hemoglobin and hemoglobinopathies

Hemoglobin & hemoglobinopathies

A 15 year old Filipino female is noted to have a hemoglobin of 10.6g/dl with an MCV of 65 on routine testing. She reports regular menses lasting 4-5 days each cycle.She has no specific complaints. She is unaware of a family history of anemia. By history, her diet appears to be adequate. PE is normal; specifically there is no hepatospleenomegaly, jaundice or scleral icterus. What is the most probable diagnosis.

IntroductionHaemoglobin (Hb), protein constituting 1/3 of the red blood cells• 65% at erythroblast stage• 35% at reticulocyte stage

Normal concentration of Hb in the blood:• adult males 13.5 – 16.5 g/dL• adult females 12.5 – 15 g/dl

Approximately 6.25 G (90 mg/kg) of Hb are produced and destroyed in the body each day.

Two parts- Heme- Globin

BIOMEDICAL IMPORTANCEHaem-proteins are characteristic of aerobic life. Hb is important in O2-binding and its transport and delivery to tissues which is required for metabolism.

• 2,3-biphosphoglycerate (BPG) produced in RB cells by Rapoport-Luebering shunt is necessary for stabilization of Hb-conformation at quaternary level by holding salt bridges and is important for understanding of high-altitude sickness and changes that take place in adaptation at high altitudes.

• Cyanide poisoning and carbon monoxide poisoning are fatal because they combine and inhibit heme protein cytochrome oxidase in electron transport chain and stops cellular respiration.

• Conversion of Hb to methaemoglobin by NaNO2/or sodium thiosulphate forms the basis of treatment of cyanide poisoning, as cyanide combines readily withmethaemoglobin to form cyanmethaemoglobin which is not toxic and thus spares the cytochrome oxidase.

• Study of Hb chemistry provides an insight into the molecular basis of genetic diseases such as haemoglobinopathies and abnormal Haemoglobins.

STRUCTURE OF Heme Heme• It is a Fe-porphyrin compound. The porphyrinsare complex compounds with a tetrapyrrole structure

Four such pyrroles called I to IV, are combined through –CH= bridges, called as methyne bridges to form a porphyrin nucleus.

• The outer carbons of the four pyrrole rings, which are not linked with the methylidene bridges, are numbered 1 to 8.

• The methylidene bridges are referred to as α, β, γ and δ respectively.

• In addition, the hydrogens at positions 1 to 8 are substituted by different groups in different compounds.

• In the protoporphyrin IX, which forms parent compoundof heme, the positions 1 to 8 in pyrroles are substitutedby methyl (–CH3), vinyl (–CH=CH2), methyl, vinyl,methyl, propionic acid (–CH2–CH2–COOH), propionic acid and methyl groups in that order respectively.

The Fe molecule in HemeThe two hydrogen atoms in the –NH groups of pyrrolerings (II and IV) are replaced by ferrous iron (Fe++) whichoccupy the centre of the compound ring structure andestablish linkages with all the four nitrogens of all thepyrrole rings.

• The Fe, besides its linkages to four nitrogens of the pyrrole rings, is also linked internally (5th linkage) to the nitrogen of the imidazole ring of histidine (His) of the polypeptide chains

• the sixth valence is directed outwards from the molecule and is linked to a molecule of H2O in deoxygenated Hb. When Hb is oxygenated, the H2O is displaced by O2.

The propionic acid COOH groups of 6 and 7 positions of haem, of III and IV pyrroles, are also linked to the basic groups of amino acids Arg and Lys of the polypeptide chains

Structure and function of MYOGLOBIN • Myoglobin, a hemeprotein present in heart and skeletal

muscle, functions both as a reservoir for oxygen, and as an oxygen carrier that increases the rate of transport of oxygen within the muscle cell.

• Myoglobin consists of a single polypeptide chain that is structurally similar to the individual subunit polypeptide chains of the hemoglobin molecule.

• This homology makes myoglobin a useful model forinterpreting some of the more complex properties of hemoglobin.

1. α-Helical content: Myoglobin is a compact molecule, with approximately 80% of its polypeptide chain folded into eight stretches ofα-helix.

2. These α-helical regions, are terminated either by the presence of proline, whose five-membered ring cannot be accommodated in an α-helix orby β-bends and loops stabilized by hydrogen bonds and ionicbonds

3. Binding of the heme group: The heme group of myoglobin sits in a crevice in the molecule, which is lined with nonpolar amino acids.

