Hemoglobin: Red Blood Cells & Oxygen Transport PDF
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Marie-Pierre Hasne, PhD
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This document covers lecture notes on the topic of hemoglobin and red blood cells, including their function, interactions, and related biochemistry. The lecture format provides a fundamental explanation of this crucial topic.
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HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT Block: Foundations Block Director: James Proffitt, PhD Session Date: Monday, August 05, 2024 Time: 2:30 – 4:00 pm Instructor: Marie-Pierre Hasne, PhD Department: Chemistry & Biochemist...
HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT Block: Foundations Block Director: James Proffitt, PhD Session Date: Monday, August 05, 2024 Time: 2:30 – 4:00 pm Instructor: Marie-Pierre Hasne, PhD Department: Chemistry & Biochemistry Email: [email protected] INSTRUCTIONAL METHODS Primary Method: IM13: Lecture ☐ Flipped Session ☐ Clinical Correlation Resource Types: RE18: Written or Visual Media (or Digital Equivalent) INSTRUCTIONS Please read lecture objectives and notes prior to attending session. READINGS REQUIRED Reading: The Big Picture: Medical Biochemistry. BOOK / E-BOOK: Janson L, Tischler M. The Big Picture: Medical Biochemistry. 1st ed. New York: McGraw-Hill, 2012. e- book: Access Medicine. Required: Chapter 14, [stop at 'Iron'], p. 201-210 LEARNING OBJECTIVES: 1. Differentiate between reticulocytes and erythrocytes, and discuss how reticulocyte and erythrocyte count may be used diagnostically. 2. Describe the role of band 3 protein (anion exchange protein), and cytoskeletal proteins associated with the red blood cell membrane. 3. Describe similarities and differences between Myoglobin and Hemoglobin. 4. Describe the beta and alpha globin gene clusters, and identify the different globin chain compositions that comprise HbA1, HbA2 and HbF. 5. Discuss how the R(elaxed) and T(ense) configurations of Hb relate to the positioning of the heme iron, the role of the proximal histidine, and the binding or releasing of oxygen. 6. Describe how cooperative binding of oxygen by hemoglobin improves its effectiveness as an oxygen transporter. 7. Discuss the physiological significance of protons (Bohr Effect), carbon dioxide, BPG (2,3-bisphosphoglycerate) and the presence of the -subunit rather than the -subunit in HbF on the affinity of Hb for oxygen and under what circumstances these effects are important. 8. Discuss how carbon monoxide (CO) competes with oxygen for binding to Hb and how its greater affinity relative to O2 is reduced by the distal histidine. 9. Describe the relationship between hemoglobin and acid-base homeostasis. 10. Describe the molecular genetic basis of the various thalassemias, the genotype- phenotype relationships in these diseases and the aberrant hemoglobins that are produced. Block: Foundations | HASNE [1 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT CURRICULAR CONNECTIONS Below are the competencies, educational program objectives (EPOs), block goals, disciplines and threads that most accurately describe the connection of this session to the curriculum. Related Related Competency\EPO Disciplines Threads COs LOs CO-01 LO #1 MK-01: Core of basic sciences Biochemistry N/A CO-01 LO #2 MK-01: Core of basic sciences Biochemistry N/A Physiology CO-01 LO #3 MK-01: Core of basic sciences Biochemistry N/A MK--03: The molecular, cellular Physiology and biochemical mechanisms of homeostasis CO-01 LO #4 MK-01: Core of basic sciences Biochemistry N/A CO-01 LO #5 MK--03: The molecular, cellular Physiology N/A and biochemical mechanisms of homeostasis CO-01 LO #6 MK-01: Core of basic sciences Biochemistry N/A CO-01 LO #7 MK-01: Core of basic sciences Biochemistry N/A Physiology CO-01 LO #8 MK-01: Core of basic sciences Biochemistry N/A MK--03: The molecular, cellular Physiology and biochemical mechanisms of homeostasis CO-01 LO #9 MK-01: Core of basic sciences Biochemistry N/A CO-01 LO #10 MK--03: The molecular, cellular Physiology N/A and biochemical mechanisms of homeostasis CONTEXT: Understanding the basis of oxygen transport is critical to appreciating the links between respiration and cell metabolism. Hemoglobin also plays a role in pH control in the blood and hence ties in intimately to acid-base balance in the body. It is positioned here as a follow up to the preceding lecture in which the basic structure of hemoglobin is discussed. Relevance to later blocks includes gas transport in CPR, aerobic metabolism and oxidative phosphorylation earlier in this block and in DMH, and exercise and physical activity in MSS and CPR. RETICULOCYTES AND ERYTHROCYTES Reticulocyte Features: Erythrocytes are the most abundant single cell type in the human body, comprising approximately 35% of the volume of whole blood. Erythrocytes