1 Instructor : Dr. Madeline A. Shea, Prof . of Biochemistry 4-450 BSB, 335-7885, [email protected] 99:163 Medical Biochemistry Allosteric Regulatory Proteins Lectures 5 & 6 2 I. Outline, Study Guide, Reading List II. Sc ientific T op ic s A. Allosteric Regulation - Definitions B. Myoglobin - Heme Group, Ligand Binding C. Hemoglobin 1. Subunits and 4˚ Structure, “T” to “R” Transition 2. Oxygen Binding: Sigmoidal vs. Hyperbolic 3. Mechanism of Cooperat ive Binding 4. Allosteric Effector s: pH, C O 2 , 2,3-BPG 5. Genes for Subunits , Fetal vs. Adult 6. Pathology: Sickle Cell, V alue of Heterozygote D. Classic Models of Allostery 1. Concerted (MWC) vs. Sequential (KNF) model 2. Hill Plots and Cooperativity E. Review of Binding Basics (See Review Session Handout ) Reading Suggestions  Berg et al.,  Biochemistry 5e. Ch. 10: 261-262, 267-275 Devlin, Biochemistry 4e Ch. 3: 114-124, Ch.4: 151-154 Lipincott Review 3rd Ed., Ch. 3, Lehninger Ch. 4 and 5  Allosteric Regulatory Proteins Topics

Allosteric Regulatory Proteins

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    Instructor: Dr. Madeline A. Shea, Prof. of Biochemistry 4-450 BSB, 335-7885, [email protected]

    99:163 Medical Biochemistry Allosteric Regulatory Proteins

    Lectures 5 & 6


    I. Outline, Study Guide, Reading List II. Scientific Topics

    A. Allosteric Regulation - Definitions B. Myoglobin - Heme Group, Ligand Binding C. Hemoglobin

    1. Subunits and 4 Structure, T to R Transition 2. Oxygen Binding: Sigmoidal vs. Hyperbolic 3. Mechanism of Cooperat ive Binding 4. Allosteric Effectors: pH, CO2, 2,3-BPG 5. Genes for Subunits , Fetal vs. Adult 6. Pathology: Sickle Cell, Value of Heterozygote

    D. Classic Models of Allostery 1. Concerted (MWC) vs. Sequential (KNF) model 2. Hill Plots and Cooperativity

    E. Review of Binding Basics (See Review Session Handout)

    Reading Suggestions

    Berg et al., Biochemistry 5e. Ch. 10: 261-262, 267-275

    Devlin, Biochemistry 4e Ch. 3: 114-124, Ch.4: 151-154

    Lipincott Review 3rd Ed., Ch. 3, Lehninger Ch. 4 and 5

    Allosteric Regulatory Proteins Topics

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    1. List the major features of the secondary and tertiary structures of myoglobin and hemoglobin. How do they differ? How are they similar? What prosthetic group do they share? What does it bind?

    2. How do the oxygen binding curves for Mb and Hb differ in midpoint and shape? How do they compare on a log scale vs. arithmetic scale? What is meant by positive cooperativity in oxygen binding to hemoglobin? What is its physiological significance?

    3. How does 2,3-BPG influence oxygen binding to hemoglobin? Describe how pH and C02 affect hemoglobin.

    4. What are the developmentally programmed changes in expression of Hb subunits? What causes Sickle Cell disease? What causes Thalassemias?

    5. Compare and contrast the KNF and MWC models of hemoglobin cooperativity.

    Study Questions & Concepts


    Allosteric Regulation: Effector alters other site

    Allostery = when the activity of a protein is regulated by the binding of a factor (small molecule or protein) that binds at a site other than the active site or primary ligand binding site. The allosteric effector changes the behavior of the protein (which may have multiple subunits). Stoichiometry must be determined experimentally.

    Sometimes effector is X itself.

    ProteinE + k kE




  • 3


    Domains: Covalently linked sequences of polypeptide that have independent & stable 3 structure, easily manufactured from gene duplication events. (4 Fibrinection Type 3 modules/domains)

    Subunits: Discrete sequences of separate polypeptides that have independent & stable 3 structure and associate via non-covalent interactions. Allows Mix & Match to modulate function. Hb: 2 of 2

    4 (Quaternary) Structure: Stoichiometry & Arrangement of Subunits

    Stryer 5e


    Allosteric Regulation Common in Multimeric Proteins ( 2 Subunits)

    Allostery = cAMP (allosteric effector ) binding to the R (regulatory) subunit of a tetrameric enzyme causes release of C (catalytic) subunits, freeing them for catalysis of available substrate molecules.

  • 4


    HbA = Adult Hemoglobin: Oxygen Delivery in Bloodstream

    1 2

    1 2

    Hb = Closed-Form Tetramer = 2 + 2 = 2 pairs of Mb-like monomers packed together (4 hemes)

    Lehninger, Ch. 5


    Legend: Hb Stoichiometry

    At the core of the molecule is a heterocyclic ring, known as a heme or porphyrin which holds an iron atom; this iron atom is the site of oxygen binding.

    In adult humans, hemoglobin is a tetramer, consisting of two alpha and two beta subunits non-covalently bound.

    The subunits are structurally similar and about the same number of amino acids (alpha = 141 aa, beta = 146 residues).

    Each subunit of hemoglobin contains a single heme, so that the overall binding capacity of adult human hemoglobin for oxygen is four oxygen molecules.

