Hemoglobin Metabolism

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Hemoglobin Metabolism

Mary Coleman

Objectives

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

1. Describe the primary structure of the globin chains found in hemoglobin.

2. Describe the quaternary structure of hemoglobin.

3. Describe the biosynthesis of heme and globin.

4. Differentiate steps in heme synthesis that occur in the mitochondria and the cytoplasm.

5. Identify the ontogeny of hemoglobin with emphasis on the hemoglobin of newborns and adults.

6. Identify the three types of normal hemoglobin in adults and their reference intervals.

7. Describe the regulatory effects of hemoglobin metabolism.

8. Identify the important role that hemoglobin plays in maintaining body functions.

9. Describe the mechanism by which hemoglobin carries oxygen to the tissue.

10. Describe the Bohr effect.

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

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

13. Identify the P50 value (amount of oxygen needed to saturate 50% of hemoglobin) as it pertains to a normal oxygen dissociation curve.

14. Differentiate T and R forms of hemoglobin.

15. Identify the source of production of 2,3-bisphosphoglycerate and describe its impact on hemoglobin oxygenation.

16. Identify the oxygen affinity of fetal hemoglobin.

17. Compare and contrast the composition of the chemically modified hemoglobins—methemoglobin, carboxyhemoglobin, and sulfhemoglobin—and their affinity for oxygen.

18. Compare and contrast oxygenated, deoxygenated, and oxidized hemoglobin and ferric versus ferrous iron.

19. Describe how hemoglobin is routinely measured in the laboratory.

20. Identify how different kinds of hemoglobins are identified by laboratory tests.

21. Identify the gene locations of the globins that make up the hemoglobin molecule, including the number of genes for each globin chain (for Hb A, A2, and F) and their general arrangement on chromosomes.

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).

image
Figure 10-1 Hemoglobin: a tetramer of four heme molecules, each attached to a globin polypeptide chain.

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.

image
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 10-1).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.

image
Figure 10-3 A β-globin chain: a polypeptide with helical and nonhelical segments. (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. 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

image
Figure 10-4 Hemoglobin molecule illustrating tertiary folding and quaternary assembly. Heme is suspended between the E and F helices of the polypeptide chain. Pink represents α1 (left) and α2 (right); yellow represents non-α2 (left) and non-α1 (right).

Hemoglobin Biosynthesis

Heme Biosynthesis

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

image
Figure 10-5 Hemoglobin assembly begins with glycine and succinyl coenzyme A (CoA), which assemble in the mitochondria catalyzed by aminolevulinic acid (ALA) synthase to form ALA. In the cytoplasm, ALA undergoes several transformations to form coproporphyrin 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 ferrochetolase to form heme. In the cytoplasm, heme assembles with α chain and non-α chain globins to form dimers and ultimately hemoglobin tetramers.

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.

Globin Biosynthesis

Six structural genes control the synthesis of the six globin chains. The α and ζ genes are on chromosome 16; the γ, β, δ, and ε genes are linked on chromosome 11. In the human genome, there is one copy of each globin gene per chromatid for a total of two genes per person with the exception of α and γ. There are two copies of the α and γ genes per chromatid for a total of four genes per person.

The production of globin chains takes place in RBC precursors from the pronormoblast through the circulating polychromatic (polychromatophilic) erythrocyte, but not in the mature RBC.8,9 Globin proteins arise via transcription of the genetic code to messenger ribonucleic acid (mRNA) and translation of mRNA to the globin polypeptide chain. A slight excess of α-globin mRNA is present in pronormoblasts (proerythroblasts); however, β-globin mRNA is translated more efficiently than α-globin mRNA. This results in synthesis of sets of chains in approximately equal amounts. When synthesized, the chains are released from the ribosomes in the cytoplasm.7

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