hemoglobina y mioglobina

Harvey F

errier

Lippincott’s Illustrated Reviews

LWW.com

5th edition


Richard HarveyDenise Ferrier

Series Editor:

Richard A. Harvey

5th e

dition

Series Editor:

Biochemistry—USMLE Review

Figure 3.20Molecular and cellular events leading to sickle cell crisis.

Biochemistry 5th edition

Richard A. HarveyDenise R. Ferrier


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Lippincott’s Illustrated Reviews—Seeing is Understanding

I. OVERVIEW

The previous chapter described the types of secondary and tertiarystructures that are the bricks-and-mortar of protein architecture. Byarranging these fundamental structural elements in different combina-tions, widely diverse proteins can be constructed that are capable ofvarious specialized functions. This chapter examines the relationshipbetween structure and function for the clinically important globularhemeproteins. Fibrous structural proteins are discussed in Chapter 4.

II. GLOBULAR HEMEPROTEINS

Hemeproteins are a group of specialized proteins that contain heme asa tightly bound prosthetic group. (See p. 54 for a discussion of pros-thetic groups.) The role of the heme group is dictated by the environ-ment created by the three-dimensional structure of the protein. Forexample, the heme group of a cytochrome functions as an electron car-rier that is alternately oxidized and reduced (see p. 76). In contrast, theheme group of the enzyme catalase is part of the active site of theenzyme that catalyzes the breakdown of hydrogen peroxide (seep. 148). In hemo globin and myoglobin, the two most abundant heme -proteins in humans, the heme group serves to reversibly bind oxygen.

A. Structure of heme

Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+)(Figure 3.1). The iron is held in the center of the heme molecule bybonds to the four nitrogens of the porphyrin ring. The heme Fe2+

can form two additional bonds, one on each side of the planar por-phyrin ring. In myo globin and hemoglobin, one of these positions iscoordinated to the side chain of a histidine residue of the globinmolecule, whereas the other position is available to bind oxygen(Figure 3.2). (See p. 278 for a discussion of the synthesis anddegradation of heme.)

25

GlobularProteins 3

Figure 3.1A. Hemeprotein (cytochrome c). B. Structure of heme.

COO-

CH3CH

CH

C

NC

NC N C

N

CC

CC

CC

CC

C

HC

CC

CH3C

CH3

CH CH2

CH3

CH

H2C

CH2

CH2CH2

CH2

COO-

Fe

C

COCOCOCOCOOOOOO-

CH3CH

NNNNNNNNNNNNNCC NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN CC

CCCC

CCCCCCC

C

HC

CH3C

CH2

CH2CHCHHHH222

CHCHHH2222

COOOOOOOOO-

FFeFeFeFeFeFe

Iron can form six bonds:four with porphyrin nitrogens, plus two additional bonds, one above and one below the planar porphyrin ring

A

B

168397_P025-042.qxd7.0:03 Hemoglobin 5-20-04 2010.4.4 1:04 PM 5

B. 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 carrierthat increases the rate of transport of oxygen within the muscle cell.Myoglobin consists of a single polypeptide chain that is structurallysimilar to the individual subunit polypeptide chains of the hemo -globin 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 approxi-mately 80% of its polypeptide chain folded into eight stretches ofα-helix. These α-helical regions, labeled A to H in Figure 3.2A,are terminated either by the presence of proline, whose five-mem-bered ring cannot be accommodated in an α-helix (see p. 16), orby β-bends and loops stabilized by hydrogen bonds and ionicbonds (see p. 17).

2. Location of polar and nonpolar amino acid residues: The interiorof the myoglobin molecule is composed almost entirely of nonpo-lar amino acids. They are packed closely together, forming astructure stabilized by hydrophobic interactions between theseclustered residues (see p. 19). In contrast, charged amino acidsare located almost exclusively on the surface of the molecule,where they can form hydrogen bonds, both with each other andwith water.

3. Binding of the heme group: The heme group of myoglobin sits ina crevice in the molecule, which is lined with nonpolar aminoacids. Notable exceptions are two histidine residues (Figure3.2B). One, the proximal histidine (F8), binds directly to the iron ofheme. The second, or distal histidine (E7), does not directly inter-act with the heme group, but helps stabilize the binding of oxygento the ferrous iron. The protein, or globin, portion of myoglobinthus creates a special microenvironment for the heme that permits the reversible binding of one oxygen molecule (oxygena-tion). The simultaneous loss of electrons by the ferrous iron (oxi-dation) occurs only rarely.

26 3. Globular Proteins

Figure 3.2A. Model of myoglobin showing helices A to H. B. Schematic diagram of the oxygen-binding site of myoglobin.

A B

Heme

CB

A

E

F

G

H

D

Oxygenmolecule(O2)

Heme

F Helix E Helix

Proximalhistidine(F8)

Distalhistidine(E7)

Fe

168397_P025-042.qxd7.0:03 Hemoglobin 5-20-04 2010.4.4 1:04 PM Page 26

C. Structure and function of hemoglobin

Hemoglobin is found exclusively in red blood cells (RBCs), where itsmain function is to transport oxygen (O2) from the lungs to the capil-laries of the tissues. Hemoglobin A, the major hemoglobin in adults,is composed of four polypeptide chains—two α chains and twoβ chains—held together by noncovalent interactions (Figure 3.3).Each subunit has stretches of α-helical structure, and a heme-bind-ing pocket similar to that described for myoglobin. However, thetetrameric hemoglobin molecule is structurally and functionally morecomplex 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 interac-tion with allosteric effectors (see p. 29).

Obtaining O2 from the atmosphere solely by dif-fusion greatly limits the size of organisms.Circulatory systems overcome this, but transportmolecules such as hemoglobin are also requiredbecause O2 is only slightly soluble in aqueoussolutions such as blood.

1. Quaternary structure of hemoglobin: The hemoglobin tetramercan be envisioned as being composed of two identical dimers,(αβ)1 and (αβ)2, in which the numbers refer to dimers one andtwo. The two polypeptide chains within each dimer are held tightlytogether, primarily by hydrophobic interactions (Figure 3.4). [Note:In this instance, hydrophobic amino acid residues are localizednot only in the interior of the molecule, but also in a region on thesurface of each subunit. Interchain hydrophobic interactions formstrong associations between α-subunits and β-subunits in the

II. Globular Hemeproteins 27

Figure 3.3A. Structure of hemoglobin showing the polypeptide backbone. B. Simplified drawing showing the helices.