    Hb + 4O2 ok Hb(O2)4

    Lehninger, Ch. 5

  • 5


    Prosthetic Group: Heme Protoporphyrin IX, Iron (Fe)

    Lehninger, Ch. 5



    Legend: Heme Group

    A heme is a cofactor that consists of an iron atom in the center of a large heterocyclic organic ring called a porphyrin. Although porphyrins do not necessarily contain iron, a substantial fraction of porphyrin-containing metalloproteins do in fact have heme as their prosthetic subunit.

    Hydrophobic interactions between the tetrapyrrole ring and hydrophobic amino acid R groups on the interior of the cleft in the protein strongly stabilize the heme protein conjugate. In addition a nitrogen atom from a histidine R group located above the plane of the heme ring is coordinated with the iron atom further stabilizing the interaction between the heme and the protein. In oxymyoglobin the remaining bonding site on the iron atom (the 6th coordinate position) is occupied by the oxygen, whose binding is stabilized by a second histidine residue.

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    Recall Properties of Myoglobin Protein Fold (combo of 2 and 3 Structure)

    Mostly alpha-helical Water soluble: polar on the outside Compact: excludes water from interior Interior is hydrophobic - nonpolar Heme (Fe + protoporphyrin IX)

    cross-section surface

    To make Hb from Mb:Change Surface of Mb-like subunits

    to stay associated circulating in blood.Lehninger



    Hb Chains (Subunits) share the Globin Fold - the first known scaffold for any protein

    The globin fold is an all-alpha fold consistingof 6-8 helices that cradlethe heme which canbind a variety of ligands including: oxygen (O2), carbon monoxide (CO), cyanide (CN).

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    A + B ABKassn = [AB]/[A][B]

    Oxygen (X) Binding to Mb

    Adapted from Biochemistry, by Garrett & Grisham

    Binding Isotherm

    Y = MbHemes with Oxygen BoundAll MbHemes

    Fractional Saturation[MbX]

    [Mb] + [MbX]Substitute using Kassn

    Ka [X]1 + Ka [X]


    Legend: Oxygen Binding to Mb

    Y = fractional saturation (occupancy); curve has hyperbolic shape. Dose response for 1 site.

    Ka = association equilibrium constant If Y = 1 (100%), then every heme iron of

    every myoglobin is bound to an Oa. If Y = 0.5 (50%), then half the Mb molecules

    have oxygen bound, and half are empty. At Y = 0.5, [X]0.5 = 1/Ka = Kd , where

    Kd = dissociation eq. constant (MX k M + X)

  • 8








    1 0-12 1 0-10 1 0- 8 1 0- 6 1 0- 4 1 0- 2 1 00



    l Sa






    l Sat









    0 100 2 10- 5 4 10- 5 6 10- 5 8 10- 5 1 10- 4



    l Sa






    l Sat



    Review of Binding: Binary Reactions

    Kassn = 106 M-1 Kdssn = 10-6 M = 1 M*

    [X] on linear scale [X] on log scale


    Hb: Dimer of Dimers

    Ackers et al (2002) PNAS 99:9777-9783

  • 9


    Hemoglobin Dimer-Dimer Interactions Depend on Oxygenation State of Hemes

    Dimer-Dimer Interface between 11 & 22Deoxy Hb is much more stable than Oxy Hb At 21.5 C, pH 7.4 the half-time of Hb dissociation is ~ 10 hrs. for deoxy < 1 sec. for oxy

    k Ligand binding & Subunit Association are linked Illustration from

    Introduction to Protein Structure By Branden & Tooze


    Oxygen binds to Heme (Prosthetic Group) Fe (iron) Protoporphyrin IX




  • 10


    Oxygen Induces Molecular Rearrangement

    Propagated movement one helix adjusts and

    nudges the adjacent dimer.

    Cooperative transition takes place, making if more likely to bind second oxygen on the same dimer!



    Quaternary Transition of Hb: T (taut/deoxy) to R (relaxed/oxy)

    The quaternary structure of low affinity, deoxy(genated) hemoglobin (Hb) is called the taut (T) state.

    The quaternary structure of the fully oxy(genated) high affinity form of hemoglobin (Hb(O2)4) is known as the relaxed (R) state (15 rotation).


  • 11


    Oxygen-Induced Dimer Rotation Cracks Deoxy Hb Crystals

    Individual dimers are rigid (i.e., the 11 interface seems hard).

    One dimer rotates (and twists) across the other.

    The dimer-dimer interface is the conduit for inter-subunit communication k cooperativity!

    What are the rules for switching? Are any or all intermediates populated?



    Oxygen Binding Curves Coooperative vs. Non-Cooperative

    [PX][P] + [PX]

    Expected if sites do NOT


    Hyperbola [HbXi=1,4][Hb] + ([HbXi=1,4])

    Sigmoid Shape

    Lehninger, Ch. 5

  • 12


    Oxygen Binding Curves Coooperative vs. Non-Cooperative



    Legend: Oxygen Binding The binding of oxygen by hemoglobin is cooperative; the protein is not 4 Mb

    (i.e., it is not four independent oxygen-binding subunits). As hemoglobin binds successive oxygens, the oxygen affinity of the subunits increases. The affinity for the fourth oxygen to bind is approximately 300 times that for the first.

    This cannot be explained by four independent subunits with intrinsically different affinities. If this were the case, then oxygen would bind to the subunit with the highest intrinsic affinity first. Then the affinity of the molecule would subsequently decrease rather than increase.