are formed in the bone marrow from reticulocytes. Reticulocytes are formed from their erythropoietic precursor cells. These precursor cells contain a cell nucleus and very active protein synthesis machinery. Reticulocytes do not have a nucleus but do have some ribosomes. Once in the blood stream, they mature into erythrocytes in about a day by losing their Block: Foundations | HASNE [2 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT mitochondria and ribosomes. Hence, erythrocytes can neither make new proteins nor carry out aerobic metabolism. Reticulocytes usually make up about 1% of the total population of RBCs. In hemolytic anemias, the destruction of erythrocytes leads to increased compensatory production of reticulocytes resulting in significantly higher reticulocyte count (see clinical correlate). Erythrocyte Features: Human erythrocytes contain no intracellular organelles and thus the only membrane found in erythrocytes is the plasma membrane. Mature erythrocytes are discoid, with a long axis of 8 microns and a height of 2 microns. Their major purpose is to transport oxygen from the lungs to the tissues and to return carbon dioxide from the tissue to the lungs. Both oxygen and carbon dioxide bind to the major soluble protein of erythrocytes, hemoglobin (Hb). For the carbon dioxide and glucose to enter the erythrocyte across the membrane, specialized membrane transport proteins are required. As erythrocytes wind their way from the lungs to the tissues, they must pass from large arteries at speeds of up to 1 meter per second, down to a single file through the smallest of blood vessels, capillaries, which can be as small as 3 microns in diameter. A typical blood flow rate through these capillaries is 1 mm/sec. Thus, erythrocytes have to be able to withstand extremes of shear stress and deformability, while maintaining their surface area and integrity. Much of this ability is due to the nature of the membrane itself. The mature erythrocyte is largely a gel of proteins, the primary one being hemoglobin. Other important proteins include the enzymes of glycolysis, which is the sole metabolic pathway in erythrocytes that can produce ATP. Some proteins are needed for the maintenance of ion gradients and others form cytoskeletal elements for shape changes during circulation. Total hemoglobin in blood is used diagnostically and normal values can range from 14-18 g/dL. Values below this range are indicative of anemia. Nutritional Correlate: Iron-deficiency anemia is the most common type of anemia and is generally caused by a dietary deficiency of iron. It is estimated that 20% to 25% of women of child-bearing age have iron deficiency anemia due to blood loss during menses. Iron-deficiency anemia is associated with hemoglobin values below 10 for females. The condition is associated with paleness, due to reduced amounts of oxygenated hemoglobin, fatigue and weakness. Block: Foundations | HASNE [3 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT BIOLOGICAL MEMBRANES Erythrocyte Membrane Features: Anion exchange protein- band 3- Cl-/HCO3- antiporter: A Cl-/HCO3- antiporter also called band 3 protein is a transmembrane protein that enables the RBC to export bicarbonate (HCO3-) and import chloride ion (Cl-) in the cell. In peripheral tissues, bicarbonate is derived from carbon dioxide as a product of aerobic metabolism. In the lungs this bicarbonate is exchanged for chloride ion to allow CO 2 to be exhaled. Flexibility of the red blood cell: Spectrin is the primary protein that comprises the cytoskeletal structure of the erythrocyte membrane. Spectrin binds several other proteins including ankyrin, actin and band 4.1 protein. These interactions help connect the cytoskeleton and the lipid bilayer. This overall structure provides the shape and flexibility of the red blood cell allowing it to squeeze through the narrow diameter of capillaries. From: The Inherited Metabolic Diseases by A.J. Grimes and N.C.P. Slater, Figure 14.1, p. 526, 1994 Figure 1. The erythrocyte membrane and associated proteins. Block: Foundations | HASNE [4 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT RED BLOOD CELLS AND METABOLISM: GLYCOGEN PROTEIN TRIACYLGLYCEROLS [e] [i] [g] GLUCOSE AMINO ACIDS Glucose-6-P FATTY ACIDS [a] [a] BPG [f] [b] H+ LACTATE H+ [c] Pyruvate Acetyl-CoA LEGEND [h] [a]: glycolysis [b]: anaerobic glucose metabolism [d] KETONE [c]: aerobic glucose metabolism [d]: citric acid (Krebs) cycle BODIES [e]: glycogenolysis H+ [f]: beta-oxidation CO2 ATP [g]: lipolysis [h]: ketone oxidation [i]: protein synthesis Figure 2. Overview of metabolic pathways that produce energy from glucose, fatty acids, or ketone bodies (see3 also Introduction to Metabolism in week 1 of the block). The production of regulators of oxygen binding (i.e., CO2, H+, BPG) is shown in red. The primary purpose of blood is to deliver nutrients to tissues and eliminate