Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 10. Hemoglobin metabolism

Elaine M. Keohane*


Hemoglobin Structure

Heme Structure

Globin Structure

Complete Hemoglobin Molecule

Hemoglobin Biosynthesis

Heme Biosynthesis

Globin Biosynthesis

Hemoglobin Assembly

Hemoglobin Ontogeny

Regulation of Hemoglobin Production

Heme Regulation

Globin Regulation

Systemic Regulation of Erythropoiesis

Hemoglobin Function

Oxygen Transport

Carbon Dioxide Transport

Nitric Oxide Transport





Hemoglobin Measurement


After completion of this chapter, the reader will be able to:

1. Describe the components and structure of hemoglobin.

2. Describe steps in heme synthesis that occur in the mitochondria and the cytoplasm.

3. Name the genes and the chromosome location and arrangement for the various polypeptide chains of hemoglobin.

4. Describe the polypeptide chains produced and the hemoglobins they form in the embryo, fetus, newborn, and adult.

5. List the three types of normal hemoglobin in adults and their reference intervals.

6. Describe mechanisms that regulate hemoglobin synthesis.

7. Describe the mechanism by which hemoglobin transports oxygen to the tissues and transports carbon dioxide to the lungs.

8. Explain the importance of maintaining hemoglobin iron in the ferrous state (Fe2+).

9. Explain the significance of the sigmoid shape of the oxygen dissociation curve.

10. Correlate right and left shifts in the oxygen dissociation curve with conditions that can cause shifts in the curve.

11. Differentiate T and R forms of hemoglobin and the effect of oxygen and 2,3-bisphosphoglycerate on those forms.

12. Explain the difference between adult Hb A and fetal Hb F and how that difference impacts oxygen affinity.

13. Compare and contrast the composition and the effect on oxygen binding of methemoglobin, carboxyhemoglobin, and sulfhemoglobin.


After studying the material in this chapter, the reader should be able to respond to the following case study:

Hemoglobin and hemoglobin fractionation and quantification using high performance liquid chromatography (HPLC) were performed on a mother and her newborn infant, both presumed to be healthy. The assays were part of a screening program to establish reference intervals. The mother’s hemoglobin concentration was 14 g/dL, and the newborn’s was 20 g/dL. The mother’s hemoglobin fractions were quantified as 97% Hb A, 2% Hb A2, and 1% Hb F by HPLC. The newborn’s results were 88% Hb F and 12% Hb A.

1. Were these hemoglobin results within expected reference intervals?

2. Why were the mother’s and the newborn’s hemoglobin concentration so different?

3. What is the difference between the test to determine the hemoglobin concentration and the test to analyze hemoglobin by HPLC?

4. Why were the mother’s and newborn’s hemoglobin fractions so different?

Hemoglobin (Hb) is one of the most studied proteins in the body due to the ability to easily isolate it from red blood cells (RBCs). It comprises approximately 95% of the cytoplasmic content of RBCs.1 The body very efficiently carries hemoglobin in RBCs, which provides protection from denaturation in the plasma and loss through the kidneys. Free (non-RBC) hemoglobin, generated from RBCs through hemolysis, has a short half-life outside of the RBCs. When released into the plasma, it is rapidly salvaged to preserve its iron and amino acid components; when salvage capacity is exceeded, it is excreted by the kidneys (Chapter 23). The concentration of hemoglobin within RBCs is approximately 34 g/dL, and its molecular weight is approximately 64,000 Daltons.2 Hemoglobin’s main function is to transport oxygen from the lungs to tissues and transport carbon dioxide from the tissues to the lungs for exhalation. Hemoglobin also contributes to acid-base balance by binding and releasing hydrogen ions and transports nitric oxide (NO), a regulator of vascular tone.13

This chapter covers the structure, biosynthesis, ontogeny, regulation, and function of hemoglobin. The formation, composition, and characteristics of several dyshemoglobins—namely, methemoglobin, carboxyhemoglobin, and sulfhemoglobin—are also discussed at the end of the chapter.

Hemoglobin structure

Hemoglobin is the first protein whose structure was described using x-ray crystallography.4 The hemoglobin molecule is a globular protein consisting of two different pairs of polypeptide chains and four heme groups, with one heme group imbedded in each of the four polypeptide chains (Figure 10-1).


FIGURE 10-1 Hemoglobin: a tetramer of four globin polypeptide chains, with a heme molecule attached to each chain.

Heme structure

Heme consists of a ring of carbon, hydrogen, and nitrogen atoms called protoporphyrin IX, with a central atom of divalent ferrous iron (Fe2+) (Figure 10-2). Each of the four heme groups is positioned in a pocket of the polypeptide chain near the surface of the hemoglobin molecule. The ferrous iron in each heme molecule reversibly combines with one oxygen molecule. When the ferrous irons are oxidized to the ferric state (Fe3+), they no longer can bind oxygen. Oxidized hemoglobin is also called methemoglobin and is discussed later in this chapter.


FIGURE 10-2 Heme is protoporphyrin IX that carries a central ferrous ion (Fe2+).

Globin structure

The four globin chains comprising each hemoglobin molecule consist of two identical pairs of unlike polypeptide chains, 141 to 146 amino acids each. Variations in amino acid sequences give rise to different types of polypeptide chains. Each chain is designated by a Greek letter ().Table 10-113

TABLE 10-1

Globin Chains



Number of Amino Acids








Gamma A

146 (position 136: alanine)


Gamma G

146 (position 136: glycine)













Each globin chain is divided into eight helices separated by seven nonhelical segments ().Figure 10-33 The helices, designated A to H, contain subgroup numberings for the sequence of the amino acids in each helix and are relatively rigid and linear. Flexible nonhelical segments connect the helices, as reflected by their designations: NA for the sequence between the N-terminus and the A helix, AB between the A and B helices, and so forth, with BC, CD, DE, EF, FG, GH, and finally HC between the H helix and the C-terminus.3


FIGURE 10-3 A β-globin chain: a polypeptide with helical (labeled A through H) and nonhelical segments. Heme (protoporphyrin IX with a central iron atom) is suspended in a pocket between the E and F helices. The iron atom of heme is linked to the F8 proximal histidine on one side of the heme plane (solid line). Oxygen binds to the iron atom on the other side of the plane and is close (but not linked) to the E7 distal histidine (dotted line). Source: (Modified from Huisman TH, Schroder WA: New aspects of the structure, function, and synthesis of hemoglobins, Boca Raton, Fla, 1971, CRC Press; modified from Stamatoyannopoulos G: The molecular basis of blood diseases, ed 2, Philadelphia, 1994, Saunders.)