    A cooperative binding mechanism is more efficient at collecting oxygen in lungs where it is in high concentration, and supplying it where it is needed. Tissues can be relatively mildly deficient in oxygen for hemoglobin to give up a much more significant amount compared to a non-cooperative protein such as myoglobin.

    CO has > 200x affinity for Hb than O2. It locks Hb into oxy form, thereby making it harder for CO to dissociate and more likely to pick up more CO.


  • 13


    How is the whole > sum of the parts?

    Regulatory proteins engage in intramolecular communication: The cooperative oxygen-binding properties of hemoglobin rely on specific quaternary interactions between the subunits. The allosteric mechanism of hemoglobin enable the affinity for oxygen to be adjusted by interactions with a number of reagents.

    Why doesnt the Hbtetramer act like

    4 Mb monomers?4 identical suitcases?



    The Allosteric Pathway of Hb 3 of 8 Possible Intermediates are Abundant

    Ackers et al (2000) Proteins S4:23-43

    Ackers et al (2002) PNAS 99:9777-9783

    Hb with 2 or 3 Ligands

  • 14


    Standard: Leftmost curve Bohr Effect: Middle curve Deoxy Hb binds protons w/higher affinity than oxy Hb. Acidity (lower pH) promotes deoxy form or dissociation of O2 from Hb tetramer. Effect of C02: Right curve increased dissolved carbon dioxide creates bicarbonate ion and H+, and binds to Hb as carbamate. Stabilizes T (deoxy form) - dissociation of O2 from Hb tetramer.

    Promotion of O2 release improves O2 delivery to tissues.

    Allosteric Effectors: Cellular Conditions Affect Oxygen Binding



    Legend: Hb Allosteric Effectors

    In the tetrameric form of normal adult hemoglobin, the binding of oxygen is a cooperative process, due to allosteric regulation.

    Affinity of hemoglobin for oxygen is decreased in the presence of carbon dioxide and at lower pH. Carbon dioxide reacts with water to give bicarbonate, via the reaction:

    CO2 + H2O ok HCO3- + H+ So blood with high carbon dioxide levels is also lower in pH (more

    acidic). Conversely, when the carbon dioxide levels in the blood decrease (i.e. around the lungs), carbon dioxide is released, increasing the oxygen affinity of the protein. This control of affinity for oxygen by pH is known as the Bohr effect.

    The binding of oxygen is also affected by molecules such as carbon monoxide (e.g. from tobacco smoking) or 2,3-diphosphoglycerate, which lowers the affinity of hemoglobin for oxygen.

  • 15


    Amino Acids Responsible for Bohr Effect: Histidines Are only AA Titrating @ pH 7


    Allosteric Effector of Hb Function 2,3-Bisphosphoglycerate (BPG) binds to Beta Cleft

    1 Hb tetramer binds1 molecule of 2,3-BPG which stabilizes deoxy Hb &lowers oxygen affinity, promoting oxygen release in tissues


  • 16


    Effect of BPG on Oxygen Binding



    Hemoglobin Pathologies: Mostly Mutations or Shortages

    Mutations can affect the stability of a protein molecule, its ligand binding affinity, and its ability to form oligomers.

    Mutations can be deletions, insertions or single point changes causing a.a. substitution.

    Many substitutions on the surface have little effect on protein stability, but may affect function if that surface is an important interface for interactions.

    Over 1000 mutations in globin genes are known. About 1 in 300 persons has a mutant form. Few of these known mutations lead to abnormal Hb and pathology.

  • 17


    Multiple Genes for Hb Subunits: Expression controlled Developmentally

    From Hemoglobin Synthesissickle.bwh.harvard.edu

    Embryonic first 12 weeks Epsilon, Zeta

    Fetal Hb Gamma, Alpha

    Adult HbA0 Beta, Alpha Diabetic Hb A1C Glycosylated

    Adult HbA2 < 3%

    Delta, Alpha

    Thalassemias (shortage of alpha or beta subunits) result in anemias because subunit proportions are not even.


    Developmentally programmed Variants in Primary Sequence of Hb

    Oxygen Affinity of Maternal vs. Fetal Hb (Hb F) depends on 2,3-BPG which stabilizes the deoxy form (more open) & lowers O2 affinity. 2,3-BPG binds more tightly to adult than fetal, promoting transfer to fetal.

    Example of Allosteric Regulation The change in primary sequence 22 provides for higher oxygen affinity with no change heme chemistry. Economical because alpha subunit is the same in adult and fetal. Stryer

  • 18


    E6V Glu6 to Val

    Fiber Formation Distorts RBC

    See Clinical Case handout First Molecular Disease

    Ingram & Hunt, 1956

    HbS: Sickle Cell Hb is normal in oxygen affinity & dimer-tetramer equilibrium


    Legend: Sickle Cell Disease

    The sickle cell mutation reflects a single change in the beta subunit, an amino acid change from glutamic acid to valine at position 6.

    The hemoglobin molecule picks up oxygen and releases it when appropriate.

    Normally, the hemoglobin molecules exist as single, isolated units in the red cell, whether they have oxygen bound or not. Normal red cells maintain a basic disc shape.