metabolic by-products (waste). Under well-fed conditions, amino acids are primarily used for the de-novo synthesis of proteins (Figure 2 [i]), while glucose ([a] to [d]), and fats ([d]; [f] to [h]) are used as fuel. Fats are transported in the blood either bound to proteins such as albumin, or as smaller soluble units, the ketone bodies. Carbohydrates are transported as simple sugars, such as the monosaccharide, glucose, which is normally ~5 mM, or 90 mg/dL in humans. In the presence of oxygen and mitochondria, fats and sugars are step-wise oxidized to CO2, and the chemical energy is converted to ATP (Figure 1). In the absence of oxygen, as in vigorous exercise, wounding, ischemia, and cancer, fats cannot be metabolized and all energy is derived from the anaerobic conversion of glucose to lactic acid (Figure 2). In the blood oxygen is carried in red blood cells (RBCs) by hemoglobin (Hb). Block: Foundations | HASNE [5 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT In tissues, glucose is converted to pyruvate + 2 ATP through a process known as glycolysis (literally, splitting of glucose). A by-product of glycolysis is 2,3- bisphosphoglycerate, which is important in causing oxygen dissociation from Hb (see later in these notes). In either aerobic or anaerobic metabolism, the by-products are acids, i.e. they produce hydrogen ions, H+. Pyruvate and lactate are both acids, and CO2 is hydrated to form carbonic acid, H2CO3. Both CO2 and H+ are transported, partially by Hb, back to the lungs and kidneys, where they are eliminated (Figure 3). RBCs lack mitochondria and hence, they are dependent on glycolysis for their energy. Under normal physiological conditions, RBCs are the major source of lactic acid in the blood. Figure 3. Hb transports O2 from lungs to peripheral tissues, and H+ AND CO2 from peripheral tissues to lungs. MYOGLOBIN AND HEMOGLOBIN Myoglobin and hemoglobin are highly related globular proteins that bind molecular oxygen (O2). Myoglobin is found in muscle tissues where it stores O2. Hemoglobin (Hb) is found in the blood, especially in red blood cells. Hb carries O2 from the lungs to the tissues and carbon dioxide (CO2) from the tissues to the lungs. Block: Foundations | HASNE [6 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT Heme: Figure 4. Ferrous iron in the heme pocket with 4 electrons liganded to the heme group, a 5th electron coordinated to the proximal histidine in the globin protein, and the 6th electron bound to molecular oxygen, which also interacts with a distal histidine. Both myoglobin and hemoglobin contain heme prosthetic groups (Figure 4). The heme group belongs to a larger family of compounds called porphyrins. Heme contains an iron atom liganded to four nitrogen atoms in a pyrrole structure. The iron in active heme is the reduced form, Fe+2, called ferrous iron. If ferrous iron becomes oxidized (losing an electron to oxygen) it becomes Fe+3, or ferric iron. Ferrihemoglobin (Fe+3), an oxidized form of hemoglobin is also called methemoglobin. Ferrous iron (Fe+2) has six valence electrons available for bonding. It’s liganded to the heme with four valence electrons, and liganded to a histidine in the globin protein with a 5th valence electron. The 6th valence electron is available to bind molecular oxygen (O2). In contrast, ferric iron is penta-coordinated, lacking the 6th valence electron, which prevents binding of O2. Myoglobin and Hemoglobin Function and Structure: Myoglobin (Mb) and hemoglobin (Hb) are oxygen-binding proteins. They are kinetically similar to enzymes in that they have an affinity for a substrate, in this case, oxygen. The surface of Mb or Hb has a crevice that forms an active site. Oxygen enters (by diffusion) and binds to the heme ferrous iron at the bottom of this crevice. Block: Foundations | HASNE [7 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT Proximal His Heme Figure 5. A model of myoglobin showing the -helical domains and positioning of the heme group (circled) and its interaction with the proximal histidine. Myoglobin was the first protein whose structure was solved to high resolution. This protein is extremely compact with most of the atoms in direct contact with each other. About 75% of the structure is comprised of -helices and is strikingly similar to that of Hb monomers. These globin proteins contain eight distinct -helical regions (A to H) (Figure 5). The designation in Figure 3 of the distal and proximal histidines indicates the - helical domain, E or F, where each is found. The interior amino acids are primarily non- polar. This structure is driven and stabilized by hydrophobic forces. The only exceptions are two histidine residues exposed to the heme and play a part in the active site. Unlike Mb, hemoglobin is a tetrameric protein, i.e. it is comprised of four polypeptide chains. The four chains of Hb are derived from two families. One family includes