Complete hemoglobin molecule

The hemoglobin molecule can be described by its primary, secondary, tertiary, and quaternary protein structures. The primary structure refers to the amino acid sequence of the polypeptide chains. The secondary structure refers to chain arrangements in helices and nonhelices. The tertiary structure refers to the arrangement of the helices into a pretzel-like configuration.

Globin chains loop to form a cleft pocket for heme. Each chain contains a heme group that is suspended between the E and F helices of the polypeptide chain (Figure 10-3).23 The iron atom at the center of the protoporphyrin IX ring of heme is positioned between two histidine radicals, forming a proximal histidine bond within F8 and, through the linked oxygen, a close association with the distal histidine residue in E7.3 Globin chain amino acids in the cleft are hydrophobic, whereas amino acids on the outside are hydrophilic, which renders the molecule water soluble. This arrangement also helps iron remain in its divalent ferrous form regardless of whether it is oxygenated (carrying an oxygen molecule) or deoxygenated (not carrying an oxygen molecule).

The quaternary structure of hemoglobin, also called a tetramer, describes the complete hemoglobin molecule. The complete hemoglobin molecule is spherical, has four heme groups attached to four polypeptide chains, and may carry up to four molecules of oxygen (Figure 10-4). The predominant adult hemoglobin, Hb A, is composed of two α-globin chains and two β-globin chains. Strong α1–β1 and α2–β2 bonds hold the dimers in a stable form. The α1–β2 and α2–β1 bonds are important for the stability of the quaternary structure in the oxygenated and deoxygenated forms (Figure 10-1).12


FIGURE 10-4 Hemoglobin molecule illustrating tertiary folding of the four polypeptide chains. Heme is suspended between the E and F helices of each polypeptide chain. Pink represents α1 (left) and α2 (right); yellow represents non-α2 (left) and non-α1(right). The polypeptide chains first form α1 - non-α1 and α2-non-α2 dimers, and then assemble into a tetramer (quaternary structure) with α1-non-α2 and α2-non-α1 bonds.

A small percentage of Hb A is glycated. Glycation is a posttranslational modification formed by the nonenzymatic binding of various sugars to globin chain amino groups over the life span of the RBC. The most characterized of the glycated hemoglobins is Hb A1c, in which glucose attaches to the N-terminal valine of the β chain.1 Normally, about 4% to 6% of Hb A circulates in the A1c form. In uncontrolled diabetes mellitus, the amount of A1c is increased proportionally to the mean blood glucose level over the preceding 2 to 3 months.

Hemoglobin biosynthesis

Heme biosynthesis

Heme biosynthesis occurs in the mitochondria and cytoplasm of bone marrow erythrocyte precursors, beginning with the pronormoblast through the circulating polychromatic (also known as polychromatophilic) erythrocyte (Chapter 8). As they lose their ribosomes and mitochondria (location of the citric/tricarboxylic acid cycle), mature erythrocytes can no longer make hemoglobin.5

Heme biosynthesis begins in the mitochondria with the condensation of glycine and succinyl coenzyme A (CoA) catalyzed by aminolevulinate synthase to form aminolevulinic acid (ALA) (Figure 10-5).5 In the cytoplasm, aminolevulinic acid dehydratase (also known as porphobilinogen synthase) converts ALA to porphobilinogen (PBG). PBG undergoes several transformations in the cytoplasm from hydroxylmethylbilanetocoproporphyrinogen III. This pathway then continues in the mitochondria until, in the final step of production of heme, Fe2+ combines with protoporphyrin IX in the presence of ferrochelatase (heme synthase) to make heme.5


FIGURE 10-5 Hemoglobin assembly begins with glycine and succinyl coenzyme A (CoA), which assemble in the mitochondria catalyzed by aminolevulinate synthase to form aminolevulinic acid (ALA). In the cytoplasm, ALA undergoes several transformations from porphobilinogen (PBG) to coproporphyrinogen III, which, catalyzed by coproporphyrinogen oxidase, becomes protoporphyrinogen IX. In the mitochondria, protoporphyrinogen IX is converted to protoporphyrin IX by protoporphyrinogen oxidase. Ferrous (Fe2+) ion is added, catalyzed by ferrochelatase to form heme. In the cytoplasm, heme assembles with an α chain and non-α chain, forming a dimer, and ultimately two dimers join to form the hemoglobin tetramer.

Transferrin, a plasma protein, carries iron in the ferric (Fe3+) form to developing erythroid cells (Chapter 11). Transferrin binds to transferrin receptors on erythroid precursor cell membranes and the receptors and transferrin (with bound iron) are brought into the cell in an endosome (Figure 11-5). Acidification of the endosome releases the iron from transferrin. Iron is transported out of the endosome and into the mitochondria where it is reduced to the ferrous state, and is united with protoporphyrin IX to make heme. Heme leaves the mitochondria and is joined to the globin chains in the cytoplasm.

Globin biosynthesis

Six structural genes code for six globin chains. The α- and ζ-globin genes are on the short arm of chromosome 16; the ε-, γ-, δ-, and β-globin gene cluster is on the short arm of chromosome 11 (Figure 28-1). In the human genome, there is one copy of each globin gene per chromatid, for a total of two genes per diploid cell, with the exception of α and γ. There are two copies of the α- and γ-globin genes per chromatid, for a total of four genes per diploid cell.