Notable exceptions are two histidine residues. One, the proximal histidine (F8), binds directly to the iron of heme. The second, or distal histidine (E7), does not directly interact with the heme group, but helps stabilize the binding of oxygen to the ferrous iron.

Evolutionary Vignette:

Structure and function of HEMOGLOBIN • Hemoglobin is found exclusively in red blood cells

(RBCs), where its main function is to transport oxygen (O2) from the lungs to the capillaries of the tissues.

• Hemoglobin A, the major hemoglobin in adults,is composed of four polypeptide chains—two α chains and two β chains—held together by noncovalent interactions.

• Each subunit has stretches of α-helical structure, and a heme-binding pocket similar to that of myoglobin.

• The tetrameric hemoglobin molecule is structurally and functionally more complex than myoglobin. For example, hemoglobin can transport H+ and CO2 from the tissues to the lungs, and can carry four moleculesof O2 from the lungs to the cells of the body.

• Furthermore, the oxygen-binding properties of hemoglobin are regulated by interaction with allosteric effectors

Quaternary structure of hemoglobin: The hemoglobin tetramer can be envisioned as being composed of two identical dimers, (αβ)1 and (αβ)2, in which the numbers refer to dimers one and two. The two polypeptide chains within each dimer are held tightly together, primarily by hydrophobic interactions

Schematic diagram showing structural changes resulting from oxygenation and deoxygenation of hemoglobin

a. T form: The deoxy form of hemoglobin is called the “T,” or taut (tense) form. In the T form, the two αβ dimers interact through a network of ionic bonds and hydrogen bonds that constrain the movement of the polypeptide chains. The T form is the low oxygen-affinity form of hemoglobin.

b. R form: The binding of oxygen to hemoglobin causes the rupture of some of the ionic bonds and hydrogen bonds between the αβ dimers. This leads to a structure called the “R,” or relaxed form, in which the polypeptide chains have more freedom of movement. The R form is the high oxygen-affinity form of hemoglobin.

Formation of haem-pockets: The Hb molecule and itssubunits contain mostly hydrophobic amino acids internally and hydrophilic amino acids on their surfaces. Thus, the Hb molecule is waxy inside and soapy outside making it soluble in water, but impermeable to water.

Each subunit contains one haem moiety hidden within a waxy pocket of the subunit. The haem pockets of α-subunits are of size just adequate for entry of an O2

molecule, but the entry of O2 into the haem-pockets of the β-subunits is blocked by a valine residue.

Binding of oxygen to myoglobin and hemoglobin

Myoglobin can bind only one molecule of oxygen, because it contains only one heme group.In contrast, hemoglobin can bind four oxygen molecules—one at each of its four heme groups.The degree of saturation (Y) of these oxygen-binding sites on all myoglobin or hemoglobin molecules can vary between zero (all sites are empty) and 100%

1. Oxygen dissociation curve: A plot of Y measured at different partial pressures of oxygen (pO2) is called the oxygen dissociation curve.

2. The curves for myoglobin and hemoglobin show importantdifferences

Oxygen dissociation curves for myoglobin and hemoglobin (Hb).

- This graph illustrates that myoglobinhas a higher oxygen affinity at all pO2 values than does hemoglobin.

-The partial pressure of oxygen needed to achieve half-saturation of the binding sites (P50) is approximately 1 mm Hg for myoglobin and 26 mm Hg for hemoglobin.

- The higher the oxygen affinity (that is, the more tightly oxygen binds), the lower the P50.

Myoglobin (Mb): The oxygen dissociation curve for myoglobin has a hyperbolic shape. This reflects the factthat myoglobin reversibly binds a single molecule of oxygen.

Thus, oxygenated (MbO2) and deoxygenated (Mb) myoglobinexist in a simple equilibrium:

Mb + O2 MbO2

The equilibrium is shifted to the right or to the left as oxygen is added to or removed from the system.

[Note: Myoglobin is designed to bind oxygen released by hemoglobin at the low pO2 found in muscle. Myoglobin, in turn, releases oxygen within the muscle cell in response to oxygen demand.]

Hemoglobin (Hb): The oxygen dissociation curve for hemoglobin is

sigmoidal in shape, indicating that the subunits cooperate in binding

oxygen.