the (alpha) and (zeta) chains. The other family includes the (beta), (delta), (gamma) and (epsilon) chains. Each of these chains contains a heme prosthetic group. Normal adult hemoglobin (HbA1) is α2β2 (Figure 6). Although these different chains have different primary sequences, their tertiary structures are all similar to each other and to Mb. This is significant, since they only have 17% sequence homology! Hence, there must be some generic tertiary structure for an oxygen carrier that can be derived from a variety of primary structures. Only nine residues are conserved when comparing Mb and Hb across species. Four of these conserved amino acids (including the two above- mentioned histidines) contact the heme. Block: Foundations | HASNE [8 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT Figure 6. Structure of adult Hb showing two and two subunits. Image courtesy of University of Miami Biology bio.miami.edu/~cmallery/150/chemistry/hemoglobin.jpg HEMOGLOBIN GENES The and subunits of adult Hb are encoded by two gene families that exist as gene clusters. The gene cluster is shown in Figure 7a with 1 and 2 being duplicated genes, both of which are transcribed. The gene is only expressed at the embryonic stage (Table 1). The gene cluster is shown in Figure 7b. The gene is found in 2-5% of adult Hb, the HbA2 form. G and A refer to duplicated fetal genes that differ in a single amino acid and are found in fetal Hb, HbF, which is an 22 tetramer. Both forms of the gene are expressed. The gene is only expressed at the embryonic stage. 5’ end---------------------------2---------1-------------3’ end Figure 7a. Alpha gene cluster The gene cluster in Figure 6b contains: 5’ end--------------------G----A----------------------------------3’ end Figure 7b. Beta gene cluster Hb tetramers can be formed between any two chains of the -family and any two chains of the -family. The -family has higher affinity for O2 than the -family. In certain disease states a tetramer of four chains of the -family can exist and is problematic because these Hb forms have aberrant oxygen dissociation characteristics. However, all of the following hemoglobin tetramers can be formed from native (i.e. non-mutated) globin chains (Table 1). Table 1. Native globin tetramers found in the embryo, fetus and adult. Block: Foundations | HASNE [9 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT Hb form Structure Notes HbA1 22 Normal adult form; usually 95-98% HbA2 2 2 Normal adult form; usually 2-5% HbF 2 2 Fetal form. Disappears by 12 months after birth Hb Gower 1 22 Embryonic form. Disappears by 8 weeks gestation Hb Gower 2 22 Embryonic form. Disappears by 8 weeks gestation Hb Portland 22 Embryonic form. Disappears by 8 weeks gestation THE TWO STATES OF HEMOGLOBIN: Multimeric enzymes generally have a site, other than the active site, which binds an "activity modulator". This other site is termed the allosteric site and is distant from the active site. Hemoglobin is an example where allosteric regulation is understood at the molecular and functional levels. In the examples illustrated below, we will consider the allosteric regulation of hemoglobin function by 2,3-bis-phosphoglycerate (BPG), pH and CO2. Comparative studies of oxyhemoglobin and deoxyhemoglobin show that these two forms of hemoglobin have distinct quaternary structures. Imagine the hemoglobin tetramer as a dimer of dimers: one pair sits on top of the other pair. In deoxyhemoglobin a gap exists between the pairs. When oxygen binds, one pair of subunits rotates 15o relative to the other pair compacting the structure and closing the gap (Figure 8). R (oxy-) FORM T (deoxy-) FORM TOP VIEW SIDE VIEW TOP VIEW SIDE VIEW Figure 8. Rotation of hemoglobin. The upper pair is rotated by 15 degrees between the two forms. An animation illustrating this is at: http://www.chembio.uoguelph.ca/educmat/chm356/Hbrotation.html The deoxy form of hemoglobin is tense (or T). Another feature besides the cleft is that the structure is stabilized by eight salt bridges between the carboxyl terminal residues of all four subunits. The oxy form of hemoglobin, on the other hand, does not have salt bridges and is relaxed (or R). Hence binding of oxygen ruptures the salt bridges leading to a less stable structure. At the molecular level, the R- and T-states of hemoglobin can be defined by the position of the iron relative to the plane of the heme (Figure 9). Note that, in the T-state, the iron is pulled away from the plane of the heme, making it more difficult for oxygen to bind. In the R-state, the iron is “pulled” (by oxygen) into the plane, making it easier for oxygen to bind. Hence, the R-state of Hb has a higher affinity for Block: Foundations | HASNE [10 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT oxygen than does the T-state, whether or not oxygen is present. O2 stabilizes the R state, whereas, as described below, the T