The production of globin chains takes place in erythroid precursors from the pronormoblast through the circulating polychromatic erythrocyte, but not in the mature erythrocyte.5 Transcription of the globin genes to messenger ribonucleic acid (mRNA) occurs in the nucleus, and translation of mRNA to the globin polypeptide chain occurs on ribosomes in the cytoplasm. Although transcription of the α-globin genes produces more mRNA than the β-globin gene, there is less efficient translation of the α-globin mRNA.2 Therefore, the α and β chains are produced in approximately equal amounts. After translation is complete, the chains are released from the ribosomes in the cytoplasm.

Hemoglobin assembly

After their release from ribosomes, each globin chain binds to a heme molecule, then forms a heterodimer (Figure 10-5). The non-α chains have a charge difference that determines their affinity to bind to the α chains. The α chain has a positive charge and has the highest affinity for a β chain due to its negative charge.12 The γ-globin chain has the next highest affinity, followed by the δ-globin chain.2 Two heterodimers then combine to form a tetramer. This completes the hemoglobin molecule.

Two α and two β chains form Hb A, the major hemoglobin present from 6 months of age through adulthood. Hb A2 contains two α and two δ chains. Owing to a mutation in the promoter region of the δ-globin gene, production of the δ chain polypeptide is very low.6 Consequently, Hb A2 comprises less than 3.5% of total hemoglobin in adults. Hb F contains two α and two γ chains. In healthy adults, Hb F comprises 1% to 2% of total hemoglobin, and it is present only in a small proportion of the RBCs (uneven distribution). These RBCs with Hb F are called F or A/F cells.12

The various amino acids that comprise the globin chains affect the net charge of the hemoglobin tetramer. Electrophoresis and high performance liquid chromatography (HPLC) are used for fractionation, presumptive identification, and quantification of normal hemoglobins and hemoglobin variants (Chapter 27). Molecular genetic testing of globin gene DNA provides definitive identification of variant hemoglobins.

Hemoglobin ontogeny

Hemoglobin composition differs with prenatal gestation time and postnatal age. Hemoglobin changes reflect the sequential activation and inactivation (or switching) of the globin genes, progressing from the ζ- to the α-globin gene on chromosome 16 and from the ε- to the γ-, δ-, and β-globin genes on chromosome 11. The ζ- and ε-globin chains normally appear only during the first 3 months of embryonic development. These two chains, when paired with the α and γ chains, form the embryonic hemoglobins (). Duringthe second and third trimesters of fetal life and at Figure 10-6 birth, Hb F (α2γ2) is the predominant hemoglobin. By 6 months of age and through adulthood, Hb A (α2β2) is the predominant hemoglobin, with small amounts of Hb A2 (α2δ2) and Hb F.2 Table 10-2 presents the reference intervals for the normal hemoglobin fractions at various ages.


FIGURE 10-6 Timeline of globin chain production from intrauterine life to adulthood. See also Table 10-2.

TABLE 10-2

Normal Hemoglobins


Globin Chain



Early embryogenesis (product of yolk sac erythroblasts)

ζ2 + ε2 

α2 + ε2 

ζ2 + γ2




Begins in early embryogenesis; peaks during third trimester and begins to decline just before birth

α2 + γ2




α2 + γ2

F, 60% to 90%


α2 + β2

A, 10% to 40%

Two Years through Adulthood


α2 + γ2

F, 1% to 2%


α2 + δ2

A2, < 3.5%


α2 + β2

A, > 95%

Mechanisms that control the switching from γ chain production to β chain production (γ-β switching) are discussed in the next section.

Regulation of hemoglobin production

Heme regulation

The key rate-limiting step in heme synthesis is the initial reaction of glycine and succinyl CoA to form ALA, catalyzed by ALA synthase (Figure 10-5). Heme inhibits the transcription of the ALA synthase gene, which leads to a decrease in heme production (a negative feedback mechanism). Heme inhibits other enzymes in the biosynthesis pathway, including ALA dehydrase and PBG deaminase. A negative feedback mechanism by heme or substrate inhibition by protoporphyrin IX is believed to inhibit the ferrochelatase enzyme.5 Conversely, an increased demand for heme induces an increased synthesis of ALA synthase.5

Globin regulation

Globin synthesis is highly regulated so that there is a balanced production of globin and heme. This is critical because an excess of globin chains, protophophyrin IX, or iron can accumulate and damage the cell, reducing its life span.

Globin production is mainly controlled at the transcription level by a complex interaction of deoxyribonucleic acid (DNA) sequences (cis-acting promoters, enhancers, and silencers) and soluble transcription factors (trans-acting factors) that bind to DNA or to one another to promote or suppress transcription.2 Initiation of transcription of a particular globin gene requires (1) the promoter DNA sequences immediately before the 5’ end or the beginning of the gene; (2) a key transcription factor called Krüppel-like factor 1 (KLF1); (3) a number of other transcription factors (such as GATA1, Ikaros, TAL1, p45-NF-E2, and LDB1); and (4) an enhancer region of DNAse 1 hypersensitive nucleic acid sequences located more than 20 kilobases upstream (before the 5’ start site of the gene) from the globin gene called the locus control region or LCR.7For example, to activate transcription of the β-globin gene in the β-globin gene cluster on chromosome 11, the LCR, the promoter for the β-globin gene, and various transcription factors join together to form a three-dimensional active chromosome hub (ACH), with KLF1 playing a key role in connecting the complex.78 Because the LCR is located a distance upstream from the β-globin gene complex, a loop of DNA is formed when the LCR and β-globin gene promoter join together in the chromosome hub.8 The other globin genes in the cluster (ε-, γ-, and δ-) are maintained in the inactive state in the DNA loop, so only the β-globin gene is transcribed.78

Krüppel-like factor 1 also plays a key regulatory role in the switch from γ chain to β chain production (γ-β switching) that begins in late fetal life and continues through adulthood. The KLF1 is an exact match for binding to the DNA promoter sequences of the β-globin gene, while the γ-globin gene promoter has a slightly different sequence.7 This results in a preferential binding to and subsequent activation of transcription of the β-globin gene.7 KLF1 also regulates the expression of repressors of γ-globin gene transcription, such as BCL11A and MYB.79

Globin synthesis is also regulated during translation when the mRNA coding for the globin chains associates with ribosomes to produce the polypeptide. Many protein factors are required to control the initiation, elongation, and termination steps of translation. Heme is an important regulator of globin mRNA translation at the initiation step by promoting the activation of a translation initiation factor and inactivating its repressor.5 Conversely, when the heme level is low, the repressor accumulates and inactivates the initiation factor, thus blocking translation of the globin mRNA.25

Systemic regulation of erythropoiesis

When there is an insufficient quantity of hemoglobin or if the hemoglobin molecule is defective in transporting oxygen, tissue hypoxia occurs. The hypoxia is detected by the peritubular cells of the kidney, which respond by increasing the production of erythropoietin (EPO). EPO increases the number of erythrocytes produced and released into the periphery; it also accelerates the rate of synthesis of erythrocyte components, including hemoglobin (Chapter 8).