    Sickle hemoglobin (Hb S) behave as normal isolated tetramers in the red cells when they have oxygen bound. When sickle hemoglobin releases oxygen in the peripheral tissues, however, the molecules tend to stick together and form long chains or polymers. These rigid polymers distort the cell and cause it to bend out of shape. This can clog capillaries. If the red cell is able to return to the lungs and pick up oxygen again, it can depolymerize.

    A single red cell may traverse the circulation four times in one minute. Sickle hemoglobin undergoes repeated episodes of polymerization and depolymerization.

    Despite being the first molecular disease, there is no effective safe cure.

  • 19


    Varying Degrees of Illness: Dimer-Tetramer Equilibrium Helps Heterozygotes

    If mutation does not affect equilibrium constant for association of dimers into tetramers, binomial distribution of tetrameric forms will result (1:2:1 ratio). Thus, only 25% is pure mutant.

    This is the case for Hb S (sickle cell anemia) mutation in Beta chain position 6: Glu to Val. Dimer-dimer interface is not affected. Thus, fibers of deoxy HbS are naturally interrupted by mass action - binding of normal or mixed tetramers that cannot continue the chain.

    Suggests cure is to Stimulate Gamma production?

    A S A* S S S A A

    25% 50% 25%

    *A = normal Adult Hb; S = sickle mutation in beta


    Embryonic 9 days before birth

    Binomial Distribution

    of combinations of subunits

    changes over time.

    Subunit Equilibrium found in Isozymes: Embryonic and Adult Lactate Dehydrogenase


  • 20


    Structural similarity provides clues to function & a foundation for testing hypotheses

    Extrapolating from Myoglobin to Hemoglobin Safe Bet Heme iron binds oxygen Hard to Predict Cooperativity, BPG Binding


    A Structure is a Start, not an END


    Deoxy Hb Oxygen-saturated Hb Dickerson & Geis, Hemoglobin

    Quaternary Rearrangement Models

  • 21


    Elegant symmetric models for

    multi-site cooperative ligand binding

    MWC: postulates 2 quaternary structuresonly the number and not position of ligand determines energy of state; model only accounts for positive cooperativity

    KNF: allows sequential change of individual subunits of multimeric complexcan account for negative cooperativity

    Classic Models of Allostery: MWC (Monod, Wyman, Changeux) & KNF (Koshland, Nemethy, Filmer)

    42 Adapted from Biochemistry, M. Campbell

    Concerted (MWC) Model

  • 22


    Legend: MWC Model

    According to the theory of Monod, Wyman, and Changeux (MWC), the protein exists in an equilibrium between two forms - the taut (T) state (deoxy for Hb), in which all subunits in each molecule are in the weak-binding conformation, and the relaxed (R) state (oxy in Hb), in which all subunits are in the strong-binding form.

    An equilibrium between the T and R states is presumed to exist, and partial saturation shifts that equilibrium toward the R state. The shift is a concerted one, so that intermediates with some subunits in the weak-binding state and some in the strong-binding state are specifically excluded.

    44 Adapted from Biochemistry, M. Campbell

    Multi-step Process of Binding

  • 23

    45 Adapted from Biochemistry, M. Campbell

    Allosteric Effectors acting on a Concerted Transition: R k T

    46 Adapted from Biochemistry, M. Campbell

    Inhibitors bind selectively to R or T

  • 24

    47 Adapted from Biochemistry, M. Campbell

    Sequential (KNF) model


    Legend: KNF Model

    A prototype for models that describe allosteric transitions is the sequential model of Koshland, Nemethy, and Filmer (KNF). The KNF model assumes that the subunits can change their tertiary conformation one at a time in response to binding of oxygen.

    Cooperativity arises because the presence of some subunits carrying ligand favors the strong-binding state in adjacent subunits whose sites are not yet filled. Thus, as binding progresses, almost all the sites become strong-binding.

    Such models are characterized by the existence of molecules with some subunits in the weak-binding state and some in the strong. It allows negative interactions.

  • 25

    49 Adapted from Biochemistry, M. Campbell

    Sequential Model, contd.


    Adapted from Biochemistry, Garrett & Grisham

    Textbooks Emphasize Hill Plots & Coefficients: Macroscopic Measures of Heterogeneity and Cooperativity

    Students are NOT responsible for Hill Plots

  • May 2003: HemoglobinRed Blood, Blue BloodEver wondered why blood vessels appear blue? Oxygenated blood is bright red:when you are cut, the blood you see is brilliant red oxygenated blood.Deoxygenated blood is deep purple: when you donate blood or give a bloodsample at the doctor's office, it is drawn into a storage tube away from oxygen,so you can see this dark purple color. However, deep purple deoxygenatedblood appears blue as it flows through our veins, especially in people with fairskin. This is due to the way that different colors of light travel through skin:blue light is reflected in the surface layers of the skin, whereas red lightpenetrates more deeply. The dark blood in the vein absorbs most of this redlight (as well as any blue light that makes it in that far), so what we see is theblue light that is reflected at the skin's surface. Some organisms like snails andcrabs, on the other hand, use copper to transport oxygen, so they truly haveblue blood.Hemoglobin is the protein that makesblood red. It is composed of fourprotein chains, two alpha chains andtwo beta chains, each with a ring-likeheme group containing an iron atom.Oxygen binds reversibly to these ironatoms and is transported throughblood. Each of the protein chains issimilar in structure to myoglobin(presented in the January 2000Molecule of the Month), the proteinused to store oxygen in muscles andother tissues. However, the fourchains of hemoglobin give it someextra advantages.Use and Abuse of HemoglobinAside from oxygen transport, hemoglobin can bind and transport othermolecules like nitric oxide and carbon monoxide. Nitric oxide affects the wallsof blood vessels, causing them to relax. This in turn reduces the bloodpressure. Recent studies have shown that nitric oxide can bind to specificcysteine residues in hemoglobin and also to the irons in the heme groups, asshown in PDB entry 1buw. Thus, hemoglobin contributes to the regulation ofblood pressure by distributing nitric oxide through blood. Carbon monoxide,on the other hand, is a toxic gas. It readily replaces oxygen at the hemegroups, as seen in PDB entry 2hco and many others, forming stable complexesthat are difficult to remove. This abuse of the heme groups blocks normaloxygen binding and transport, suffocating the surrounding cells.