state is stabilized by H +, CO2 and BPG. A. Tense B. Relaxed F-Helix +O2 His F-Helix His Fe Porphyrin plane Fe O O His His E-Helix E-Helix Figure 9. Effect of oxygen on the position of iron relative to the plane of the porphyrin ring of the heme. CARBON MONOXIDE: Carbon monoxide (CO) also binds to the ferrous iron in myoglobin and hemoglobin. In contrast to the binding of O2, the binding of CO occurs with 200 times higher affinity than that of O2. However, its affinity would be 10,000 times higher, except that it is sterically hindered from binding by the position of the distal histidine on the E-helix (Figure 8). The atomic structure of CO makes it bind more efficiently straight on (i.e. at an angle of 0) whereas O2 can bind efficiently at an angle. The pocket formed by the distal histidine forces the CO to bind less efficiently, i.e. at an angle relative to heme Fe. CO is endogenously produced during the breakdown of heme. As a consequence, at steady state, approximately 2% of adult Hb is carbonmonoxy Hb. This inactive form of Hb is efficiently catabolized and recycled. COOPERATIVITY & THE BINDING CURVE: O2 binds covalently to iron in its 6 th coordination position. When O2 binds to one hemoglobin subunit, it induces a conversion from the T form to the R form in the other subunits. This happens because the O2 "pulls" the iron atom into the plane of the heme (Figure 9). This movement of the iron also pulls on the proximal histidine to which it is liganded. This movement of the histidine is transmitted to other parts of the same polypeptide, and ruptures the salt links of the T form between the subunit to which O2 has bound and the other Hb ‘deoxy’ subunits. This rupture of the salt bridges "frees up" another subunit, which converts to an "R-like" state with an increased affinity for O2, Block: Foundations | HASNE [11 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT which will then readily bind. This binding of a second O2 pulls on the heme and histidine of that subunit, breaking further salt links, etc. In this way the binding of O2 to hemoglobin is highly cooperative. It is analogous to pulling apart a set of 4 postage stamps in a square. Pulling the first requires breaking two borders, while pulling the second and third requires only the breaking of one border each, and pulling off the last does not require breaking any borders. Cooperativity can be depicted in a saturation (or binding) curve, wherein the percent of globin that is oxygen-bound is plotted against the concentration of oxygen. The saturation curves for myoglobin and hemoglobin differ markedly (Figure 10). Note that the Hb curve has a distinct sigmoid shape. This sigmoid shape means that Hb has a very low affinity for O2 at a low O2 pressure (pO2). As the pO2 increases, the slope gets steeper, reflecting an increased affinity. Myoglobin, on the other hand, is a monomer and does not exhibit cooperativity. Hence, it has a high affinity at all pO2 levels. This feature ensures that oxygen will always be transferred from the blood (Hb) to the tissues (Mb), where it is needed. The pO2 when the globin is 50% saturated, when half of the globin has bound O2, is known as the P50. The P50 is a rough measure of affinity in that the lower the P50, the higher is the affinity. Mb has a P50 of about 3 mm Hg and native HbA1 has a P50 of 26 mm Hg. 100 80 Percent Saturation 60 Sigmoid shape for Hb = positive cooperativity 40 20 0 Oxygen pressure – pO2 (mm Hg) Figure 10. Comparison of O2-binding curves for Mb and Hb. In the lungs, arterial pO2 is about 100 mm Hg so that Hb is saturated. In the periphery, capillary pO2 is about 20 mm Hg, which favors oxygen dissociation. Block: Foundations | HASNE [12 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT THE BOHR EFFECT: Protons are a product of metabolism. The higher the rate of metabolism, the more H + is produced, and the greater is the demand for oxygen. To meet this demand, the binding of H+ induces an R to T conversion in the hemoglobin molecule. Binding of H+ is enhanced by the increased concentration of H+ (lower pH) in the capillaries due to tissue metabolism. This R to T conversion "squeezes" O2 off Hb by lowering the affinity of Hb for O2. This process is reversed in the lungs, where the pH is higher (lower H+ concentration). This increased affinity of Hb for O2 in the lungs enhances its uptake. The transition from R to T states caused by the lowering of the pH is mediated by a histidine residue (β1 His146). Recall that the side chain of His has a pKa of about 6.0. Hence, higher H+ concentration leads to His protonation, giving it a positive charge (HisO + H+ → His+). This positive charge "pulls" on an adjacent negatively charged aspartate residue (β1 Asp 94), creating an Asp---+His salt bridge. This salt bridge is characteristic of the T configuration. Thus, binding of H+ reduces the affinity of Hb for O2 and is termed the Bohr effect (Figure 11). This effect helps to ensure that, during active metabolism, O2 dissociates from Hb for delivery to tissues. 