Although each laboratory must establish its own reference intervals based on their instrumentation, methodology, and patient population, in general, reference intervals for hemoglobin concentration are as follows:


14 to 18 g/dL (140 to 180 g/L)


12 to 15 g/dL (120 to 150 g/L)


16.5 to 21.5 g/dL (165 to 215 g/L)

Reference intervals for infants and children vary according to age group. Individuals living at high altitudes have slightly higher levels of hemoglobin as a compensatory mechanism to provide more oxygen to the tissues in the oxygen-thin air. Tables on the inside front cover of this text provide reference intervals for all age-groups.

Hemoglobin function

Oxygen Transport

The function of hemoglobin is to readily bind oxygen molecules in the lung, which requires high oxygen affinity; to transport oxygen; and to efficiently unload oxygen to the tissues, which requires low oxygen affinity. During oxygenation, each of the four heme iron atoms in a hemoglobin molecule can reversibly bind one oxygen molecule. Approximately 1.34 mL of oxygen is bound by each gram of hemoglobin.1

The affinity of hemoglobin for oxygen relates to the partial pressure of oxygen (PO2), often defined in terms of the amount of oxygen needed to saturate 50% of hemoglobin, called the P 50 value. The relationship is described by the oxygen dissociation curve of hemoglobin, which plots the percent oxygen saturation of hemoglobin versus the PO2 (Figure 10-7). The curve is sigmoidal, which indicates low hemoglobin affinity for oxygen at low oxygen tension and high affinity for oxygen at high oxygen tension.


FIGURE 10-7 Oxygen dissociation curves. A, Normal hemoglobin-oxygen dissociation curve. P50 is the partial pressure of oxygen (O2) needed for 50% O2 saturation of hemoglobin. B, Left-shifted curve with reduced P50 can be caused by decreases in 2,3-bisphosphoglycerate (2,3-BPG), H+ ions (raised pH), partial pressure of carbon dioxide (PCO2), and/or temperature. A left-shifted curve is also seen with hemoglobin F and hemoglobin variants that have increased oxygen affinity. C, Right-shifted curve with increased P50 can be caused by elevations in 2,3-BPG, H+ ions (lowered pH), PCO2, and/or temperature. A right-shifted curve is also seen with hemoglobin variants that have decreased oxygen affinity. Myoglobin, a muscle protein, produces a markedly left-shifted curve indicating a very high oxygen affinity. It is not effective in releasing oxygen at physiologic oxygen tensions.

Cooperation among hemoglobin subunits contributes to the shape of the curve. Hemoglobin that is completely deoxygenated has little affinity for oxygen. However, with each oxygen molecule that is bound, there is a change in the conformation of the tetramer that progressively increases the oxygen affinity of the other heme subunits. Once one oxygen molecule binds, the remainder of the hemoglobin molecule quickly becomes fully oxygenated.2 Therefore, with the high oxygen tension in the lungs, the affinity of hemoglobin for oxygen is high, and hemoglobin becomes rapidly saturated with oxygen. Conversely, with the relatively low oxygen tension in the tissues, the affinity of hemoglobin for oxygen is low, and hemoglobin rapidly releases oxygen.

Normally, a PO2 of approximately 27 mm Hg results in 50% oxygen saturation of the hemoglobin molecule. If there is a shift of the curve to the left, 50% oxygen saturation of hemoglobin occurs at a PO2 of less than 27 mm Hg. If there is a shift of the curve to the right, 50% oxygen saturation of hemoglobin occurs at a PO2 higher than 27 mm Hg.

The reference interval for arterial oxygen saturation is 96% to 100%. If the oxygen dissociation curve shifts to the left, a patient with arterial and venous PO2 levels in the reference intervals (80 to 100 mm Hg arterial and 30 to 50 mm Hg venous) will have a higher percent oxygen saturation and a higher affinity for oxygen than a patient for whom the curve is normal. With a shift in the curve to the right, a lower oxygen affinity is seen.

In addition to the PO2, shifts of the curve to the left or right occur if there are changes in the pH of the blood. In the tissues, a lower pH shifts the curve to the right and reduces the affinity of hemoglobin for oxygen, and the hemoglobin more readily releases oxygen. A shift in the curve due to a change in pH (or hydrogen ion concentration) is termed the Bohr effect. It facilitates the ability of hemoglobin to exchange oxygen and carbon dioxide (CO2) and is discussed later.

The concentration of 2,3-bisphosphoglycerate (2,3-BPG, formerly 2,3-diphosphoglycerate) also has an effect on oxygen affinity. In the deoxygenated state, the hemoglobin tetramer assumes a tense or Tconformation that is stabilized by the binding of 2,3-BPG between the β-globin chains (Figure 10-8). The formation of salt bridges between the phosphates of 2, 3-BPG and positively charged groups on the globin chains further stabilizes the tetramer in the T conformation.1 The binding of 2, 3-BPG shifts the oxygen dissociation curve to the right, favoring the release of oxygen.1 In addition, a lower pH and higher PCO2 in the tissues further shifts the curve to the right, favoring the release of oxygen.1


FIGURE 10-8 Tense (T) and relaxed (R) forms of hemoglobin. The tense form incorporates one 2,3-bisphosphoglycerate (2,3-BPG) molecule, bound between the β-globin chains with salt bridges. It is unable to transport oxygen. As hemoglobin binds oxygen molecules, the α1β1 and α2β2 dimers rotate 15º relative to each other as a result of the change in hydrophobic interactions at the α1β2 contact point, disruption of salt bridges, and release of 2, 3-BPG.