BA β2

α2 α1

β1


dimers.] Ionic and hydrogen bonds also occur between the mem-bers of the dimer. In contrast, the two dimers are able to movewith respect to each other, being held together primarily by polarbonds. The weaker interactions between these mobile dimersresult in the two dimers occupying different relative positions indeoxyhemoglobin as compared with oxyhemoglobin (see Figure3.4). [Note: The binding of O2 to the heme iron pulls the iron intothe plane of the heme. Because the iron is also linked to the proxi-mal histidine (F8), there is movement of the globin chains thatalters the interface between the αβ dimers.]

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 througha network of ionic bonds and hydrogen bonds that constrainthe 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 rup-ture of some of the ionic bonds and hydrogen bonds betweenthe αβ dimers. This leads to a structure called the “R,” orrelaxed form, in which the polypeptide chains have more freedom of movement (see Figure 3.4). The R form is the high-oxygen-affinity form of hemoglobin.

D. Binding of oxygen to myoglobin and hemoglobin

Myoglobin can bind only one molecule of oxygen, because it con-tains only one heme group. In contrast, hemoglobin can bind fouroxygen molecules —one at each of its four heme groups. Thedegree of saturation (Y) of these oxygen-binding sites on all myo-globin or hemoglobin molecules can vary between zero (all sites areempty) and 100% (all sites are full, Figure 3.5).


O2

O2O2

Figure 3.4Schematic diagram showing structural changes resulting from oxygenation and deoxygenation of hemoglobin. .

Weak ionic and hydrogen bonds occurbetween αβ dimer pairs

Some ionic and hydrogen bonds between αβ dimers are broken in the oxygenated state.

"R," or relaxed, structure of oxyhemoglobin"T," or taut, structure of deoxyhemoglobin

αβ

αβ αβ dimer 1

αβ dimer 2

Strong interactions,primarily hydrophobic, between α and β chains form stable αβ dimers.


Figure 3.5Oxygen dissociation curves formyoglobin and hemoglobin (Hb).

00

40 80 120

P50 = 1 P50 = 26

The oxygen dissociation curve for Hb is steepest at the oxygen concentrations that occur in the tissues. This permits oxygen delivery to respond to small changes in pO2.

Partial pressure of oxygen (pO2)(mm Hg)

Figure 3.6Hemoglobin (Hb) binds oxygen with increasing affinity.

O2

O2

O2

O2

Hb

Hb

Hb

Hb

Hb

O2 O2

O2 O2

O2

O2

O2 O2

O2

O2Increasingaffinity

forO2

1. Oxygen dissociation curve: A plot of Y measured at different par-tial pressures of oxygen (pO2) is called the oxygen dissociationcurve. The curves for myoglobin and hemoglobin show importantdifferences (see Figure 3.5). This graph illustrates that myoglobinhas a higher oxygen affinity at all pO2 values than doeshemoglobin. The partial pressure of oxygen needed to achievehalf-saturation of the binding sites (P50) is approximately 1 mm Hgfor myoglobin and 26 mm Hg for hemoglobin. The higher the oxygen affinity (that is, the more tightly oxygen binds), the lowerthe P50. [Note: pO2 may also be represented as PO2.]

a. Myoglobin (Mb): The oxygen dissociation curve for myoglobinhas a hyperbolic shape (see Figure 3.5). 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 isadded to or removed from the system. [Note: Myoglobin isdesigned to bind oxygen released by hemoglobin at the lowpO2 found in muscle. Myoglobin, in turn, releases oxygenwithin the muscle cell in response to oxygen demand.]

b. Hemoglobin (Hb): The oxygen dissociation curve for hemo -globin is sigmoidal in shape (see Figure 3.5), indicating thatthe subunits cooperate in binding oxygen. Cooperative bindingof oxygen by the four subunits of hemoglobin means that thebinding of an oxygen molecule at one heme group increasesthe oxygen affinity of the remaining heme groups in the samehemoglobin molecule (Figure 3.6). This effect is referred to asheme-heme interaction (see below). Although it is more diffi-cult for the first oxygen molecule to bind to hemoglobin, thesubsequent binding of oxygen occurs with high affinity, asshown by the steep upward curve in the region near 20–30mm Hg (see Figure 3.5).

E. Allosteric effects

The ability of hemoglobin to reversibly bind oxygen is affected by thepO2 (through heme-heme interactions as described above), the pHof the environment, the partial pressure of carbon dioxide, pCO2,and the availability of 2,3-bisphosphoglycerate. These are collec-tively called allosteric (“other site”) effectors, because their interac-tion at one site on the hemoglobin molecule affects the binding ofoxygen to heme groups at other locations on the molecule. [Note:The binding of oxygen to myoglobin is not influenced by allostericeffectors.]

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.

→←



a. Loading and unloading oxygen: The cooperative binding ofoxygen allows hemoglobin to deliver more oxygen to the tissues in response to relatively small changes in the partialpressure of oxygen. This can be seen in Figure 3.5, which indi-cates pO2 in the alveoli of the lung and the capillaries of thetissues. For example, in the lung, the concentration of oxygenis high and hemoglobin becomes vir tually saturated (or“loaded”) with oxygen. In contrast, in the peripheral tissues,oxyhemoglobin releases (or “unloads”) much of its oxygen foruse in the oxidative metabolism of the tissues (Figure 3.7).

b. Significance of the sigmoidal oxygen dissociation curve: Thesteep slope of the oxygen dissociation curve over the range ofoxygen concentrations that occur between the lungs and thetissues permits hemoglobin to carry and deliver oxygen effi-ciently from sites of high to sites of low pO2. A molecule with ahyperbolic oxygen dissociation curve, such as myoglobin, couldnot achieve the same degree of oxygen release within thisrange of partial pressures of oxygen. Instead, it would havemaximum affinity for oxygen throughout this oxygen pressurerange and, therefore, would deliver no oxygen to the tissues.

2. Bohr effect: The release of oxygen from hemoglobin is enhancedwhen the pH is lowered or when the hemoglobin is in the pres-ence of an increased pCO2. Both result in a decreased oxygenaffinity of hemoglobin and, therefore, a shift to the right in the oxy-gen dissociation curve (Figure 3.8), and both, then, stabilize the Tstate. 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 oxy-gen dissociation curve, and stabilization of the R state.

a. Source of the protons that lower the pH: The concentration ofboth CO2 and H+ in the capillaries of metabolically active tis-sues is higher than that observed in alveolar capillaries of thelungs, where CO2 is released into the expired air. [Note:Organic acids, such as lactic acid, are produced during anaer-obic metabolism in rapidly contracting muscle (see p. 103).] Inthe tissues, CO2 is converted by carbonic anhydrase to carbonic acid:

CO2 + H2O H2CO3

which spontaneously loses a proton, becoming bicarbonate(the major blood buffer):

H2CO3 HCO3– + H+

The H+ produced by this pair of reactions contributes to the low-ering of pH. This differential pH gradient (lungs having a higherpH, tissues a lower pH) favors the unloading of oxygen in theperipheral tissues, and the loading of oxygen in the lung. Thus,the oxygen affinity of the hemoglobin molecule responds tosmall shifts in pH between the lungs and oxygen-consuming tis-sues, making hemoglobin a more efficient transporter of oxygen.

