Case Study

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

Hemoglobin and hemoglobin electrophoresis testing 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 values. The mother’s hemoglobin value was 14 g/dL (140 g/L), and the infant’s was 20 g/dL (200 g/L). The mother’s hemoglobin electrophoresis results were 97% Hb A, 2% Hb A₂, and 1% Hb F. The newborn’s results were 88% Hb F and 12% Hb A.

Hemoglobin Structure

Hemoglobin (Hb) is the first protein whose structure was described using x-ray crystallography.1–4 The hemoglobin molecule is a conjugated globular protein consisting of four heme groups and two heterogenous pairs of polypeptide chains (Figure 10-1).

Hemoglobin is the main cytoplasmic component of erythrocytes (red blood cells, or RBCs). Free (non-RBC) hemoglobin, generated from RBCs through hemolysis, has a short half-life, is rapidly salvaged, and is catabolized or excreted renally. The concentration of hemoglobin within RBCs is approximately 34 g/dL, and its molecular weight is 64,000 D (Daltons). Hemoglobin’s main function is to transport oxygen from the lungs to tissues. Hemoglobin also modulates vascular dilation by transporting nitric oxide (NO) and transports carbon dioxide from the tissues to the lungs for exhalation.

Heme Structure

Heme consists of a ring of carbon, hydrogen, and nitrogen atoms called protoporphyrin IX with an atom of divalent ferrous iron (Fe²⁺) attached (ferroprotoporphyrin, 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. Each heme molecule combines reversibly with one oxygen molecule. Owing to its double bonds, heme renders blood red.

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 10-1).⁵

TABLE 10-1

Globin Chains

Symbol	Name	No. of Amino Acids
α	Alpha	141
β	Beta	146
γA	Gamma A	146 (position 136: alanine)
γG	Gamma G	146 (position 136: glycine)
δ	Delta	146
ε	Epsilon	Unknown
ζ	Zeta	141
θ	Theta	Unknown

Each globin chain is divided into eight helices and seven nonhelical segments (Figure 10-3). 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. The 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.

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. 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. The distal histidine appears to swing in and out of position to permit the passage of oxygen into and out of the hemoglobin molecule. 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 oxygen molecules) or deoxygenated (not carrying oxygen molecules).

The quaternary structure of hemoglobin, also called a tetrameric molecule, describes the complete hemoglobin molecule. The complete hemoglobin molecule is spherical, has four heme groups attached to four polypeptide chains, and may carry four molecules of oxygen. It is composed of two α globin chains and two non-α globin chains. Each globin chain has a heme group attached. Each heme molecule is capable of carrying one molecule of oxygen. Strong α₁,– non-α₁ and α₂,–non-α₂ dimeric bonds hold the molecule in a stable form. Tetrameric α₁–non-α₂ and α₂,–non-α₁ bonds also contribute to the stability of the structure (Figure 10-4).^6,7

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

Heme biosynthesis begins with the condensation of glycine and succinyl coenzyme A (CoA) catalyzed by aminolevulinate synthase (ALAS) to form aminolevulinic acid (ALA). ALA dehydratase (also known as ALA dehydrase, porphobilinogen synthase) in the presence of ALA catalyzes the formation of porphobilinogen. Porphobilinogen deaminase, also known as hydroxymethylbilane synthase, in the presence of porphobilinogen catalyzes the formation of hydroxyl methylbilane. This pathway continues until, in the final step of production of heme, Fe²⁺ combines with protoporphyrin IX in the presence of ferrochelatase/heme synthase to make heme (Figure 10-5).⁶

Transferrin, a plasma protein, carries iron in the ferric (Fe³⁺) form to developing RBCs. Iron passes through the RBC membrane to the mitochondria and is united with protoporphyrin IX to make heme. Heme leaves the mitochondria and is joined to the globin chains in the cytoplasm.

Hemoglobin Ontogeny

Hemoglobin composition differs with prenatal gestation time and postnatal age. Hemoglobin changes reflect changes in the activation and inactivation or switching of the globin genes, progressing from the ζ to the α gene on chromosome 16 and from the ε to the γ, δ, or β genes on chromosome 11. The ζ and ε genes normally appear only during the first 3 months of embryonic development. These two chains in addition to the α and γ chains are constituents of embryonic hemoglobins (Figure 10-6). At birth, Hb F is the predominant hemoglobin. Normal adult hemoglobin is predominately Hb A (α₂β₂) with small amounts of Hb A₂ (α₂δ₂) and Hb F (α₂γ₂).⁷ Table 10-2 presents adult reference intervals.

A small percentage of Hb A is glycated. Glycation is a posttranslational modification formed by nonenzymatic binding of various sugars with globin chain amino groups. The most glycated hemoglobin is Hb A_1c, in which glucose attaches to the N-terminal valine of the β chain. Normally, about 4% to 6% of Hb A circulates in the A_1c form. In uncontrolled diabetes mellitus, the amount of A_1c is increased. Other posttranslational modifications seem to be of little importance.⁷

The key rate-limiting step in heme synthesis is the initial reaction of glycine and succinyl CoA to form ALA, catalyzed by ALAS. Transcription of the ALAS gene is inhibited by heme, which leads to a decrease in heme production (a negative feedback mechanism). Other enzymes in the heme pathway inhibited by heme are ALA dehydrase and porphobilinogen deaminase/hydroxymethylbilane synthase. An increased demand for heme induces an increased synthesis of ALAS.⁹

Ferrochelatase (also known as heme synthase) also plays a regulatory role in heme biosynthesis. A negative feedback mechanism by heme or substrate inhibition by protoporphyrin IX is believed to inhibit the ferrochelatase/heme synthase enzyme.⁹

Hemoglobin Function

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 ions in a hemoglobin molecule can reversibly bind one oxygen molecule. Approximately 1.34 mL of oxygen is bound by each gram of hemoglobin.