As hemoglobin binds oxygen molecules, a change in conformation of the hemoglobin tetramer occurs with a change in hydrophobic interactions at the α1β2 contact point, a disruption of the salt bridges, and release of 2, 3-BPG.1 A 15-degree rotation of the α1β1 dimer, relative to the α2β2 dimer, occurs along the α1β2 contact point.2 When the hemoglobin tetramer is fully oxygenated, it assumes a relaxed or R state (Figure 10-8).

Clinical conditions that produce a shift of the oxygen dissociation curve to the left include a lowered body temperature due to external causes; multiple transfusions of stored blood with depleted 2,3-BPG; alkalosis; and the presence of hemoglobin variants with a high affinity for oxygen. Conditions producing a shift of the curve to the right include increased body temperature; acidosis; the presence of hemoglobin variants with a low affinity for oxygen; and an increased 2,3-BPG concentration in response to hypoxic conditions, such as high altitude, pulmonary insufficiency, congestive heart failure, and severe anemia (Chapter 19).

The sigmoidal oxygen dissociation curve generated by normal hemoglobin contrasts with myoglobin’s hyperbolic curve (Figure 10-7). Myoglobin, present in cardiac and skeletal muscle, is a 17,000-Dalton, monomeric, oxygen-binding heme protein. It binds oxygen with greater affinity than hemoglobin. Its hyperbolic curve indicates that it releases oxygen only at very low partial pressures, which means it is not as effective as hemoglobin in releasing oxygen to the tissues at physiologic oxygen tensions. Myoglobin is released into the plasma when there is damage to the muscle in myocardial infarction, trauma, or severe muscle injury, called rhabdomyolysis. Myoglobin is normally excreted by the kidney, but levels may become elevated in renal failure. Serum myoglobin levels aid in diagnosis of myocardial infarction in patients who have no underlying trauma, rhabdomyolysis, or renal failure. Myoglobin in the urine produces a positive result on the urine dipstick test for blood; this must be differentiated from a positive result caused by hemoglobin.

Hb F (fetal hemoglobin, the primary hemoglobin in newborns) has a P50 of 19 to 21 mm Hg, which results in a left shift of the oxygen dissociation curve and increased affinity for oxygen relative to that of Hb A. This increased affinity for oxygen is due to its weakened ability to bind 2,3-BPG.2 There is only one amino acid difference in a critical 2,3-BPG binding site between the γ chain and the β chain that accounts for this difference in binding.2

In fetal life, the high oxygen affinity of Hb F provides an advantage by allowing more effective oxygen withdrawal from the maternal circulation. At the same time, Hb F has a disadvantage in that it delivers oxygen less readily to tissues. The bone marrow in the fetus and newborn compensates by producing more RBCs to ensure adequate oxygenation of the tissues. This response is mediated by erythropoietin (Chapter 8). Consequently, the RBC count, hemoglobin concentration, and hematocrit of a newborn are higher than adult values (values are on the inside front cover), but they gradually decrease to normal physiologic levels by 6 months of age as the γ-β switching is completed and most of the Hb F is replaced by Hb A.

Carbon Dioxide Transport

A second crucial function of hemoglobin is the transport of carbon dioxide. In venous blood, the carbon dioxide diffuses into the red blood cells and combines with water to form carbonic acid (H2CO3). This reaction is facilitated by the RBC enzyme carbonic anhydrase. Carbonic acid then dissociates to release H+ and bicarbonate (HCO3) (Figure 10-9).


FIGURE 10-9 Transport and release of oxygen (O2) and carbon dioxide (CO2) in the tissues and lungs. A, In the tissues, CO2 diffuses into the red blood cell and combines with water (H2O) to form carbonic acid (H2CO3). This reaction is catalyzed by carbonic anhydrase. H2CO3 disassociates to hydrogen (H+) and bicarbonate (HCO3) ions. H+ binds to oxyhemoglobin (HbO2). resulting in the release of O2 due to the Bohr effect. The O2 diffuses out of the cell into the tissues. The HCO3 diffuses out of the cell as its concentration increases and is replaced by chloride (Cl–) to maintain electroneutrality (chloride shift). Some CO2 directly binds to the globin chains of hemoglobin. B, In the lungs, O2 binds to deoxygenated hemoglobin (HHb) due to the high oxygen tension. The H+ dissociates from HbO2, combines with HCO3 to form H2CO3, which then dissociates into CO2 and H2O. The CO2 diffuses out of the red blood cells and is exhaled by the lungs.

The H+ from the second reaction binds oxygenated hemoglobin (HbO2), and the oxygen is released from the hemoglobin due to the Bohr effect. The oxygen then diffuses out of the cell into the tissues. As the concentration of the negatively charged bicarbonate increases, it diffuses across the RBC membrane into the plasma. Chloride (Cl), also negatively charged, diffuses from the plasma into the cell to maintain electroneutrality across the membrane; this is called the chloride shift (Figure 10-9).

In the lungs, oxygen diffuses into the cell and binds to deoxygenated hemoglobin (HHb) due to the high oxygen tension. H+ is released from hemoglobin and combines with bicarbonate to form carbonic acid. Carbonic acid is converted to water and CO2; the latter diffuses out of the cells and is expelled by the lungs. As more bicarbonate diffuses into the cell to produce carbonic acid, chloride diffuses back out into the plasma. Approximately 85% of the CO2 produced in the tissues is transported by hemoglobin as H+.1 In this capacity, hemoglobin provides a buffering effect by binding and releasing H+.1 A small percentage of CO2 remains in the cytoplasm and the remainder binds to the globin chains as a carbamino group.