    PDB Molecule of the Monthhttp://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/alphabetical_list.html

  • May 2003: HemoglobinArtificial BloodBlood transfusions have saved countless lives. However, the need for matchingblood type, the short life of stored blood, and the possibility of contaminationare still major concerns. An understanding of how hemoglobin works, based ondecades of biochemical study and many crystallographic structures, hasprompted a search for blood substitutes and artificial blood. The most obviousapproach is to use a solution of pure hemoglobin to replace lost blood. Themain challenge is keeping the four protein chains of hemoglobin together. Inthe absence of the protective casing of the red blood cell, the four chainsrapidly fall apart.To avoid this problem, novel hemoglobinmolecules have been designed where twoof the four chains are physically linkedtogether, as shown in PDB entry 1c7d. Inthat structure, two additional glycineresidues form a link between two of thechains, preventing their separation insolution.

    Hemoglobin CousinsLooking through the PDB, you will find many different hemoglobin molecules.You can find Max Perutz's groundbreaking structure of horse hemoglobin inentry 2dhb, shown in the picture here. There are structures of humanhemoglobins, both adult and fetal. You can also find unusual hemoglobins likeleghemoglobin, which is found in legumes. It is thought to protect the oxygen-sensitive bacteria that fix nitrogen in leguminous plant roots. In the past fewyears some hemoglobin cousins called the "truncated hemoglobins" have beenidentified, such as the hemoglobin in PDB entry 1idr, which have severalportions of the classic structure edited out. The only feature that is absolutelyconserved in this subfamily of proteins is the histidine amino acid that binds tothe heme iron.

    PDB Molecule of the Monthhttp://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/alphabetical_list.html

  • May 2003: HemoglobinCooperation Makes It EasierHemoglobin is a remarkable molecular machine that uses motion and smallstructural changes to regulate its action. Oxygen binding at the four heme sitesin hemoglobin does not happen simultaneously. Once the first heme bindsoxygen, it introduces small changes in the structure of the correspondingprotein chain. These changes nudge the neighboring chains into a differentshape, making them bind oxygen more easily. Thus, it is difficult to add thefirst oxygen molecule, but binding the second, third and fourth oxygenmolecules gets progressively easier and easier. This provides a great advantagein hemoglobin function. When blood is in the lungs, where oxygen is plentiful,oxygen easily binds to the first subunit and then quickly fills up the remainingones. Then, as blood circulates through the body, the oxygen level drops whilethat of carbon dioxide increases. In this environment, hemoglobin releases itsbound oxygen. As soon as the first oxygen molecule drops off, the proteinstarts changing its shape. This prompts the remaining three oxygens to bequickly released. In this way, hemoglobin picks up the largest possible load ofoxygen in the lungs, and delivers all of it where and when needed.

    In this figure, the heme group of one subunit is shown in the little circularwindow. The oxygen molecule is shown in blue green. As it binds to the ironatom in the center of the heme, it pulls a histidine amino acid upwards on thebottom side of the heme. This shifts the position of an entire alpha helix,shown here in orange below the heme. This motion is propagated throughoutthe protein chain and on to the other chains, ultimately causing a large rockingmotion of the two subunits shown in blue. The two structures shown are PDBentries 2hhb and 1hho.

    PDB Molecule of the Monthhttp://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/alphabetical_list.html

  • May 2003: HemoglobinTroubled Hemoglobins

    The genes for the protein chains ofhemoglobin show small differenceswithin different human populations, sothe amino acid sequence ofhemoglobin is slightly different fromperson to person. In most cases thechanges do not affect protein functionand are often not even noticed.However, in some cases thesedifferent amino acids lead to majorstructural changes. One such exampleis that of the sickle cell hemoglobin,where glutamate 6 in the beta chain ismutated to valine. This change allowsthe deoxygenated form of thehemoglobin to stick to each other, asseen in PDB entry 2hbs, producingstiff fibers of hemoglobin inside redblood cells. This in turn deforms thered blood cell, which is normally asmooth disk shape, into a C or sickleshape. The distorted cells are fragileand often rupture, leading to loss ofhemoglobin. This may seem like auniformly terrible thing, but in onecircumstance, it is actually anadvantage. The parasites that causethe tropical disease malaria, whichspend part of their life cycle inside redblood cells, cannot live in the fiber-filled sickle cells. Thus people withsickle cell hemoglobin are somewhatresistant to malaria.

    Other circumstances leading to troubled hemoglobins arise from a mismatch inthe production of the alpha and beta proteins. The structure requires equalproduction of both proteins. If one of these proteins is missing, it leads toconditions called Thalassemia.