- Cooperative binding of oxygen by the four subunits of hemoglobin

means that the binding of an oxygen molecule at one heme group

increases the oxygen affinity of the remaining heme groups in the

same hemoglobin molecule. This effect is referred to as

heme-heme interaction.

- Although it is more difficult for the first oxygen molecule to bind to

hemoglobin, the subsequent binding of oxygen occurs with high

affinity, as shown by the steep upward curve in the region near 20–

30 mm Hg

Allosteric effects 1. Heme-heme interactions: The sigmoidal oxygen dissociationcurve reflects specific structural changes that are initiated at oneheme group and transmitted to other heme groups in thehemoglobin tetramer.

The net effect is that the affinity ofhemoglobin for the last oxygen bound is approximately 300 timesgreater than its affinity for the first oxygen bound.

Hemoglobin (Hb) binds oxygen with increasing affinity.

Transport of oxygen and carbondioxide by hemoglobin.

1.a. Loading and unloading oxygen: The cooperative binding of oxygen allows hemoglobin to deliver more oxygen to the tissues in response to relatively small changes in the partialpressure of oxygen, which indicates pO2 in the alveoli of the lung and the capillaries of the tissues.

For example, in the lung, the concentration of oxygen is high and hemoglobin becomes virtually saturated (or “loaded”) with oxygen. In contrast, in the peripheral tissues,oxyhemoglobin releases (or “unloads”) much of its oxygen for use in the oxidative metabolism of the tissues

1.b. Significance of the sigmoidal oxygen dissociation curve: The steep slope of the oxygen dissociation curve over the range of oxygen concentrations that occur between the lungs and the tissues permits hemoglobin to carry and deliver oxygen efficiently from sites of high to sites of low pO2.

A molecule with a hyperbolic oxygen dissociation curve, such as myoglobin, could not achieve the same degree of oxygen release within this range of partial pressures of oxygen. Instead, it would have maximum affinity for oxygen throughout this oxygen pressure range and, therefore, would deliver no oxygen to the tissues.

2. Bohr effect:The release of oxygen from hemoglobin is enhanced when the pH is lowered or when the hemoglobin is in the presence of an increased pCO2. - Both result in a decreased oxygen

affinity of hemoglobin and, therefore, a shift to the right in the oxygen dissociation curve, and both, then, stabilize the T state.

- This change in oxygen binding is called the Bohr effect.

- Conversely, raising the pH or lowering the concentration of CO2 results in a greater affinity for oxygen, a shift to the left in the oxygen dissociation curve, and stabilization of the R state.

2.a. Source of the protons that lower the pH: The concentration of both CO2 and H+ in the capillaries of metabolically active tissues is higher than that observed in alveolar capillaries of the lungs, where CO2 is released into the expired air.[Note: Organic acids, such as lactic acid, are produced during anaerobic metabolism in rapidly contracting muscle. In the tissues, CO2 is converted by carbonic anhydrase to carbonic acid: which spontaneously loses a proton, becoming bicarbonate (the major blood buffer):

The H+ produced by this pair of reactions contributes to the lowering of pH. This differential pH gradient (lungs having a higher pH, tissues a lower pH) favors the unloading of oxygen in the peripheral tissues, and the loading of oxygen in the lung.

Thus, the oxygen affinity of the hemoglobin molecule responds to small shifts in pH between the lungs and oxygen-consuming tissues, making hemoglobin a more efficient transporter of oxygen.

2.b. Mechanism of the Bohr effect: The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for protons than does oxyhemoglobin. This effect is caused by ionizable groups, such as specific histidine side chains that have higher pKas in deoxyhemoglobin than in oxyhemoglobin.

Therefore, an increase in the concentration of protons (resulting in a decrease in pH) causes these groups to become protonated (charged) and able to form ionic bonds (also called salt bridges). These bonds preferentially stabilize the deoxy form of hemoglobin, producing a decrease in oxygen affinity

3. Effect of 2,3 bisphosphoglycerate on oxygen affinity:2,3-Bisphosphoglycerate (2,3-BPG) is an important regulator of the binding of oxygen to hemoglobin.

It is the most abundant organicphosphate in the RBC, where its concentration is approximatelythat of hemoglobin.

2,3-BPG is synthesized from an intermediate of the glycolytic pathway

Synthesis of 2,3-bisphosphoglycerate. [Note: is a phosphoryl group. In older literature 2,3-bisphosphoglycerate (2,3-BPG) may be referred to as 2,3-diphosphoglycerate (2,3-DPG).