→←

→←


Figure 3.8Effect of pH on the oxygen affinity of hemoglobin. Protons are allostericeffectors of hemoglobin.


00

50

Decrease in pH results in decreased oxygen affinity of hemoglobin and, therefore, a shift to the right in the oxygen dissociation curve.

At lower pH, agreater pO2 isrequired to achieve any given oxygensaturation.

Figure 3.7Transport of oxygen and carbondioxide by hemoglobin.

Fe2+ Fe2+

Fe2+ Fe2+

O2

O2

O2

O2

Oxyhemoglobin

Fe2+ Fe2+

Fe2+ Fe2+

NHCOO–

NHCOO–

Carbaminohemoglobin

CO2 binds tohemoglobin

O2 is released from hemoglobin

O2 binds to hemoglobin

CO2 is releasedfrom hemoglobin

O2CO2

TISSUES

LUNGS

O2CO2


b. Mechanism of the Bohr effect: The Bohr effect reflects the factthat the deoxy form of hemoglobin has a greater affinity forprotons than does oxyhemoglobin. This effect is caused byionizable groups, such as specific histidine side chains thathave higher pKas in deoxy hemoglobin than in oxyhemoglobin.There fore, an increase in the concentration of protons (result-ing in a decrease in pH) causes these groups to become pro-tonated (charged) and able to form ionic bonds (also calledsalt bridges). These bonds preferentially stabilize the deoxyform of hemoglobin, producing a decrease in oxygen affinity.

The Bohr effect can be represented schematically as:

HbO2 + H+ HbH + O2

oxyhemoglobin deoxyhemoglobin

where an increase in protons (or a lower pO2) shifts the equi-librium to the right (favoring deoxyhemoglobin), whereas anincrease in pO2 (or a decrease in protons) shifts the equilib-rium to the left.

3. Effect of 2,3-bisphosphoglycerate on oxygen affinity: 2,3-Bis -phospho glycerate (2,3-BPG) is an important regulator of the bind-ing 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 intermediateof the glycolytic pathway (Figure 3.9; see p. 101 for a discussionof 2,3-BPG synthesis in glycolysis).

a. Binding of 2,3-BPG to deoxyhemoglobin: 2,3-BPG decreasesthe oxygen affinity of hemoglobin by binding to deoxy -hemoglobin but not to oxyhemoglobin. This preferential bindingstabilizes the taut conformation of deoxyhemoglobin. Theeffect of binding 2,3-BPG can be represented schematicallyas:

HbO2 + 2,3-BPG Hb–2,3-BPG + O2

oxyhemoglobin deoxyhemoglobin

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 thedeoxyhemoglobin tetramer (Figure 3.10). This pocket containsseveral positively charged amino acids that form ionic bondswith the negatively charged phosphate groups of 2,3-BPG.[Note: A mutation of one of these residues can result inhemoglobin variants with abnormally high oxygen affinity.]2,3-BPG is expelled on oxygenation of the hemoglobin.

c. Shift of the oxygen dissociation curve: Hemoglobin fromwhich 2,3-BPG has been removed has a high affinity for oxy-gen. However, as seen in the RBC, the presence of 2,3-BPGsignificantly reduces the affinity of hemoglobin for oxygen, shift-ing the oxygen dissociation curve to the right (Figure 3.11). Thisreduced affinity enables hemoglobin to release oxygen effi-ciently at the partial pressures found in the tissues.

→←

→←


Figure 3.9Synthesis of 2,3-bisphospho-glycerate. [Note: is a phosphorylgroup.] In older literature 2,3-bis-phosphoglycerate (2,3-BPG) may be referred to as 2,3-diphospho-glycerate (2,3-DPG).

2,3-Bisphospho-glycerate

H

CH OCH OCO

O–

PP

1,3-Bisphospho-glycerate

Glucose

3-Phospho-glycerate

Pyruvate

H2O

PO42–

Glycolysis

Lactate

P

––

––

Figure 3.10Binding of 2,3-BPG by deoxy-hemoglobin.

β2 α2

A single molecule of 2,3-BPG binds to a positively chargedcavity formed by the β-chains of deoxyhemoglobin.

α1


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 diffi-culty receiving sufficient oxygen. Intracellular levels of 2,3-BPGare also elevated in chronic anemia, in which fewer than nor-mal RBCs are available to supply the body’s oxygen needs.Elevated 2,3-BPG levels lower the oxygen affinity of hemo -globin, permitting greater unloading of oxygen in the capillariesof the tissues (see Figure 3.11).

e. Role of 2,3-BPG in transfused blood: 2,3-BPG is essential forthe normal oxygen transport function of hemoglobin. However,storing blood in the currently available media results in adecrease in 2,3-PBG. Stored blood displays an abnormallyhigh oxygen affinity, and fails to unload its bound oxygen prop-erly in the tissues. Hemoglobin deficient in 2,3-BPG thus actsas an oxygen “trap” rather than as an oxygen transport sys-tem. Transfused RBCs are able to restore their depleted sup-plies 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. [Note: The maximum storagetime for red cells has been doubled (21 to 42 days, withmedian time of 15 days) by changes in H+, phosphate andhexose sugar concentration, and by the addition of adenine(see p. 291). Although the content of 2,3-BPG was not greatlyaffected by these changes, ATP production was increased andimproved RBC survival.]

4. Binding of CO2: Most of the CO2 produced in metabolism ishydrated and transported as bicarbonate ion (see p. 9). However,some CO2 is carried as carbamate bound to the N-terminal aminogroups of hemoglobin (forming carbaminohemoglobin, see Figure3.7), which can be represented schematically as follows:

Hb – NH2 + CO2 Hb – NH – COO– + H+

The binding of CO2 stabilizes the T (taut) or deoxy form ofhemoglobin, resulting in a decrease in its affinity for oxygen (seep. 28) 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 monoxy hemo -globin (or carboxyhemoglobin). When CO binds to one or more ofthe four heme sites, hemoglobin shifts to the relaxed conforma-tion, causing the remaining heme sites to bind oxygen with highaffinity. This shifts the oxygen dissociation curve to the left, andchanges the normal sigmoidal shape toward a hyperbola. As aresult, the affected hemoglobin is unable to release oxygen to thetissues (Figure 3.12). [Note: The affinity of hemoglobin for CO is220 times greater than for oxygen. Consequently, even minuteconcentrations of CO in the environment can produce toxic con-centrations of carbon monoxyhemoglobin in the blood. For exam-

→←

32 3. Globular Proteins%

Sat

ura

tio

n w

ith

O2

(Y)

Figure 3.11Allosteric effect of 2,3-BPG on the oxygen affinity of hemoglobin.