    PDB Molecule of the Monthhttp://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/alphabetical_list.html

  • May 2003: Hemoglobin

    Exploring the Structure

    You can look at the binding of oxygen up close in two structures of humanhemoglobin. PDB entry 2hhb shows hemoglobin with no oxygen bound. In thispicture, the heme is seen edge-on with the iron atom colored in gold. You cansee the key histidine reaching up on the bottom side to bind to the iron atom.In PDB entry 1hho, oxygen has bound to the iron, pulling it upwards. This inturn, pulls on the histidine below, which then shifts the location of the entireprotein chain. These changes are transmitted throughout the protein,ultimately causing the big shift in shape that changes the binding strength ofthe neighboring sites.

    PDB Molecule of the Monthhttp://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/alphabetical_list.html

  • Hemoglobin Overview http://sickle.bwh.harvard.edu/hemoglobin.html


    revised April 10, 2002

    An Overview of Hemoglobin

    This brief overview of hemoglobin is not meant to be comprehensive. The goal is to provide sufficient background to make this Web site useful to peopleunfamiliar with the area. More detailed sources are listed at the end of this file.

    A.) What is hemoglobin?

    Hemoglobin is a protein that is carried by red cells. It picks up oxygen in the lungs and delivers it to the peripheral tissues to maintain the viability of cells.Hemoglobin is made from two similar proteins that "stick together". Both proteins must be present for the hemoglobin to pick up and release oxygennormally. One of the component proteins is called alpha, the other is beta. Before birth, the beta protein is not expressed. A hemoglobin protein found onlyduring fetal development, called gamma, substitutes up until birth.

    B.) How is hemoglobin made?

    Like all proteins, the "blueprint" for hemoglobin exists in DNA (the material that makes up genes). Normally, an individual has four genes that code for thealpha protein, or alpha chain. Two other genes code for the beta chain. (Two additional genes code for the gamma chain in the fetus). The alpha chain and thebeta chain are made in precisely equal amounts, despite the differing number of genes. The protein chains join in developing red blood cells, and remaintogether for the life of the red cell.

    C.) How do abnormal hemoglobins arise?

    The composition of hemoglobin is the same in all people. The genes that code for hemoglobin are identical throughout the world. Occasionally, however,one of the genes is altered by any of a variety of "accidents" that can occur in nature. These alterations in the genes (called "mutations") are very rare. Sincegenes are inherited, and they contain the information needed to make a protein, if a mutation produces an abnormal hemoglobin gene in a person, the genewill be passed on to his or her children. The children will produce a modified hemoglobin identical to that of the parent. Most mutations in hemoglobinproduce no problem. Occasionally, however, the alteration in the protein changes aspects of its behavior. The types of disorders that can result include sicklecell disease and thalassemia.

    D.) What about all the different blood types?

    Blood cells are made up of two components. The hemoglobin is in solution inside the cell. The cell is surrounded by a membrane that holds in thehemoglobin. A rough analogy would be a rubber water balloon. The rubber would be the membrane, and the water would be the hemoglobin. The bloodtypes that most of us know, A, B, O, and Rh, are properties of the membrane. The hemoglobin inside the red cells of a person with type O blood and thatinside the red cells of a person with type A blood are identical. The analogy would be of water balloons made from blue and red balloons. The color of theballon would differ, but the material inside (water) would be the same.

    E.) How many types of abnormal hemoglobins are there?

    Although the changes that produce abnormal hemoglobins are rare, several hundred abnormal (or more precisely, "variant") hemoglobins exist. These haveaccumulated over the millions of years of human existence. Most variant hemoglobins function normally, and are only found through specialized research techniques. Some hemoglobins, however, do not function normally and can produce clinical disorders, such as sickle cell disease.

    F.) What happens if a hemoglobin gene "burns out"?

    Genes can suffer damage to an extent that they no longer produce normal amounts of hemoglobin. Usually, only one of the sets of hemoglobin genes isaffected, that is the alpha gene set or the beta gene set. For example, one of the two beta globin genes may fail to produce a normal quantity of beta chainprotein. The alpha globin gene set will continue to produce a normal quantity of alpha chain protein. An imbalance develops in the amount of alpha chain andbeta chain protein in the cell. There is too much alpha chain for the amount of beta chain that is present. This imbalance is called "thalassemia ". In this example, it would be beta thalassemia, because it is the beta chain gene that has failed. An analogy would be cars coming out of the factory. Engines andbodies must be made in equal numbers to have functional automobiles. If the engine plant goes on strike (thalassemia), the bodies produced by the bodyplant are useless.

    How can I find out more about hemoglobin disorders?

    The best source of information about hemoglobin disorders in general are textbooks of medicine. Textbooks of hematology tend to be very detailed, andconfusing for people not conversant with the area. Some text books of medicine are:

    Harrison's Principles of Internal Medicine, McGraw-Hill

    The Principles and Practice of Medicine, Appleton, Century, Croftis

    "Hemoglobin: molecular, genetic, and clinical aspects", HF Bunn and B Forget, Saunders, 1986.