3.a. Binding of 2,3-BPG to deoxyhemoglobin:2,3-BPG decreases the oxygen affinity of hemoglobin by binding to deoxyhemoglobin but not to oxyhemoglobin.

This preferential binding stabilizes the taut conformation of deoxyhemoglobin. The effect of binding 2,3-BPG can be represented schematicallyas:

3.b. Binding site of 2,3-BPG:One molecule of 2,3-BPG binds to apocket, formed by the two β-globin chains, in the center of the deoxyhemoglobin tetramer.

This pocket contains several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG.

[Note: A mutation of one of these residues can result in hemoglobin variants with abnormally high oxygen affinity.]

2,3-BPG is expelled on oxygenation of the hemoglobin.

Binding of 2,3-BPG by deoxyhemoglobin.

3.c. Shift of the oxygen dissociation curve:Hemoglobin from which 2,3-BPG has been removed has a high affinity for oxygen.

However, as seen in the RBC, the presence of 2,3-BPG significantly reduces the affinity of hemoglobin for oxygen, shifting the oxygen dissociation curve to the right.

This reduced affinity enables hemoglobin to release oxygen efficiently at the partial pressures found in the tissues.

Allosteric effect of 2,3-BPG on theoxygen affinity of hemoglobin.

3.d. Response of 2,3-BPG levels to chronic hypoxia or anemia:

The concentration of 2,3-BPG in the RBC increases inresponse to chronic hypoxia, such as that observed in chronicobstructive pulmonary disease (COPD) like emphysema, or athigh altitudes, where circulating hemoglobin may have difficulty receiving sufficient oxygen.

Intracellular levels of 2,3-BPG are also elevated in chronic anemia, in which fewer than normal RBCs are available to supply the body’s oxygen needs.

Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of oxygen in the capillariesof the tissues

3. e. Role of 2,3-BPG in transfused blood:

2,3-BPG is essential for the normal oxygen transport function of hemoglobin. However, storing blood in the currently available media results in a decrease in 2,3-PBG.

Stored blood displays an abnormally high oxygen affinity, and fails to unload its bound oxygen properly in the tissues.

Hemoglobin deficient in 2,3-BPG thus acts as an oxygen “trap” rather than as an oxygen transport system.

Transfused RBCs are able to restore their depleted supplies of 2,3-BPG in 6–24 hours. However, severely ill patientsmay be compromised if transfused with large quantities ofsuch 2,3-BPG–“stripped” blood.

4. Binding of CO2: Most of the CO2 produced in metabolism is hydrated and transported as bicarbonate ion.However, some CO2 is carried as carbamate bound to the N-terminal amino groups of hemoglobin (forming carbaminohemoglobin, which can be represented schematically as follows:

The binding of CO2 stabilizes the T (taut) or deoxy form ofhemoglobin, resulting in a decrease in its affinity for oxygen and a right shift in the oxygen dissociation.

In the lungs, CO2 dissociates from the hemoglobin, and is released in thebreath.

5. Binding of CO:Carbon monoxide (CO) binds tightly (butreversibly) to the hemoglobin iron, forming carbon monoxyhemoglobin (or carboxyhemoglobin).

When CO binds to one or more of the four heme sites, hemoglobin shifts to the relaxed conformation, causing the remaining heme sites to bind oxygen with high affinity.

This shifts the oxygen dissociation curve to the left, and changes the normal sigmoidal shape toward a hyperbola. As a result, the affected hemoglobin is unable to release oxygen to thetissues

Carbon monoxide poisoning is treated with 100%oxygen at high pressure (hyperbaric oxygen therapy), which facilitates the dissociation of CO from the hemoglobin.

Effect of carbon monoxide on theoxygen affinity of hemoglobin.CO-Hb = carbon monoxyhemoglobin.

ORGANIZATION OF THE GLOBIN GENES

The genes coding for the α-globin-like and β-globin-like subunits of the hemoglobin chains occur in two separate gene clusters (or families) located on two different chromosomes

α-Gene family - The α-gene cluster on chromosome 16 contains two genes for the α-globin chains.- It also contains the ζ gene that is expressed early in development as a component of embryonic hemoblobin.

HEMOGLOBINOPATHIES Hemoglobinopathies, a family of genetic disorders caused by production of a structurally abnormalhemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin, or, rarely, both.