The affinity of hemoglobin for oxygen depends on the partial pressure of oxygen (Po₂), often defined in terms of the amount of oxygen needed to saturate 50% of hemoglobin, called the P₅₀ 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.

Cooperation among hemoglobin subunits contributes to the shape of the curve. Heme units do not undergo simultaneous oxygenation or deoxygenation; rather, the state of each heme unit in comparison with the other units influences further binding. That is, hemoglobin that is completely deoxygenated has little affinity for oxygen, but with each oxygen molecule that is bound, the avidity increases, and the hemoglobin molecule quickly becomes fully oxygenated.⁷ Shifts of the curve to the left or right occur if there are changes in the pH of the blood. This shift in the curve as a result of pH is termed the Bohr effect.

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 concentrations 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. Hemoglobin absorbs less oxygen in the lungs but delivers more oxygen to the tissues than it normally would, as in the presence of Hb S (see Chapter 26).

The effect of 2,3-bisphosphoglycerate (2,3-BPG, formerly 2,3-diphosphoglycerate), on oxygen affinity is complex. The hemoglobin molecule is allosteric; that is, its function and structure are influenced by other molecules. In the presence of large amounts of 2,3-BPG, the hemoglobin molecule changes from a relaxed (R) oxygenated molecule to a tense (T) deoxygenated molecule. The T structure is stabilized by salt bridges, which become broken as the molecule switches to the R structure. When the salt bridges are broken, the hemoglobin molecule is able to bind to oxygen (Figure 10-8). Carbon dioxide, hydrogen ions, and chloride ions all decrease the affinity of hemoglobin for oxygen by strengthening the salt bridges that lock the molecule into its T conformation. Some abnormal hemoglobins with a high oxygen affinity and low P₅₀ occur as a result of amino acid substitutions that lead to loss of bonds that would have stabilized the tetramer in the T conformation. Without these binding sites, the hemoglobin molecule holds on more tightly to oxygen—hence a higher oxygen affinity.⁷

Clinical conditions that produce a shift 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 methemoglobin, carboxyhemoglobin, or some other hemoglobin variants. Conditions producing a shift of the curve to the right include increased body temperature, increased 2,3-BPG concentrations, increased H⁺ concentration (decreased pH), and abnormal hemoglobins with a low affinity for oxygen. Clinical conditions that produce a right shift include a high fever, acidosis, and circumstances that produce hypoxia, such as high altitude, pulmonary insufficiency, congestive heart failure, severe anemia, and cardiac right-to-left shunt.⁷

The sigmoidal oxygen curve generated by normal hemoglobin contrasts with myoglobin’s hyperbolic curve (see Figure 10-7). Myoglobin, present in cardiac and skeletal muscle, is a 17,000-D 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 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 through the kidney, but levels may become elevated in renal failure. Testing for serum myoglobin aids in detecting myocardial infarction in patients who have no underlying trauma, rhabdomyolysis, or renal failure. An elevated myoglobin level generates a positive result on the urine hemoglobin dipstick test that must be differentiated from a response caused by hemoglobin. In contrast to myoglobinuria, hemoglobinuria is seen in intravascular hemolytic disorders.⁷

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 relative to that of Hb A. Consequently, fetal circulation extracts oxygen from maternal blood. Hb F delivers oxygen less readily to tissues, however. To achieve adequate tissue oxygenation, fetal hemoglobin concentration is high. The concentration approximates adult values by 6 months as Hb F production is reduced.

A second crucial function of hemoglobin is the transport of carbon dioxide. In the blood, carbon dioxide undergoes a pair of reactions in which it combines with water to form carbonic acid. Carbonic acid then disassociates to release H⁺ and bicarbonate:

< ?xml:namespace prefix = "mml" />CO2+H2O→H2CO3

H2CO3→H++HCO3−

The first of these reactions is facilitated by the RBC enzyme carbonic anhydrase. The H⁺ from the second reaction binds deoxygenated hemoglobin. The bicarbonate diffuses across the RBC membrane, and a portion is exchanged with plasma Cl⁻; this is called the chloride shift. The plasma bicarbonate travels to the lungs, where it is expired. About 70% to 85% of tissue carbon dioxide is processed in this manner.¹⁰ Approximately 10% to 20% of carbon dioxide binds to the N-terminal amino group of each globin chain as carbaminohemoglobin. Carbaminohemoglobin has a lower affinity for oxygen than does hemoglobin in the absence of carbon dioxide.¹¹

Variant Hemoglobins

The variant hemoglobins methemoglobin, sulfhemoglobin, and carboxyhemoglobin are hemoglobins whose structure has been modified by drugs or environmental chemicals.

Hemoglobin Measurement

The cyanmethemoglobin method is the reference method for hemoglobin assay.¹⁸ 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 (see 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 (see Chapter 39).

Hemoglobin electrophoresis is used to separate the different types of hemoglobins such as Hb A, A₂, and F (see Chapter 26).

Summary

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