  • A Brief History of Sickle Cell Disease http://sickle.bwh.harvard.edu/scd_history.html


    revised April 10, 2002

    A Brief History of Sickle Cell DiseaseSickle Cell Disease in African Tradition

    Sickle cell disease has been known to the peoples of Africa for hundreds of years. In West Africavarious ethnic groups gave the condtion different names:

    Ga tribe: chwechweechweFaute tribe: nwiiwiiEwe tribe: nuiduduiTwi tribe: ahotutuo

    Sickle Cell Disease in the Western Literature

    Description of Sickle Cell DiseaseIn the western literature, the first description of sickle cell disease was by a Chicago physician, James B.Herrick, who noted in 1910 that a patient of his from the West Indies had an anemia characterized by unusualred cells that were "sickle shaped.".

    Relationship of Red Cell Sickling to OxygenIn 1927, Hahn and Gillespie showed that sickling of the red cells was related to low oxygen.

    Deoxygenation and HemoglobinIn 1940, Sherman (a student at Johns Hopkins Medical School) noted a birefingence of deoxygenated red cells,suggesting that low oxygen altered the structure of the hemoglobin in the molecule.

    Protective Role of Fetal Hemoglobin in Sickle Cell DiseaseJanet Watson, a pediatric hematolist in New York, suggested in 1948 that the paucity of sickle cells in theperipheral blood of newborns was due to the presence of fetal hemoglobin in the red cells, which consequentlydid not have the abnormal sickle hemoglobin seen in adults.

    Abnormal Hemoglobin in Sickle Cell DiseaseUsing the new technique of protein electrophoresis, Linus Pauling and Harvey Itano showed in 1948 that the hemoglobin from patients with sickle cell disease is different than that of normals. This made sickle cell diseasethe first disorder in which an abnormality in a protein was known to be at fault.

    Amino Acid Substitution in Sickle HemoglobinIn 1956, Vernon Ingram, then at the MRC in England, and J.A. Hunt sequenced sickle hemoglobin andshowed that a glutamic acid at position 6 was replaced by a valine in sickle cell disease. Using the knowninformation about amino acids and the codons that coded for them, he was able to predict the mutation in sicklecell disease. This made sickle cell disease the first genetic disorder whose molecular basis was known.

    Cure of Sickle Cell DiseaseIn 1984, bone marrow transplantion in a child with sickle cell disease produced the first reported cure of thedisease. The transplantation was done to treat acute leukemia. The child's sickle cell disease was cured as aside-event. The procedure nonetheless set the precedence for later transplantion efforts directed specifically atsickle cell disease.

    Preventive Treatment for Sickle Cell DiseaseHydroxyurea became the first (and only) drug proven to prevent complications of sickle cell disease in theMulticenter Study of Hydroxyurea in Sickle Cell Anemia which was completed in 1995.

    For more information, see "Hemoglobin: molecular, genetic, and clinical aspects", Bunn and Forget, Saunders, 1986.

  • CHA

    PTER 10

    Regulatory StrategiesA Close Call for a Boy Scout



    The Patient Is Admitted with a Painful Crisis

    A 14-year-old African American boy known to have sickle-cell disease was admitted tothe hospital because of pain in the hip and lower back. The patient had relatively milddisease during childhood with three prior painful crises precipitated by upper respira-tory infections requiring hospital admission and narcotic analgesics for pain control. Hehad received penicillin orally from two months of age until he was six years old and amultivalent streptococcal vaccine at two years of age with a booster at five.

    Two days before admission, he was hiking with his scout troop but had nosymptoms at that time. The day prior to admission he developed lower back painand left hip pain. He had a low-grade fever of 38C, tenderness over the lumbarspine, and pain with flexion of the left hip. Upon admission, his chest was clearto auscultation and abdominal exam was remarkable for enlargement of both theliver and spleen (hepatosplenomegaly). His hematocrit was 24% (normal >40%)on admission; he typically had a hematocrit of 2530%. Blood chemistries revealeda mild elevation in the serum bilirubin. Urinalysis revealed microscopic hematuria(blood in the urine but not enough to be visible to the naked eye) with evidencefor urinary tract infection. Radiographs of the hips and lower back revealed evi-dence for avascular necrosis of the left femoral head but no significant abnormal-ities in the lumbar spine. He was treated with oxygen delivered by a mask andintravenous hydromorphone (Dilaudid) 1 mg every four to six hours for pain con-trol, and he began to improve.

    What causes sickle-cell disease?

    Why is hepatosplenomegaly common in those with sickle-cell disease?

    Is the avascular necrosis of the femoral head observed in theradiograph likely to have arisen from sickle-cell disease?

    What might be the cause of the anemia (low red blood cell count)and elevated bilirubin levels found in the serum?

    Clinical Correlation with Protein Structure & FunctionTaken from Internet-available sample of

    A Clinical Companion to Accompany Biochemistry Fifth EditionBy Kirstie Saltsman, Jeremy Berg and Gordon Tomaselli

    W. H. Freeman and Co., New York

  • 2 CHAPTER 10

    His Condition Deteriorates

    On the second hospital day he developed a cough, experienced a rigor, and became agitated,complaining of increasing back and hip pain and pain in his chest with breathing (pleuriticpain). His temperature was 38.8C, and pulse was 150 bpm (normal range 60100 bpm), andthe respirations were 32 per minute (normal range 1520 per minute). The blood pressure wasslightly elevated at 135/85 mm Hg. The chest examination now suggested fluid in the left lung.The hematocrit had fallen from 24% to 18%. The arterial oxygen saturation was 91% while thepatient was breathing oxygen through a mask at a rate of 4 liters per minute. Radiographs ofthe chest showed cardiomegaly (enlarged heart) and a small area of consolidation (opacifica-tion of the lung due to fluid collection) in the left lower lobe of the lung. An electrocardiogram(ECG) revealed a sinus tachycardia at a rate of 150 and findings suggestive of left ventricularhypertrophy (muscular enlargement of the wall of the left ventricle of the heart).