Nitric Oxide Transport

A third function of hemoglobin involves the binding, inactivation, and transport of nitric oxide (NO).10 Nitric oxide is secreted by vascular endothelial cells and causes relaxation of the vascular wall smooth muscle and vasodilation.11 When released, free nitric oxide has a very short half-life, but some enters the RBCs and can bind to cysteine in the β chain of hemoglobin, forming S-nitrosohemoglobin.1011 Some investigators propose that hemoglobin preserves and transports nitric oxide to hypoxic microvascular areas, which stimulates vasodilation and increases blood flow (hypoxic vasodilation).10 In this way, hemoglobin may work with other systems in regulating local blood flow to microvascular areas by binding and inactivating nitric oxide (causing vasoconstriction and decreased blood flow) when oxygen tension is high and releasing nitric oxide (causing vasodilation and increased blood flow) when oxygen tension is low.10 This theory is not universally accepted, and the roles of hemoglobin, endothelial cells, and nitric oxide in regulating blood flow and oxygenation of the microcirculation are still being investigated.11


Dyshemoglobins (dysfunctional hemoglobins that are unable to transport oxygen) include methemoglobin, sulfhemoglobin, and carboxyhemoglobin. Dyshemoglobins form and may accumulate to toxic levels, after exposure to certain drugs or environmental chemicals or gasses. The offending agent modifies the structure of the hemoglobin molecule, preventing it from binding oxygen. Most cases of dyshemoglobinemia are acquired; a small fraction of methemoglobinemia cases are hereditary.


Methemoglobin (MetHb) is formed by the reversible oxidation of heme iron to the ferric state (Fe3+). Normally, a small amount of methemoglobin is continuously formed by oxidation of iron during the normal oxygenation and deoxygenation of hemoglobin.1112 However, methemoglobin reduction systems, predominantly the NADH-cytochrome b5 reductase 3 (NADH-methemoglobin reductase) pathway, normally limit its accumulation to only 1% of total hemoglobin (Chapter 9 and Figure 9-1).11-13

Methemoglobin cannot carry oxygen because the oxidized ferric iron cannot bind it. An increase in the methemoglobin level results in decreased delivery of oxygen to the tissues. Individuals with methemoglobin levels less than 25% are generally asymptomatic.14 If the methemoglobin level increases to more than 30% of total hemoglobin, cyanosis (bluish discoloration of skin and mucous membranes) and symptoms of hypoxia (dyspnea, headache, vertigo, change in mental status) occur.1213 Levels of methemoglobin greater than 50% can lead to coma and death.1213

An increase in methemoglobin, called methemoglobinemia, can be acquired or hereditary. The acquired form, also called toxic methemoglobinemia, occurs in normal individuals after exposure to an exogenous oxidant, such as nitrites, primaquine, dapsone, or benzocaine.1214 As the oxidant overwhelms the hemoglobin reduction systems, the level of methemoglobin increases, and the patient may exhibit cyanosis and symptoms of hypoxia.11 In many cases, withdrawal of the offending oxidant is sufficient for a recovery, but if the level of methemoglobin increases to 30% or more of total hemoglobin, intravenous methylene blue is administered. The methylene blue reduces the methemoglobin ferric iron to the ferrous state through the NADPH-methemoglobin reduction pathway that involves glutathione reductase and glucose-6-phosphate dehydrogenase (Figure 9-1).11 In life-threatening cases, exchange transfusion may be required.12

Hereditary causes of methemoglobinemia are rare and include mutations in the gene for NADH-cytochrome b5 reductase 3 (CYB5R3), resulting in a diminished capacity to reduce methemoglobin, and mutations in the α-, β-, or γ-globin gene, resulting in a structurally abnormal polypeptide chain that favors the oxidized ferric form of iron and prevents its reduction.1112 The methemoglobin produced by the latter group is called M hemoglobin or Hb M. (Chapter 27). Hb M is inherited in an autosomal dominant pattern, with methemoglobin comprising 30% to 50% of total hemoglobin.12 There is no effective treatment for this form of methemoglobinemia.1112 Cytochrome b5 reductase deficiency is an autosomal recessive disorder, and methemoglobin elevations occur in individuals who are homozygous or compound heterozygous for a CYB5R3 mutation.1112 Most individuals with Hb M or homozygous cytochrome b5 reductase deficiency maintain methemoglobin levels below 50%; they have cyanosis but only mild symptoms of hypoxia that do not require treatment.11-13 Individuals heterozygous for the CYB5R3 mutation have normal levels of methemoglobin but develop methemoglobinemia, cyanosis, and hypoxia when exposed to an oxidant drug or chemical.1214

Methemoglobin is assayed by spectral absorption analysis instruments such as the CO-oximeter. Methemoglobin shows an absorption peak at 630 nm.12 With high levels of methemoglobin, the blood takes on a chocolate brown color and does not revert back to the normal red color after oxygen exposure.1213 The methemoglobin in Hb M disease has different absorption peaks, depending on the variant.11 Hemoglobin electrophoresis, high performance liquid chromatography, and DNA mutation testing are used for identification of Hb M variants. Cytochrome b5 reductase 3 deficiency is diagnosed by enzyme assays and DNA mutation testing.11


Sulfhemoglobin is formed by the irreversible oxidation of hemoglobin by drugs (such as sulfonilamides, phenacetin, nitrites, and phenylhydrazine) or exposure to sulfur chemicals in industrial or environmental settings.1112 It is formed by the addition of a sulfur atom to the pyrrole ring of heme and has a greenish pigment.11 Sulfhemoglobin is ineffective for oxygen transport, and patients with elevated levels present with cyanosis. Sulfhemoglobin cannot be converted to normal Hb A; it persists for the life of the cell. Treatment consists of prevention by avoidance of the offending agent.