100 Percent Saturation 80 60 pH = 7.6 40 pH = 7.2 20 P50 @7.6 P50 @7.2 0 0 20 40 60 80 100 Oxygen pressure – pO2 (mm Hg) Figure 11. The Bohr Effect. The affinity of Hb for O2 is decreased at the lower pH of tissues. The decreased affinity of O2 at lower pH is reflected in the higher P50 value at pH 7.2 relative to pH 7.6. Hence, at pH 7.2, a higher pO2 (~58 mm Hg) is required to achieve 50% saturation than at pH 7.6 (~25 mm Hg). Block: Foundations | HASNE [13 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT THE CO2 EFFECT: 100 Percent Saturation 80 60 pCO2 = 20 mm pCO2 = 80 mm 40 P50 @20 20 P50 @80 0 0 20 40 60 80 100 Oxygen pressure – pO2 (mm Hg) Figure 12. CO2 effect. The affinity of Hb for O2 is decreased in the presence of high CO2. The decreased affinity of O2 at higher pCO2 is reflected in the higher P50 value at 80 mm relative to 20 mm. Hence, at 80 mm Hg, a higher pO2 (~40 mm Hg) is required to achieve 50% saturation than at 20 mm Hg (~20 mm Hg). CO2 produced by aerobic metabolism binds to the Hb molecule at its N-terminus forming a carbamino group. When CO2 binds, it induces the formation of one salt link between the N-terminus and one of the -helices. This salt link stabilizes the T form, thereby lowering affinity for O2 (Figure 12). Thus, CO2, with H+, promotes O2 dissociation from Hb in tissues. 2,3-BISPHOSPHOGLYCERATE (BPG): Red blood cells produce 2,3-bisphosphoglycerate (BPG) from the metabolism of glucose, but only under conditions of low pO2 in peripheral tissues. The "Bis-" designation is a prefix meaning that the two phosphates are bound to different carbons as opposed to the prefix “di” that indicates binding to the same carbon. Only one molecule of BPG binds per Hb tetramer and it does not bind to monomers. Therefore, BPG must interface with all four chains. Accordingly, BPG inserts itself within the "cleft" present in the T form (see above). BPG is negatively charged allowing it to interact with positively charged residues, such as lysine and histidine, which face the interior of the Hb molecule. By placing BPG in the cleft, the T-form becomes stabilized, thus keeping O2 affinity low. BPG is essential for the expression of the O2 binding cooperativity of Hb. In the absence of BPG, the O2 saturation curve of Hb becomes similar to that of Mb, indicating loss of cooperativity (Figure 13). BPG is a major compensatory mechanism for adaptation to altitude. High altitude adaptation is a complex process involving an increase in the number of erythrocytes and the Hb content per erythrocyte. This is a long process, taking approximately 1 day per 300-meter gain in altitude. A shorter-term adaptation occurs with an increase in BPG, and concomitant decrease in oxygen binding affinity, Block: Foundations | HASNE [14 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT with increasing altitude. The decreased affinity in the presence of a higher concentration of BPG allows Hb to release its oxygen more readily, assuring an adequate supply of oxygen to tissues. Loss of sigmoid shape for Hb alone = no cooperativity 100 Hb alone 80 Percent Saturation Hb + BPG 60 Hb alone 50% saturation 40 Hb + BPG 20 P50 Hb P50 Hb+BPG 0 0 20 40 60 80 100 Oxygen pressure – pO2 (mm Hg) Figure 13. Loss of positive cooperativity of O2 binding in the absence of 2,3-bisphosphoglycerate (BPG). Fetal Hemoglobin (HbF): Fetal Hb contains chains instead of chains. It therefore exists as tetramer. The presence of the subunits causes HbF to have a lower affinity for BPG than does adult Hb. This occurs because the interactions between and chains provide a weaker binding site for BPG compared to / interactions. BPG is a tetra-anion (i.e., four negative charges) allowing it to interact with six positively charged Lys and His residues in the interface between the four monomers in adult Hb. The chains contain a Ser residue instead of one of the His residues found in chains. Hence, the cleft of HbF contains only four (+) charges instead of the six found in HbA. Consequently, HbF has a higher affinity for O2 than does HbA and the O2 saturation curve of HbF is more similar to that of myoglobin. This higher affinity of HbF for O2 ensures that O2 will always be transferred from the maternal to the fetal circulation (Figure 14). Block: Foundations | HASNE [15 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT Reduced sigmoid shape for HbF = higher O2 affinity 100 Fetal Hb 80 Percent Saturation O2 flows from maternal Maternal Hb Hb to fetal Hb 60 50% saturation 40 20 P50 HbF P50 Hb+BPG 0 0 20 40 60 80 100 Oxygen pressure – pO2 (mm Hg) Figure 14. Flow of O2 from maternal to fetal Hb is favored by the decreased ability of HbF to bind BPG due to the presence of subunits instead of subunits. HEMOGLOBIN AND ACID-BASE HOMEOSTASIS