Partial pressure of oxygen(mm Hg)

00

40 80 120

100

2,3-BPG = 8 mmol/L (Blood from individual

adapted to high altitudes)

2,3-BPG = 0(Hemoglobin

stripped of 2,3-BPG)

2,3-BPG = 5 mmol/L(Normal blood)

Figure 3.12Effect of carbon monoxide on the oxygen affinity of hemoglobin. CO-Hb = carbon monoxy-hemoglobin.

O2

Co

nte

nt

(ml/1

00 m

l blo

od

)


00

40 80 120

20

Fifty percent CO-Hb

Zero percent CO-Hb

10


ple, increased levels of CO are found in the blood of tobaccosmokers. Carbon monoxide toxicity appears to result from a com-bination of tissue hypoxia and direct CO-mediated damage at thecellular level.] Carbon monoxide poisoning is treated with 100%oxygen at high pressure (hyperbaric oxygen therapy), which facili-tates the dissociation of CO from the hemoglobin. [Note: COinhibits Complex IV of the electron transport chain (see p. 76).] Inaddition to O2, CO2, and CO, nitric oxide gas (NO) also is carried byhemoglobin. NO is a potent vasodilator (see p. 151). It can be takenup (salvaged) or released from RBCs, thus modulating NO availabil-ity and influencing vessel diameter.

F. Minor hemoglobins

It is important to remember that human hemoglobin A (Hb A) is justone member of a functionally and structurally related family of pro-teins, the hemoglobins (Figure 3.13). Each of these oxygen-carryingproteins is a tetramer, composed of two α-globin polypeptides andtwo β-globin (or β-globin-like) polypeptides. Certain hemoglobins,such as Hb F, are normally synthesized only during fetal develop-ment, whereas others, such as Hb A2, are synthesized in the adult,although at low levels compared with Hb A. Hb A can also becomemodified by the covalent addition of a hexose. For example, additionof glucose forms the glucosylated hemoglobin derivative, Hb A1c.

1. Fetal hemoglobin (Hb F): Hb F is a tetramer consisting of twoα chains identical to those found in Hb A, plus two γ chains (α2γ2,see Figure 3.13). The γ chains are members of the β-globin genefamily (see p. 35).

a. Hb F synthesis during development: In the first month afterconception, embryonic hemoglobins such as Hb Gower 1,composed of two α-like zeta (ζ) chains and two β-like epsilon(ε) chains (ζ2ε2), are synthesized by the embryonic yolk sac. Inthe fifth week of gestation, the site of globin synthesis shifts,first to the liver and then to the marrow, and the primary prod-uct is Hb F. Hb F is the major hemoglobin found in the fetusand newborn, accounting for about 60% of the totalhemoglobin in the erythrocytes during the last months of fetallife (Figure 3.14). Hb A synthesis starts in the bone marrow atabout the eighth month of pregnancy and gradually replacesHb F. (Figure 3.14 shows the relative production of each typeof hemoglobin chain during fetal and postnatal life.) [Note: HbF represents less than 1% of the Hb in most adults, and isconcentrated in RBCs known as F-cells.]

b. Binding of 2,3-BPG to Hb F: Under physiologic conditions, HbF has a higher affinity for oxygen than does Hb A, as a resultof Hb F binding only weakly to 2,3-BPG. [Note: The γ-globinchains of Hb F lack some of the positively charged aminoacids that are responsible for binding 2,3-BPG in the β-globinchains.] Because 2,3-BPG serves to reduce the affinity ofhemoglobin for oxygen, the weaker interaction between2,3-BPG and Hb F results in a higher oxygen affinity for Hb Frelative to Hb A. In contrast, if both Hb A and Hb F are strippedof their 2,3-BPG, they then have a similar affinity for oxygen.


Figure 3.13Normal adult human hemoglobins.[Note: The α-chains in these hemo-globins are identical.]

HbA α2β2

FormChain

compositionFraction of

total hemoglobin

HbA1c α2β2-glucose

90%

3–9%

α2γ2HbF <2%

HbA2 α2δ2 2–5%

Figure 3.14Developmental changes in hemo-globin.

Months before and after birth

Per

cen

tag

e o

f to

tal g

lob

in c

hai

ns

-9 -6 -3 3 6 90

25

50

0

25

50

α

β

δ

ε

γ

ζ

α-Globin-like chains

β-Globin-like chains

Time of birth

0


The higher oxygen affinity of Hb F facilitates the transfer ofoxygen from the maternal circulation across the placenta tothe RBCs of the fetus.

2. Hemoglobin A2 (Hb A2): Hb A2 is a minor component of normaladult hemoglobin, first appearing shortly before birth and, ulti-mately, constituting about 2% of the total hemoglobin. It is com-posed of two α-globin chains and two δ-globin chains (α2δ2, seeFigure 3.13).

3. Hemoglobin A1c (HbA1c): Under physiologic conditions, Hb A isslowly and nonenzymically glycosylated, the extent of glycosyla-tion being dependent on the plasma concentration of a particularhexose. The most abundant form of glycosylated hemoglobin is HbA1c. It has glucose residues attached predominantly to the NH2

groups of the N-terminal valines of the β-globin chains (Figure3.15). Increased amounts of Hb A1c are found in RBCs of patientswith diabetes mellitus, because their Hb A has contact with higherglucose concentrations during the 120-day lifetime of these cells.(See p. 340 for a discussion of the use of this phenomenon inassessing average blood glucose levels in persons with diabetes.)

III. ORGANIZATION OF THE GLOBIN GENES

To understand diseases resulting from genetic alterations in the structureor synthesis of hemoglobins, it is necessary to grasp how the hemoglobingenes, which direct the synthesis of the different globin chains, are struc-turally organized into gene families and also how they are expressed.

A. α-Gene family

The genes coding for the α-globin-like and β-globin-like subunits ofthe hemoglobin chains occur in two separate gene clusters (or fami-lies) located on two different chromosomes (Figure 3.16). Theα-gene cluster on chromosome 16 contains two genes for theα-globin chains. It also contains the ζ gene that is expressed earlyin development as a component of embryonic hemoblobin. [Note:Globin gene famillies also contain globin-like genes that are notexpressed (that is, their genetic information is not used to produceglobin chains). These are called pseudogenes.]


Figure 3.16Organization of the globin gene families.