Sulfhemoglobin has a similar peak to methemoglobin on a spectral absorption instrument. The sulfhemoglobin spectral curve, however, does not shift when cyanide is added, a feature that is used to distinguish it from methemoglobin.11


Carboxyhemoglobin (COHb) results from the combination of carbon monoxide (CO) with heme iron. The affinity of carbon monoxide for hemoglobin is 240 times that of oxygen.11 Once one molecule of carbon monoxide binds to hemoglobin, it shifts the hemoglobin-oxygen dissociation curve to the left, further increasing its affinity and severely impairing release of oxygen to the tissues.1115 Carbon monoxide has been termed the silent killer because it is an odorless and colorless gas, and victims may quickly become hypoxic.15

Some carboxyhemoglobin is produced endogenously, but it normally comprises less than 2% of total hemoglobin.11 Exogenous carbon monoxide is derived from the exhaust of automobiles, tobacco smoke, and from industrial pollutants, such as coal, gas, and charcoal burning. In smokers, COHb levels may be as high as 15%.14 As a result, smokers may have a higher hematocrit and polycythemia to compensate for the hypoxia.1114

Exposure to carbon monoxide may be coincidental, accidental, or intentional (suicidal). Many deaths from house fires are the result of inhaling smoke, fumes, or carbon monoxide.15 Even when heating systems in the home are properly maintained, accidental poisoning with carbon monoxide may occur. Toxic effects, such as headache, dizziness, and disorientation, begin to appear at blood levels of 20% to 30% COHb.1114Levels of more than 40% of total hemoglobin may cause coma, seizure, hypotension, cardiac arrhythmias, pulmonary edema, and death.1115

Carboxyhemoglobin may be detected by spectral absorption instruments at 540 nm.12 It gives blood a cherry red color, which is sometimes imparted to the skin of victims.15 A diagnosis of carbon monoxide poisoning is made if the COHb level is greater than 3% in nonsmokers and greater than 10% in smokers.15 Treatment involves removal of the patient from the carbon monoxide source and administration of 100% oxygen.11 The use of hyperbaric oxygen therapy is controversial.15 It is primarily used to prevent neurologic and cognitive impairment after acute carbon monoxide exposure in patients whose COHb level exceeds 25%.15

Hemoglobin measurement

The cyanmethemoglobin method is the reference method for hemoglobin assay.16 A lysing agent present in the cyanmethemoglobin reagent frees hemoglobin from RBCs. Free hemoglobin combines with potassium ferricyanide contained in the cyanmethemoglobin reagent, which converts the hemoglobin iron from the ferrous to the ferric state to form methemoglobin. Methemoglobin combines with potassium cyanide to form the stable pigment cyanmethemoglobin. The cyanmethemoglobin color intensity, which is proportional to hemoglobin concentration, is measured at 540 nm spectrophotometrically and compared with a standard (Chapter 14). The cyanmethemoglobin method is performed manually but has been adapted for use in automated instruments.

Many instruments now use sodium lauryl sulfate (SLS) to convert hemoglobin to SLS-methemoglobin. This method does not generate toxic wastes (Chapter 15).

Hemoglobin electrophoresis and HPLC are used to separate the different types of hemoglobins such as Hb A, A2, and F (Chapters 27 and 28).


• The hemoglobin molecule is a tetramer composed of two pairs of unlike polypeptide chains. A heme group (protoporphyrin IX + Fe2+) is bound to each of the four polypeptide chains.

• Hemoglobin, contained in RBCs, carries oxygen from the lungs to the tissues. Oxygen binds to the ferrous iron in heme. Each hemoglobin tetramer can bind four oxygen molecules.

• Six structural genes code for the six globin chains of hemoglobin. The α- and ζ-globin genes are on chromosome 16; the ε-, γ-, δ-, and β-globin gene cluster is on chromosome 11. There is one copy of the δ-globin gene and one β-globin gene per chromosome, for a total of two genes per diploid cell. There are two copies of the α- and γ-globin genes per chromosome, for a total of four genes per diploid cell.

• The three hemoglobins found in normal adults are Hb A, Hb A2, and Hb F. Hb A (α2β2), composed of two αβ heterodimers, is the predominant hemoglobin of adults. Hb F (α2γ2) is the predominant hemoglobin in the fetus and newborn. Hb A2 (α2δ2) is present from birth through adulthood, but at low levels.

• Hemoglobin ontogeny describes which hemoglobins are produced by the erythroid precursor cells from the fetal period through birth to adulthood.

• Complex genetic mechanisms regulate the sequential expression of the polypeptide chains in the embryo, fetus, and adult. Heme provides negative feedback regulation on protoporphyrin and globin chain production.

• The hemoglobin-oxygen dissociation curve is sigmoid owing to cooperativity among the hemoglobin subunits in binding and releasing oxygen.

• 2,3-BPG produced by the glycolytic pathway facilitates the delivery of oxygen from hemoglobin to the tissues. The Bohr effect is the influence of pH on the release of oxygen from hemoglobin.

• In the tissues, carbon dioxide diffuses into the RBCs and combines with water to form carbonic acid (H2CO3). The carbonic acid is then converted to bicarbonate and hydrogen ions (HCO3 and H+). Most of the carbon dioxide is carried by hemoglobin as H+.

• Methemoglobin, sulfhemoglobin, and carboxyhemoglobin cannot transport oxygen. They can accumulate to toxic levels due to exposure to certain drugs, industrial or environmental chemicals, or gases. A small fraction of methemoglobinemia cases are hereditary. Cyanosis occurs in patients with increased levels of methemoglobin or sulfhemoglobin.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. A hemoglobin molecule is composed of:

a. One heme molecule and four globin chains

b. Ferrous iron, protoporphyrin IX, and a globin chain

c. Protoporphyrin IX and four globin chains

d. Four heme molecules and four globin chains

2. Normal adult Hb A contains which polypeptide chains?

a. α and β

b. α and δ

c. α and γ

d. α and ε

3. A key rate-limiting step in heme synthesis is suppression of:

a. Aminolevulinate synthase

b. Carbonic anhydrase

c. Protoporphyrin IX reductase

d. Glucose 6-phosphate dehydrogenase

4. Which of the following forms of hemoglobin molecule has the lowest affinity for oxygen?

a. Tense

b. Relaxed

5. Using the normal hemoglobin-oxygen dissociation curve in Figure 10-7 for reference, predict the position of the curve when there is a decrease in pH.