The endproducts of energy metabolism are acids. Under aerobic conditions, the endproduct is CO2, which is hydrated by an erythrocyte enzyme, carbonic anhydrase, to form bicarbonate and a proton. CO2 + H2O → HCO3- + H+ Hydrogen ions from acids and CO2 constitute a metabolic acid load. This acid load is accommodated through a number of buffering mechanisms, including the above bicarbonate equilibrium (pKa = 6.2) and phosphate buffers (pKa = 6.8). Finally, hemoglobin itself absorbs H + at His residues. This mechanism accounts for 50% of all of the aerobically produced H +. Additionally, ~15% of the metabolic CO2 binds directly to the N-termini of the globin chains and thus does not react with water to form H+. All of these passive mechanisms are effective in buffering against severe acidosis. These buffers collaborate with active buffering in the lungs (which expel CO 2) and kidneys (which regulate HCO3- levels) to maintain physiological pH within acceptable boundaries (i.e. arterial pH 7.2-7.4, venous pH 7.0-7.2). THALASSEMIAS Thalassemias are autosomal recessive diseases, meaning that both copies of a globin gene must be mutated in order for the disease to develop, which result in a reduced rate of globin chain synthesis. Loss of globin chains causes anemia due to improperly assembled and abnormal tetramers. Thalassemias are not synonymous with hemoglobinopathies, which involve structural mutations in the Hb subunits. The Block: Foundations | HASNE [16 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT thalassemias are classified according to which chain of the hemoglobin molecule is affected. In thalassemias, production of the globin chain is affected, while in thalassemia production of the globin chain is affected. Alpha () Thalassemias: There are four genetic loci for globin: two alleles of 1 and 2, one on each chromosome. Alpha thalassemias most commonly involve gene deletions. The severity of the thalassemias correlates with the number of affected globin loci; the greater the number of affected loci, the more severe will be the manifestations of the disease. The genotype/phenotype correlations are shown in Table 2, where refers to normal expression levels and O refers to deletions or non-expression of single copies. The reduced production of globin chains results in an excess of chains in adults and excess of chains in newborns. The excess β chains form unstable tetramers (called Hemoglobin H or HbH), which have abnormal oxygen dissociation features. Table 2. Genotype-phenotype relationships in -thalassemias GENOTYPE PHENOTYPE 12/12 Normal 12O/12 or Asymptomatic (silent carrier) – 1 of 4 loci 1 2/12 O abnormal 1 2 /12 or O O thalassemia Minor (trait) – 2 of 4 loci 1 2 /1 2 or O O abnormal 12 /12 or O O 1O2 /12O 1O2O/12O or thalassemia Intermedia – 3 loci abnormal 1O2O/1O2 HbH disease - 4 Hb Bart’s - 4 present at birth then declines OO/O thalassemia Major – all 4 loci abnormal Hydrops fetalis; fatal in utero 4 can be up to 80% of the Hb Key features of thalassemias are: If only one of the four loci is affected, there is minimal biological effect. Three normal -globin loci are sufficient to permit normal hemoglobin production, with no anemia or hypochromia (i.e., deficiency of hemoglobin in red blood cells) in these people. These individuals are silent (asymptomatic) carriers. When two of -globin loci are affected, the condition is known as alpha thalassemia minor (trait) and is associated with mild microcytic (i.e, small red blood cell size) hypochromic anemia. When three loci are affected, the condition is referred to as alpha thalassemia intermedia, or Hemoglobin H disease. Both HbH (β4) and Hb Barts (γ4) can be present, though γ4 declines after birth as the subunit begins to be produced (Table 2). Hb Bart’s is HbH disease at birth due to the presence of only about 25% normal a-globin chains in the fetus. Both of these conditions produce pathological hemoglobins that are unstable and have a high affinity for oxygen, resulting in poor oxygen delivery to tissues. This is accompanied by a microcytic hypochromic anemia and Heinz bodies (damaged precipitated Hb) Block: Foundations | HASNE [17 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT If all four loci are affected, the fetus cannot survive. This condition is known as alpha thalassemia major (hydrops fetalis). Although HbH and Hb Barts are formed, their high affinity for oxygen makes them very poor oxygen carrier and tissues do not receive enough oxygen to survive. Beta () Thalassemias: Beta-thalassemias can be similarly characterized as thalassemias, although the clinical symptoms are more variable because of the availability of the surrogate chains, and . Consequently, in most thalassemias, HbA2 and HbF (see Table 3) are at higher levels than normal. The molecular controls over these compensatory mechanisms are not well understood and are potentially important targets