ζ α1α2

ε δGγ β

Hb Gower 1 Hb AHb F Hb A2α2β2α2δ2α2γ2ζ2ε2

Hemoglobins are formedby combinations ofα-globin-like chains and β-globin-like chains

α-Globin-like genes(Chromosome 16)

β-Globin-like genes(Chromosome 11) Aγ

Two copies of the α-globingene are designated α1 and α2. Each can provide α-globin chains that combinewith β-globin chains.

Figure 3.15Nonenzymic addition of glucoseto hemoglobin.

CH2OH

HCOH

HCH

HCOH

HOCH

CO

NH NH

NH2 NH2

CH2OH

HCOH

HCO

HCOH

HOCH

HCOH

CH2OH

HCOH

HCH

HCOH

HOCH

CO

Glucose

Hemoglobin A1c

Hemoglobin A


B. β-Gene family

A single gene for the β-globin chain is located on chromosome 11(see Figure 3.16). There are an additional four β-globin-like genes:the ε gene (which, like the ζ gene, is expressed early in embryonicdevelopment), two γ genes (Gγ and Aγ that are expressed in Hb F),and the δ gene that codes for the globin chain found in the minoradult hemoglobin Hb A2.

C. Steps in globin chain synthesis

Expression of a globin gene begins in the nucleus of red cell precur-sors, where the DNA sequence encoding the gene is transcribed.The RNA produced by transcription is actually a precursor of themessenger RNA (mRNA) that is used as a template for the synthe-sis of a globin chain. Before it can serve this function, two noncodingstretches of RNA (introns) must be removed from the mRNA precur-sor sequence, and the remaining three fragments (exons) joined ina linear manner. The resulting mature mRNA enters the cytosol,where its genetic information is translated, producing a globin chain.(A summary of this process is shown in Figure 3.17. A more detaileddescription of protein synthesis is presented in Chapter 31, p. 431.)

IV. HEMOGLOBINOPATHIES

Hemoglobinopathies have traditionally been defined as a family ofgenetic disorders caused by production of a structurally abnormalhemoglobin molecule, synthesis of insufficient quantities of normalhemoglobin, or, rarely, both. Sickle cell anemia (Hb S), hemoglobin Cdisease (Hb C), hemoglobin SC disease (Hb S + Hb C), and the tha-lassemia syndromes are representative hemoglobinopathies that canhave severe clinical consequences. The first three conditions result fromproduction of hemoglobin with an altered amino acid sequence (qualita-tive hemoglobinopathy), whereas the thal assemias are caused bydecreased production of normal hemoglobin (quantitative hemo -globinopathy).

A. Sickle cell anemia (hemoglobin S disease)

Sickle cell anemia, the most common of the red cell sickling dis-eases, is a genetic disorder of the blood caused by a singlenucleotide alteration (a point mutation) in the gene for β-globin. It isthe most common inherited blood disorder in the United States,affecting 80,000 Americans. It occurs primarily in the African-American population, affecting one of 500 newborn African-Americaninfants in the United States. Sickle cell anemia is a homozygous,recessive disorder. It occurs in individuals who have inherited twomutant genes (one from each parent) that code for synthesis of theβ chains of the globin molecules. [Note: The mutant β-globin chain isdesignated βS, and the resulting hemoglobin, α2βS

2, is referred to asHb S.] An infant does not begin showing symptoms of the diseaseuntil sufficient Hb F has been replaced by Hb S so that sickling canoccur (see below). Sickle cell anemia is characterized by lifelongepisodes of pain (“crises”), chronic hemolytic anemia with associatedhyperbilirubinemia (see p. 284), and increased susceptibility to infec-tions, usually beginning in early childhood. [Note: The lifetime of an

IV. Hemoglobinopathies 35

Figure 3.17Synthesis of globin chains.

5'

Transcription

Splicing

Translation

mRNAprecursor

NUCLEUS

CYTOSOL

Hemoglobin

mRNA

Exon 1 Exon 2 Exon 3

Intron 2

α-Globin and β-globin gene families contain three exons (coding regions) separated by two noncoding introns.

Intron 1


erythrocyte in sickle cell anemia is less than 20 days, compared with120 days for normal RBCs; hence, the anemia.] Other symptomsinclude acute chest syndrome, stroke, splenic and renal dysfunction,and bone changes due to marrow hyperplasia. Heterozygotes, repre-senting 1 in 12 African-Americans, have one normal and one sicklecell gene. The blood cells of such heterozygotes contain both Hb Sand Hb A. These individuals have sickle cell trait. They usually do notshow clinical symptoms and can have a normal life span.

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 (Figure 3.18). Therefore, during electrophoresis atalkaline pH, Hb S migrates more slowly toward the anode (posi-tive electrode) than does Hb A (Figure 3.19). This altered mobilityof Hb S is a result of the absence of the negatively charged glutamate residues in the two β chains, thus rendering Hb S lessnegative than Hb A. [Note: Electrophoresis of hemoglobinobtained from lysed RBCs is routinely used in the diagnosis ofsickle cell trait and sickle cell disease.]

2. Sickling and tissue anoxia: The replacement of the charged gluta-mate with the nonpolar valine forms a protrusion on the β-globinthat fits into a complementary site on the β chain of anotherhemoglobin molecule in the cell (Figure 3.20). At low oxygen ten-sion, deoxyhemoglobin S polymerizes inside the RBC, forming anetwork of fibrous polymers that stiffen and distort the cell, produc-ing rigid, misshapen erythrocytes. Such sickled cells frequentlyblock the flow of blood in the narrow capillaries. This interruption inthe supply of oxygen leads to localized anoxia (oxygen deprivation)in the tissue, causing pain and eventually death (infarction) of cellsin the vicinity of the blockage. The anoxia also leads to an increasein deoxygenated Hb S. [Note: The mean diameter of RBCs is 7.5μm, whereas that of the microvasculature is 3–4 μm. Instead ofsqueezing through the microvasculature like Hb A–containingRBCs, sickled cells have a decreased ability to deform and anincreased tendency to adhere to vessel walls, and so have diffi-culty moving through small vessels.]

3. Variables that increase sickling: The extent of sickling and, there-fore, the severity of disease is enhanced by any variable thatincreases the proportion of Hb S in the deoxy state (that is,reduces the affinity of Hb S for oxygen). These variables includedecreased oxygen tension as a result of high altitudes or flying in anonpressurized plane, increased pCO2, decreased pH, dehydra-tion, and an increased concentration of 2,3-BPG in erythrocytes.