a. Shifted to the right of normal with decreased oxygen affinity

b. Shifted to the left of normal with increased oxygen affinity

c. Shifted to the right of normal with increased oxygen affinity

d. Shifted to the left of normal with decreased oxygen affinity

6. The predominant hemoglobin found in a healthy newborn is:

a. Gower-1

b. Gower-2

c. A

d. F

7. What is the normal distribution of hemoglobins in healthy adults?

a. 80% to 90% Hb A, 5% to 10% Hb A2, 1% to 5% Hb F

b. 80% to 90% Hb A2, 5% to 10% Hb A, 1% to 5% Hb F

c. > 95% Hb A, < 3.5% Hb A2, 1% to 2% Hb F

d. > 90% Hb A, 5% Hb F, < 5% Hb A2

8. Which of the following is a description of the structure of oxidized hemoglobin?

a. Hemoglobin carrying oxygen on heme; synonymous with oxygenated hemoglobin

b. Hemoglobin with iron in the ferric state (methemoglobin) and not able to carry oxygen

c. Hemoglobin with iron in the ferric state so that carbon dioxide replaces oxygen in the heme structure

d. Hemoglobin carrying carbon monoxide; hence “oxidized” refers to the single oxygen

9. In the quaternary structure of hemoglobin, the globin chains associate into:

a. α tetramers in some cells and β tetramers in others

b. A mixture of α tetramers and β tetramers

c. α dimers and β dimers

d. Two αβ dimers

10. How are the globin chain genes arranged?

a. With α genes and β genes on the same chromosome, including two α genes and two β genes

b. With α genes and β genes on separate chromosomes, including two α genes on one chromosome and one β gene on a different chromosome

c. With α genes and β genes on the same chromosome, including four α genes and four β genes

d. With α genes and β genes on separate chromosomes, including four α genes on one chromosome and two β genes on a different chromosome

11. The nature of the interaction between 2,3-BPG and hemoglobin is that 2,3-BPG:

a. Binds to the heme moiety, blocking the binding of oxygen

b. Binds simultaneously with oxygen to ensure that it stays bound until it reaches the tissues, when both molecules are released from hemoglobin

c. Binds to amino acids of the globin chain, contributing to a conformational change that inhibits oxygen from binding to heme

d. Oxidizes hemoglobin iron, diminishing oxygen binding and promoting oxygen delivery to the tissues


1.  Telen M.J. The mature erythrocyte. In: Greer J.P, Foerster J, Rodgers G.M, et al. Wintrobe’s Clinical Hematology. 12th ed. Philadelphia : Wolters Kluwer Health/Lippincott Williams & Wilkins 2009; 126-155.

2.  Steinberg M.H, Benz E.J, Jr Adewoye A.H, et al. Pathobiology of the human erythrocyte and its hemoglobins. In: Hoffman R, Benz E.J, Silberstein L.E, et al. Hematology Basic Principles and Practice. 6th ed. Philadelphia : Saunders, an imprint of Elsevier Inc 2013; 406-417.

3.  Natarajan K, Townes T.M, Kutlar A. Chapter 48. Disorders of hemoglobin structure sickle cell anemia and related abnormalities. In: Lichtman M.A, Kipps T.J, Seligsohn U, Kaushansky K, Prchal J.T. Williams Hematology. 8th ed. New York : McGraw-Hill 2010 Available at: 358& Sectionid=39835866 Accessed 11.01.14.

4.  Perutz M.F, Rossmann M.G, Cullis A.F, et al. Structure of hemoglobin a three dimensional Fourier synthesis at 5.5A resolution obtained by x-ray analysis. Nature; 1960; 185:416-422.

5.  Dessypris E.N, Sawyer S.T. Erythropoiesis. In: Greer J.P, Foerster J, Rodgers G.M, et al. Wintrobe’s Clinical Hematology. 12th ed. Philadelphia : Wolters Kluwer Health/Lippincott, Williams & Wilkins 2009; 106-125.

6.  Donze D, Jeancake P.H, Townes T.M. Activation of delta-globin gene expression by erythroid Krüpple-like factor a potential approach for gene therapy of sickle cell disease. Blood; 1996; 88:4051-4057.

7.  Tallack M.R, Perkins A.C. Three fingers on the switch Krüppel-like factor 1 regulation of α-globin to β-globin gene switching. Curr Opin Hematol; 2013; 20:193-200.

8.  Tolhuis B, Palstra R-J, Splinter E, et al. Looping and interaction between hypersensitive sites in the active β-globin locusMolecular Cell; 2002; 10:1453-1465.

9.  Zhou D, Liu K, Sun C.W, et al. KLF1 regulates BCL11A expression and gamma to beta-globin switchingNat Genet; 2010; 42:742-744.

10.  Allen B.W, Stamler J.S, Piantadosi C.A. Hemoglobin, nitric oxide and molecular mechanisms of hypoxic vasodilationTrends Mol Med; 2009; 15:452-460.

11.  Steinberg M.H. Hemoglobins with altered oxygen affinity, unstable hemoglobins, M-hemoglobins, and dyshemoglobinemias. In: Greer J.P, Foerster J, Rodgers G.M, et al. Wintrobe’s Clinical Hematology. 12th ed. Philadelphia : Wolters Kluwer Health/Lippincott Williams & Wilkins 2009; 1132-1142.

12.  Benz E.J, Jr. Ebert B.L. Hemoglobin variants associated with hemolytic anemia altered oxygen affinity, and methemoglobinemias. In: Hoffman R, Benz E.J, Silberstein L.E, et al. Hematology Basic Principles and Practice. 6th ed. Philadelphia : Saunders, an imprint of Elsevier Inc 2013; 573-580.

13.  Skold A, Cosco D.I, Klein R. Methemoglobinemia pathogenesis, diagnosis, and management. South Med J; 2011; 104:757-761.

14.  Vajpayee N, Graham S.S, Bem S. Basic examination of blood and bone marrow. In: McPherson R.A, Pincus M.R. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia : Saunders, an imprint of Elsevier Inc 2011; 509-512.

15.  Guzman J.A. Carbon monoxide poisoningCrit Care Clin,; 2012; 28:537-548.

16.  Clinical and Laboratory Standards Institute (CLSI). Reference and selected procedures for the quantitative determination of hemoglobin in blood; approved guideline. 3rd ed. Wayne, PA : CLSI document H15–A3 2000.

*The author extends appreciation to Mary Coleman, whose work in prior editions provided the foundation for this chapter.