A. Sickle cell anemia (hemoglobin S disease) Sickle cell anemia, the most common of the red cell sickling diseases, is a genetic disorder of the blood caused by a single nucleotide alteration (a point mutation) in the gene for β-globin. - AR

occurs in individuals who have inherited two mutant genes (one from each parent) that code for synthesis of the β chains of the globin molecules.

- An infant does not begin showing symptoms of the diseaseuntil sufficient Hb F has been replaced by Hb S so that sickling can occur

- Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia, and increased susceptibility to infections, usually beginning in early childhood.

1. Amino acid substitution in Hb S β chains: A molecule of Hb Scontains two normal α-globin chains and two mutant β-globinchains (βS), in which glutamate at position six has been replacedwith valine

2. 2. Sickling and tissue anoxia:- The replacement of the charged glutamate with the nonpolar valine

forms a protrusion on the β-globin that fits into a complementary site on the β chain of another hemoglobin molecule in the cell.

- At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a network of fibrous polymers that stiffen and distort the cell, producing rigid, misshapen erythrocytes.

- Such sickled cells frequently block the flow of blood in the narrow capillaries.

- This interruption in the supply of oxygen leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually death (infarction) of cells in the vicinity of the blockage.

B. Hemoglobin C disease

Like Hb S, Hb C is a hemoglobin variant that has a single amino acid substitution in the sixth position of the β-globin chain.

- In this case, however, a lysine is substituted for the

glutamate (as compared with a valine substitution in Hb S).

- Patients homozygous for hemoglobin C generally have a

relatively mild, chronic hemolytic anemia.

- These patients do not suffer from infarctive crises, and no

specific therapy is required.

• Hemoglobin E (a2bE2 , benign). This variant

results from a mutation in the hemoglobin beta chain. People with hemoglobin E disease have a mild hemolytic anemia and mild splenomegaly. Hemoglobin E is common in S.E. Asia.

• Hemoglobin Constant Spring (named after isolation in a Chinese family from the Constant Spring district of Jamaica). (severe). In this variant, a mutation in the alpha globin gene produces an alpha globin chain that is abnormally long. Both the mRNA and the alpha chain protein are unstable

D. MethemoglobinemiasOxidation of the heme component of hemoglobin to the ferric (Fe3+) state forms methemoglobin, which cannot bind oxygen.- This oxidation may be caused by the action of certain

drugs, such as nitrates, or endogenous products, such as reactive oxygen intermediates

- The methemoglobinemias are characterized by “chocolate cyanosis” (a brownish-blue coloration ofthe skin and mucous membranes) and chocolate-colored blood, as a result of the dark-colored methemoglobin.

Thalassemias - The thalassemias are hereditary hemolytic diseases in

which an imbalance occurs in the synthesis of globin chains.

- They are the most common single gene disorders in humans.

- Normally, synthesis of the α- and β-globin chains is coordinated, so that each α-globin chain has a β-globin chain partner.

- In the thalassemias, the synthesis of either the α- orthe β-globin chain is defective.

- caused by a variety of mutations, including entire gene deletions, or substitutions or deletions of one to many nucleotides in the DNA.

1. β-Thalassemias: In these disorders, synthesis of β-globin chains is decreased or

absent, typically as a result of point mutations that affect the production of

functional mRNA;

- α-globin chain synthesis is normal.

- α-Globin chains cannot form stable tetramers and, therefore, precipitate, causing

the premature death of cells initially destined to become mature RBCs.

- There are only two copies of the β-globin gene in each cell (one on each

chromosome 11).

- Therefore, individuals with β-globin gene defects have either β-thalassemia trait

(β-thalassemia minor) if they have only one defective β-globin gene

- β-thalassemia major (Cooley anemia) if both genes are defective

2. α-Thalassemias: These are defects in which the synthesis ofα-globin chains is decreased or absent, typically as a result ofdeletional mutations. Because each individual’s genome containsfour copies of the α-globin gene (two on each chromosome 16),there are several levels of α-globin chain deficiencies

- If one of the four genes is defective, the individual istermed a silent carrier of α-thalassemia, because no physicalmanifestations of the disease occur.

- If two α-globin genes are defective, the individual is designated as having α-thalassemia trait.

- If three α-globin genes are defective, the individual has HbH (β4) disease—a mildly to moderately severe hemolytic anemia.

- If all four α-globin genes are defective, Hb Bart (γ4) disease with hydrops fetalis and fetal death results, because α-globin chains are required for the synthesis of Hb F

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