4. Treatment: Therapy involves adequate hydration, analgesics,aggressive antibiotic therapy if infection is present, and trans -fusions in patients at high risk for fatal occlusion of blood vessels.Intermittent transfusions with packed red cells reduce the risk ofstroke, but the benefits must be weighed against the complicationsof transfusion, which include iron overload (hemosiderosis), blood-borne infections, and immunologic complications. Hydroxyurea, anantitumor drug, is therapeutically useful because it increases circu-lating levels of Hb F, which decreases RBC sickling. This leads todecreased frequency of painful crises and reduces mortality.


Figure 3.18Amino acid substitutions in Hb S and Hb C.

Val . His . Leu . Thr . Pro . Glu . Glu . Lys

1 2 3 4 5 6 7 8

C C

H

O

COO-

Hb A

Hb S

Val . His . Leu . Thr . Pro . Val . Glu . Lys

CH2

CH2

1 2 3 4 5 6 7 8

Val . His . Leu . Thr . Pro . Lys . Glu . Lys

C C

O

H

Hb C

CH2CH2CH2CH2NH3

+

1 2 3 4 5 6 7 8

CHN C

OCHCH3H3C

H

HN

HN

Anode

Cathode

Figure 3.19Diagram of hemoglobins A, S, and C after electrophoresis.

Power source

at thestart ofelectro- Hemoglobins are negatively

charged and migrate towardthe anode.

Hb S

Hb C

Hb A


Figure 3.20Molecular and cellular events leading to sickle cell crisis.

3 Intracellular fibers of Hb S distort the erythrocyte.

1Hydrophobic pocket

Fiber

Fibers

β-6-Valineβ Chain

A point mutationin the DNA codes for structurally altered Hb S.

2 In the deoxygenated state, Hb S polymerizesinto long, rope-like fibers.

...GTG...

...GAG...

Val.His.Leu.Thr.Pro.Glu.Glu.Lys

Val.His.Leu.Thr.Pro.Val.Glu.Lys

Microinfarcts produce tissue anoxia, resulting in severe pain.

5

Rigid erythrocytes occlude blood flow in the capillaries.

4

α1

α2

β1

β2

α1

α2

β1

β2

α1

α2

β1

β2

α1

α2

β1

β2

IV. Hemoglobinopathies 37

5. Possible selective advantage of the heterozygous state: The highfrequency of the βS mutation among black Africans, despite itsdamaging effects in the homozygous state, suggests that a selec-tive advantage exists for heterozygous individuals. For example,heterozygotes for the sickle cell gene are less susceptible tomalaria, caused by the parasite Plasmodium falciparum. Thisorganism spends an obligatory part of its life cycle in the RBC.One theory is that because these cells in individuals heterozy-gous for Hb S, like those in homozygotes, have a shorter life spanthan normal, the parasite cannot complete the intracellular stageof its development. This fact may provide a selective advantage tohetero zygotes living in regions where malaria is a major cause ofdeath. Figure 3.21 illustrates that in Africa, the geographic distri-bution of sickle cell anemia is similar to that of malaria.The mor-bidity and mortality associated with sickle cell anemia has led toits inclusion in newborn screening panels to allow prophylacticantibiotic therapy to begin soon after the birth of an affected child.

B. Hemoglobin C disease

Like Hb S, Hb C is a hemoglobin variant that has a single aminoacid substitution in the sixth position of the β-globin chain (seeFigure 3.18). In this case, however, a lysine is substituted for the glutamate (as compared with a valine substitution in Hb S). [Note:This substitution causes Hb C to move more slowly toward theanode than Hb A or Hb S does (see Figure 3.19).] Patients homo -zygous for hemoglobin C generally have a relatively mild, chronichemolytic anemia. These patients do not suffer from infarctivecrises, and no specific therapy is required.

C. Hemoglobin SC disease

Hemoglobin SC disease is another of the red cell sickling diseases.In this disease, some β-globin chains have the sickle cell mutation,whereas other β-globin chains carry the mutation found in Hb C disease. [Note: Patients with Hb SC disease are doubly hetero -



Figure 3.21A. Distribution of sickle cell in Africa expressed as a percentage of the population with disease. B. Distribution of malariain Africa.

10–20%

5–10%

1–5%

A

B

zygous (compound heterozygote) because both of their β-globingenes are abnormal, although different from each other.] Hemoglobinlevels tend to be higher in Hb SC disease than in sickle cell anemia,and may even be at the low end of the normal range. The clinicalcourse of adults with Hb SC anemia differs from that of sickle cellanemia in that symptoms such as painful crises are less frequentand less severe; however, there is significant clinical variability.

D. Methemoglobinemias

Oxidation of the heme component of hemoglobin to the ferric (Fe3+)state forms methemoglobin, which cannot bind oxygen. This oxida-tion may be caused by the action of certain drugs, such as nitrates,or endogenous products, such as reactive oxygen intermediates (seep. 148). The oxidation may also result from inherited defects, forexample, certain mutations in the α- or β-globin chain promote theformation of methemoglobin (Hb M). Furthermore, a deficiency ofNADH-cytochrome b5 reductase (also called NADH-met hemoglobinreductase), the enzyme responsible for the conversion of methe-moglobin (Fe3+) to hemoglobin (Fe2+), leads to the accumulation ofmet hemoglobin. [Note: The erythrocytes of newborns have approxi-mately half the capacity of those of adults to reduce methemoglobin.They are therefore particularly susceptible to the effects of met -hemoglobin-producing compounds.] The methemoglobin emias arecharacterized by “chocolate cyanosis” (a brownish-blue coloration ofthe skin and mucous membranes) and chocolate-colored blood, as aresult of the dark-colored methemoglobin. Symptoms are related tothe degree of tissue hypoxia, and include anxiety, headache, anddyspnea. In rare cases, coma and death can occur. Treatment is withmethylene blue, which is oxidized as Fe+3 is reduced.

E. Thalassemias

The thalassemias are hereditary hemolytic diseases in which animbalance occurs in the synthesis of globin chains. As a group, theyare the most common single gene disorders in humans. Normally, syn-thesis of the α- and β-globin chains is coordinated, so that eachα-globin chain has a β-globin chain partner. This leads to the formationof α2β2 (Hb A). In the tha l assemias, the synthesis of either the α- orthe β-globin chain is defective. A thalassemia can be caused by a vari-ety of mutations, including entire gene deletions, or substitutions ordeletions of one to many nucleo tides in the DNA. [Note: Each tha-lassemia can be classified as either a disorder in which no globinchains are produced (αo- or βo-thal assemia), or one in which somechains are synthesized, but at a reduced level (α+- or β+-thalassemia).]

