of HbF, which decreases RBC sickling. This leads to decreased frequency of painful crises and reduces mortality. [Note: The morbidity and mortality associated with sickle cell anemia has led to its inclusion in newborn screening panels to allow prophylactic antibiotic therapy to begin soon after the birth of an affected child.] Figure 3.19 Diagram of hemoglobins (HbA), (HbS), and (HbC) after electrophoresis. Figure 3.20 Molecular and cellular events leading to sickle cell crisis. HbS = hemoglobin S. 5. Possible selective advantage of the heterozygous state: The high frequency of the bS mutation among black Africans, despite its damaging effects in the homozygous state, suggests that a selective advantage exists for heterozygous individuals. For example, heterozygotes for the sickle cell gene are less susceptible to the severe malaria caused by the parasite Plasmodium falciparum. This organism spends an obligatory part of its life cycle in the RBC. One theory is that because these cells in individuals heterozygous for HbS, like those in homozygotes, have a shorter life span than normal, the parasite cannot complete the intracellular stage of its development. This fact may provide a selective advantage to heterozygotes living in regions where malaria is a major cause of death. Figure 3.21 illustrates that in Africa, the geographic distribution of sickle cell anemia is similar to that of malaria. B. Hemoglobin C disease Like HbS, HbC is a hemoglobin variant that has a single amino acid substitution in the sixth position of the β-globin chain (see Figure 3.18). In HbC, however, a lysine is substituted for the glutamate (as compared with a valine substitution in HbS). [Note: This substitution causes HbC to move more slowly toward the anode than HbA or HbS does (see Figure 3.19).] Rare patients homozygous for HbC generally have a relatively mild, chronic hemolytic anemia. These patients do not suffer from infarctive crises, and no specific therapy is required. C. Hemoglobin SC disease HbSC disease is another of the RBC sickling diseases. In this disease, some β-globin chains have the sickle cell mutation, whereas other β-globin chains carry the mutation found in HbC disease. [Note: Patients with HbSC disease are doubly heterozygous. They are called compound heterozygotes because both of their β-globin genes are abnormal, although different from each other.] Hemoglobin levels tend to be higher in HbSC disease than in sickle cell anemia and may even be at the low end of the normal range. The clinical course of adults with HbSC anemia differs from that of sickle cell anemia in that symptoms such as painful crises are less frequent and less severe. However, there is significant clinical variability. D. Methemoglobinemias Oxidation of the heme iron in hemoglobin to the ferric (Fe 3+) state forms methemoglobin, which cannot bind O2. This oxidation may be caused by the action of certain drugs, such as nitrates, or endogenous products such as reactive oxygen species (see p. 148). The oxidation may also result from inherited defects, for example, certain mutations in the α- or β-globin chain promote the formation of methemoglobin (HbM). Additionally, a deficiency of NADH-cytochrome b5 reductase (also called NADH-methemoglobin reductase), the enzyme responsible for the conversion of methemoglobin (Fe 3+) to hemoglobin (Fe 2+), leads to the accumulation of HbM. [Note: The RBC of newborns have approximately half the capacity of those of adults to reduce HbM. They are, therefore, particularly susceptible to the effects of HbM-producing compounds.] The methemoglobinemias are characterized by “chocolate cyanosis” (a brownish blue coloration of the skin and mucous membranes and brown-colored blood) as a result of the dark-colored HbM. Symptoms are related to the degree of tissue hypoxia and include anxiety, headache, and dyspnea. In rare cases, coma and death can occur. Treatment is with methylene blue, which is oxidized as Fe+3 is reduced. Figure 3.21 A. Distribution of sickle cell in Africa expressed as a percentage of the population with disease. B. Distribution of malaria in Africa. E. Thalassemias The thalassemias are hereditary hemolytic diseases in which an imbalance occurs in the synthesis o

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