for therapy. The chains are increased in expression on the same chromosome (cis) as well as the other (trans) from the defective -chain. O thalassemia results from the deletion of the genes for both chains, which are adjacent on the chromosome (Figure 7b). Furthermore, the variability in -thalassemias is complicated by the fact that the -genes can be deleted, denoted as O, or they have intermediate expression levels that range from 5% to 90% of normal, denoted +. The genotype/phenotype correlations in -thalassemias are less exact but can be generally characterized (Table 3). Table 3. Genotype-phenotype relationships in -thalassemias. is normal expression; + is reduced expression; O is lack of expression GENOTYPE PHENOTYPE / Normal / to / + O -thalassemia Minor (can be asymptomatic) O/+ to +/+ -thalassemia Intermedia (depends on severity of + O/+ to O /O -thalassemia Major (transfusion-dependent) Key features of thalassemias are: If only one globin allele bears a mutation, the disease is called thalassemia minor. This is a mild microcytic anemia. In most cases the disease is asymptomatic. Thalassemia intermedia is a condition intermediate between the major and minor forms. Affected individuals can often manage a normal life with occasional transfusions. When both alleles have thalassemia mutations, the disease is thalassemia major or Cooley's anemia. Patients are characterized by a severe microcytic, hypochromic anemia. Untreated, this progresses to death before age twenty. The disease is transfusion-dependent. In thalassemia minor, one of the globin genes is defective. The defect can be a complete absence of the globin protein (i.e. O) or a reduced synthesis of the globin protein (i.e. +). The genetic defect usually is a missense or nonsense mutation in the Block: Foundations | HASNE [18 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT globin gene, but, occasionally, defects are due to gene deletions of the globin gene and surrounding regions. In thalassemia major, the production of globin chains is severely impaired because both globin genes are mutated. Another aspect of thalassemias that differentiate them from the thalassemias is the inability of subunits to form soluble tetramers. Since there is a reduced synthesis of chains, there is a relative overabundance of chains produced. These chains can form precipitates, even in the bone marrow cells, leading to hemolysis, anemia, and the compensatory increase in hematopoiesis, resulting in bone deformation and hepatosplenomegaly. CLINICAL CORRELATE: HEMOGLOBIN S OR SICKLE CELL ANEMIA HbS is the hemoglobin found in the erythrocytes of patients with sickle cell anemia. In this disease, the erythrocytes take on a characteristic “sickled” shape - see picture at: http://www.mun.ca/biology/scarr/Hb_Val_substitution.html This sickling is caused by polymer chains of Hb that are induced in the “T-state” by a single point mutation in one or both chains. This point mutation results in a non-polar Val replacing an acidic, charged Glu residue. The movements induced by O2 binding to Hb are such that the Glu is normally more exposed to solvent in deoxyHb compared to oxyHb. In normal HbA1, the Glu interacts with water. In HbS, the Val does not interact well with water. This Val is "sticky" in that it will bind tightly to other non-polar moieties, in particular, to a hydrophobic pocket on an adjacent subunit. Since HbS has two chains, it can have up to two exposed Val residues. Each will bind to another HbS and, in this way, "chains" of deoxyhemoglobin S form in solution (Figure 15). Hemoglobin in these chains is locked in the deoxygenated form and is unavailable to bind oxygen. Patients with HbS are anemic because sickled erythrocytes are more fragile and are, hence, more easily destroyed. The chains of Hb are also responsible for the sickled appearance of the cells. "Sickle cell disease" is rare, found only in individuals who are homozygous for the altered gene. However, 8% of African Americans are heterozygous for HbS. They have the "sickle-cell trait". In parts of Africa, the incidence of the gene can be as high as 20%. The gene has persisted because it confers resistance to malaria. Although their lives are often shortened, individuals with the gene generally survive past reproductive age, which cannot be said of young malaria victims. Block: Foundations | HASNE [19 of 20] HEMOGLOBIN: RED BLOOD CELLS AND OXYGEN TRANSPORT = hydrophobic Val on -subunit replaces normal acidic Glu Hydrophobic pocket on -subunit accommodates Val on -subunit Figure 15. Hemoglobin S (HbS) contains a variant residue in the -chain (Glu→Val). HbS tetramers aggregate under certain conditions in homozygous HbS patients, forming long rod-like conglomerations. These rigid, insoluble precipitates distort the shape of red blood cells, lending to them a characteristic "sickle" shape. Block: Foundations | HASNE [20 of 20]