1. ββ-Thalassemias: In these disorders, synthesis of β-globin chainsis decreased or absent, typically as a result of point mutationsthat affect the production of functional mRNA; however, α-globinchain synthesis is normal. α-Globin chains cannot form stabletetramers and, therefore, precipitate, causing the premature deathof cells initially destined to become mature RBCs. Increase in α2γ2

(Hb F) and α2δ2 (Hb A2) also occurs. There are only two copies ofthe β-globin gene in each cell (one on each chromosome 11).Therefore, individuals with β-globin gene defects have eitherβ-thal assemia trait (β-thalassemia minor) if they have only onedefective β-globin gene, or β-thalassemia major (Cooley anemia)

Figure 3.22A. β-Globin gene mutations in the β-thalassemias. B. Hemoglobintetramers formed in β-thalassemias.

Normal

β

β

β

β

β

β

β-Thalassemiaminor

β-Thalassemiamajor

β β β β βββ

Each copy of chromosome 11 hasonly one gene for β-globin chains.

α-Chainprecipatate

Hb A

Hb F

A

Bα

β βββ

ββ

β

γ

γγ

γ δ

δ

δδ

δα

α

α

α αα

Hb A2

ααα

α αα

ααααα

αα

αα


if both genes are defective (Figure 3.22). Because the β-globingene is not expressed until late in fetal gestation, the physicalmanifestations of β-thalassemias appear only several monthsafter birth. Those individuals with β-thalassemia minor makesome β chains, and usually do not require specific treatment.However, those infants born with β-thal assemia major are seem-ingly healthy at birth, but become severely anemic, usually duringthe first or second year of life due to ineffective erythropoiesis.Skeletal changes as a result of extramedullary hematopoiesisalso are seen.These patients require regular trans fusions ofblood. [Note: Although this treatment is lifesaving, the cumulativeeffect of the transfusions is iron overload (a syndrome known ashemosiderosis). Use of iron chelation therapy has improved mor-bidity and mortality.] The increasing use of bone marrow replace-ment therapy has been a boon to these patients.

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 (Figure3.23). 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 aredefective, the individual is designated as having α -thal assemiatrait. 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 withhydrops fetalis and fetal death results, because α-globin chainsare required for the synthesis of Hb F. [Note: Hemoglobinopathiesthat result in increased O2 affinity typically are characterized byincreased production of RBCs, whereas those that result indecreased O2 affinity are characterized by anemia.]

V. CHAPTER SUMMARY

Hemoglobin A, the major hemoglobin in adults, is composed of fourpolypeptide chains (two α chains and two β chains, α2β2) held togetherby noncovalent interactions (Figure 3.24). The subunits occupy differentrelative positions in deoxyhemoglobin compared with oxyhemoglobin.The deoxy form of hemoglobin is called the “T,” or taut (tense) form. Ithas a constrained structure that limits the movement of the polypeptidechains. The T form is the low-oxygen-affinity form of hemoglobin. Thebinding of oxygen to hemoglobin causes rupture of some of the ionic andhydrogen bonds. This leads to a structure called the “R,” or relaxed form,in which the polypeptide chains have more freedom of movement. The Rform is the high-oxygen-affinity form of hemoglobin. The oxygen dissociation curve for hemo globin is sigmoidal in shape (in contrast tothat of myoglobin, which is hyperbolic), indicating that the subunitscooperate in binding oxygen. Cooperative binding of oxygen by the foursubunits of hemoglobin means that the binding of an oxygen molecule atone heme group increases the oxygen affinity of the remaining hemegroups in the same hemoglobin molecule. Hemoglobin’s ability to bind

V. Chapter Summary 39

A

Figure 3.23

α-Thalassemia trait(heterozygous form)

α-Thalassemia trait(heterozygous form)

Hemoglobin Hdisease

(clinically severe)

Hemoglobin Bart disease with hydrops fetalis (usually fatal

at birth)

Normal individuals

"Silent" carrier

Key to symbols

Chromosome16 pair

Normal gene forα-globin chain

Deleted gene forα-globin chain

Show somemild symptoms

clinically

α1

α1

α2

α2

α1

α1

α2

α2

α1

α1

α1

α1

α2

α2

α1

α1

α2

α2

α2

α2

α1

α1

α2

α2

α11 α2 α1 α2

Each copy of chromosome 16 has two adjacent genes for α-globin chains.

Hb A

Bα

β β ββ β

β

β

β ββ β

β ββ β

β ββ β

β β

ββ

γγγ

δδ

αα

Hb H(precipitate)

α

A. α-Globin gene deletions in the α-thalassemias. B. Hemoglobintetramers formed in α-thalassemias.

Hb Bart

γγγγ



Study Questions

Choose the ONE correct answer.

3.1 Which one of the following statements concerning thehemoglobins is correct?

A. Fetal blood has a higher affinity for oxygen than doesadult blood because Hb F has a decreased affinity for2,3-BPG.

B. Purified Hb F (stripped of 2,3-BPG) has a higher affin-ity for oxygen than does purified Hb A.

C. The globin chain composition of Hb F is α2δ2.D. Hb A1c differs from Hb A by a single, genetically deter-

mined amino acid substitution.E. Hb A2 appears early in fetal life.

3.2 Which one of the following statements concerning theability of acidosis to precipitate a crisis in sickle cellanemia is correct?

A. Acidosis decreases the solubility of Hb S.B. Acidosis increases the affinity of hemoglobin for

oxygen.C. Acidosis favors the conversion of hemoglobin from

the taut to the relaxed conformation.D. Acidosis shifts the oxygen dissociation curve to the

left.E. Acidosis decreases the ability of 2,3-BPG to bind to

hemoglobin.

3.3 Which one of the following statements concerning thebinding of oxygen by hemoglobin is correct?

A. The Bohr effect results in a lower affinity for oxygen athigher pH values.

B. Carbon dioxide increases the oxygen affinity ofhemoglobin by binding to the C-terminal groups of thepolypeptide chains.

C. The oxygen affinity of hemoglobin increases as thepercentage saturation increases.

D. The hemoglobin tetramer binds four molecules of 2,3-BPG.

E. Oxyhemoglobin and deoxyhemoglobin have the sameaffinity for protons (H+).

Correct answer = A. Because 2,3-BPG reducesthe affinity of hemoglobin for oxygen, theweaker interaction between 2,3-BPG and Hb Fresults in a higher oxygen affinity for Hb F rela-tive to Hb A. In contrast, if both Hb A and Hb Fare stripped of 2,3-BPG, they have a similaraffinity for oxygen. Hb F consists of α2γ2. Hb A1cis a glycosylated form of Hb A, formed nonen-zymically in red cells. Hb A2 is a minor compo-nent of normal adult hemoglobin, first appearingshortly before birth and rising to adult levels(about 2% of the total hemoglobin) by 6 monthsof age.

Correct answer = A. Hb S is significantly lesssoluble in the deoxygenated form, comparedwith oxyhemoglobin S. A decrease in pH (acido-sis) causes the oxygen dissociation curve toshift to the right, indicating a decreased affinityfor oxygen. This favors the formation of thedeoxy, or taut, form of hemoglobin, and can pre-cipitate a sickle cell crisis. The binding of 2,3-BPG is increased, because it binds only to thedeoxy form of hemoglobins.

Correct answer = C. The binding of oxygen atone heme group increases the oxygen affinity ofthe remaining heme groups in the samemolecule. Carbon dioxide decreases oxygenaffinity because it lowers the pH; moreover,binding of carbon dioxide to the N-termini stabi-lizes the taut, deoxy form. Hemoglobin bindsone molecule of 2,3-BPG. Deoxyhemoglobinhas a greater affinity for protons and, therefore,is a weaker acid.

oxygen reversibly is affected by the pO2 (through heme-heme interactions), the pH of the environment, the pCO2,and the availability of 2,3-bisphosphoglycerate (2,3-BPG). For example, the release of O2 from Hb is enhanced whenthe pH is lowered or the pCO2 is increased (the Bohr effect), such as in exercising muscle, and the oxygen dissocia-tion curve of Hb is shifted to the right. To cope long-term with the effects of chronic hypoxia or anemia, the concen-tration of 2,3-BPG in RBCs increases. 2,3-BPG binds to the Hb and decreases its oxygen affinity, and it, therefore,also shifts the oxygen-dissociation curve to the right. Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carbon monoxy hemoglobin (Hb CO). Hemoglobinopathies are disorders caused either byproduction of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normalhemoglobin subunits, or, rarely, both (Figure 3.25). The sickling diseases sickle cell anemia (Hb S disease) andhemoglobin SC disease, as well as hemoglobin C disease and the thalassemia syndromes are representativehemoglobinopathies that can have severe clinical consequences.


V. Chapter Summary 41

Figure 3.24Key concept map for hemoglobin structure and function.

Proto-porphorphyrin IX

β Subunits α Subunits

β Chains α Chains

Allosteric modifiersHydrogen ion (H+)

CO2

2,3-Bisphosphoglycerate

Heme Heme

Deoxy form(deoxyhemoglobin,

T form)

Low O2 affinity

Constrained structure

High O2 affinity

More freedomof movement

or

Four subunits

Two types

Fe++

Four O2 cooperatively

First O2 binding with low affinity

Next three O2 binding with

increasing affinity

Transition from T to R state

Sigmoid O2 binding curve

Decreased affinity for O2

“Right-shift” in O2 saturation curve

Carbon monoxide (CO)

Carboxy-hemoglobin

High affinity for CO (displaces O2)

“Left-shift” of O2 saturation curve

Hyperbolic O2 saturation curve

characterized by

Heme-heme interaction

characterized by characterized by

Different relative positions of subunits

Deoxyhemoglobin(T form)

Oxyhemoglobin(R form)

composed of

composed of composed of

composed of

composed of

characterized by

leads to

leads to

leads to

leads to

leads to

Stabilization ofthe T state

leads to

leads to

preferentially binds

leads to

leads to

Stabilization ofthe R state

leads to

leads to

exists ascomposed of

Hemoglobin

binds in binds

Hemoglobin structure Allosteric effectors Effects ofcarbon monoxideO2 binding

Study Questions (continued)

3.4 β-Lysine 82 in hemoglobin A is important for the bind-ing of 2,3-BPG. In Hb Helsinki, this amino acid hasbeen replaced by methionine. Which of the followingshould be true concerning Hb Helsinki?

A. It should be stabilized in the T, rather than the R,form.

B. It should have increased O2 affinity and, conse-quently, decreased delivery of O2 to tissues.

C. Its O2 dissociation curve should be shifted to theright relative to Hb A.

D. It results in anemia.

Correct answer is B. Substitution of lysine bymethionine decreases the ability of negativelycharged phosphate groups in 2,3-BPG to bindthe β subunits of hemoglobin. Because 2,3BPGdecreases the O2 affinity of hemoglobin, areduction in 2,3-BPG should result in increasedO2 affinity and decreased delivery of O2 to tis-sues. The R form is the high-oxygen-affinityform of hemoglobin. Increased O2 affinity(decreased delivery) results in a left shift in theO2 dissociation curve. Decreased O2 delivery iscompensated for by increased RBC production.



Figure 3.25Key concept map for hemoglobinopathies.

Hemoglobinopathies

Methemoglobinemia

Hb C Hb SCHb S

Fe2+ Fe3+

Decreased synthesis of α chains

Decreased synthesis of β chains

Accumulation of γ4 (Hb Bart) and β4

(Hb H), and β chainprecipitation

Synthesis of structurally abnormal hemoglobins

Synthesis of insufficient quantities of normal hemoglobin Other

AnemiaAnemia

Chocolate cyanosis

Accumulation of α2γ2 (Hb F) and α2δ2 (Hb A2)

for example for example for example

caused by caused by

Point mutation in both genes coding

for β chain

Decreased solubility in deoxy form

β6 Glu Val

Polymer formation

Vascular occlusion

Pain ("crises")

Different point mutations in each gene coding

for β chain

Decreased solubility in deoxy form

Polymer formation

More severe phenotype than Hb C

Pain ("crises")

Point mutation in both genes coding

for β chain

Mild hemolyticanemia

Vascular occlusion

β6 Glu Valβ6 Glu Lys β6 Glu Lys

composed of composed of composed of

results in

results in

results in

results in

results in

results in

characterized by

for example for examplefor example

characterized by

results in

results in

leads to

leads to leads to

leads toresults in

caused by caused by

result in result in

results inresults in

caused by

β-Thalassemias α-Thalassemias

Deletional mutations Point mutations

Inability to bind O2

Study Questions (continued)

3.5 A 67-year-old man presented to the emergency depart-ment with a 1 week history of angina and shortness ofbreath. He complained that his face and extremities hada “blue color.” His medical history included chronic stableangina treated with isosorbide dinitrate and nitroglycerin.Blood obtained for analysis was chocolate-colored.Which one of the following is the most likely diagnosis?

A. Sickle cell anemiaB. CarboxyhemoglobinemiaC. Methemoglobinemia.D. β-ThalassemiaE. Hemoglobin SC disease

Correct answer = C. Oxidation of the hemecomponent of hemoglobin to the ferric (Fe3+)state forms methemoglobin. This may becaused by the action of certain drugs, such asnitrates. The methemoglobinemias are charac-terized by chocolate cyanosis (a brownish-bluecoloration of the skin and mucous membranes),and chocolate-colored blood as a result of thedark-colored methemoglobin. Symptoms arerelated to tissue hypoxia, and include anxiety,headache, dyspnea. In rare cases, coma anddeath can occur.


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