    What is an ECG, how does it work, and what information does it yield?

    Why might his heart rate and respiration rate be elevated?


    In the course of a painful crisis, the patient has developed acute chest syndrome (ACS), a life-threatening complication of sickle-cell disease.

    The patient is treated with oxygen, an antibiotic, and a blood transfusion

    The inspired oxygen was increased to 100% by a nonrebreathing mask, and the patients oxy-gen saturation improved to 99%. The antibiotic, ceftriaxone (Rocephin) 50 mg/Kg was ad-ministered intravenously twice daily. An exchange transfusion was performed, with the removalof approximately 0.5 liters of the patients red cells and then transfusion of 1.5 liters of packedred cells from a donor. After transfusion the percentage of total hemoglobin that was hemo-globin A (normal adult hemoglobin) was greater than 60% and the hematocrit was 34%.

    Why was an antibiotic administered?

    His condition improves and he is discharged

    By the fourth hospital day chest, hip, and back pain had begun to subside. His blood oxy-gen saturation improved and was 99% on low-flow (2 liters/minute) oxygen delivered by anasal cannula. Blood, sputum, and urine cultures were all negative and intravenous antibi-otics were discontinued. The hematocrit was 29% and he underwent a transfusion of twounits of packed red blood cells that raised the hematocrit to 33%. Intravenous hydromor-phone was tapered and the patient was started on oxycodone 2 mg every 46 hours for paincontrol. He was discharged to home on the sixth hospital day with crutches to limit weightbearing on the left hip.

    What is a nonrebreathing mask? Why was oxygen delivered by a nasalcannula once his symptoms improved?

    The patient was treated with an exchange transfusion. What might be therationale for such a procedure?


    Sickle-Cell Disease Is a Genetic Disease

    Sickle-cell disease is caused by an inherited mutation in hemoglobin, an oxygen transport mol-ecule found in red blood cells (see Section 10.2 of Biochemistry 5e). Hemoglobin is a tetramericmolecule, consisting of two -globin and two -globin chains. Although there are several formsof the disease, the most common is caused by a point mutation in the -globin chain of he-moglobin, which results in substitution of the glutamic acid residue at the sixth position of theamino acid chain with a valine residue. This mutant version is referred to as HbS (for Sicklehemoglobin), while the wild type counterpart is termed HbA (for Adult hemoglobin).

    Heterozygotes are protected against malaria

    Individuals that inherit a single mutant copy (carriers or heterozygotes, also known as hav-ing sickle-cell trait) are usually completely asymptomatic, and in fact, are somewhat pro-tected against malaria, a disease caused by the Plamodium falciparum parasite. The relativelyhigh prevalence of the HbS allele has been attributed to this protective effect. In support ofthis premise, the occurrence of the HbS allele is highest in the low-lying areas of west andcentral Africa, where malaria is endemic.

    Homozygotes have the disease

    In contrast to heterozygotes, homozygous individuals, who have had the misfortune of in-heriting an HbS allele from both parents, are often severely affected by the disease. Thesubstitution of a valine for glutamic acid increases hemoglobins hydrophobicity, which fa-vors self-association into long, rigid fibers that distort the red blood cell into the charac-teristic crescent, or sickle shape. Sickled cells are rigid and irregularly shaped, and gettrapped in the narrow capillaries that permeate the bodily organs. Once sickled red bloodcells occlude a capillary, the obstruction can become still more impenetrable as more cellsback up behind it. Herein lies the most problematic feature of the illness: as will be de-scribed below, almost all of the symptoms and pathophysiology of the disease have theseepisodes of vaso-occlusion at their source.

    The anemia associated with the disease is a result of the increased fragility of red bloodcells in affected individuals. Sickling damages the cell membrane and renders cells more sus-ceptible to lysis, with the average half life of an erythrocyte being 1020 days in a patient withsickle-cell disease rather than the usual 120. This shortened life span results in a chronicshortage of red blood cells (anemia) and an increased turnover of the hemoglobin withinthem. Bilirubin is a breakdown product of hemoglobin, explaining the elevated levels of thecompound observed in the patient.


    Sickle-cell disease affects millions of people worldwide. It is most common in west, central,and east African countries, where the prevalence of sickle-cell trait in some regions reachesas high as 40%. Generally, the regions with the highest prevalence are low lying and wet re-gions where the Anopheles mosquito that transmits malaria is most often found. This sup-ports the assumption that sickle-cell trait persisted because it provides a survival advantagein those suffering from malaria. In the United States, approximately 8% of African Americanshave sickle-cell trait, and it is also found, but to a lesser extent, among individuals ofMediterranean, Middle Eastern, and East Indian origin.


  • 4 CHAPTER 10

    What Causes Cells to Sickle?

    Erythrocytes are packed with hemoglobin. In a healthy individual hemoglobin molecules areestimated to be separated by less than a molecules width, and the concentration is typicallyeven higher in those with sickle-cell disease due to dehydration of their red blood cells. In thiscontext, the single valine substitution in HbS is sufficient to drive the protein to self-associate.

    Formation of the double strand

    The valine residue creates a hydrophobic patch, which is well situated to interact with anothertetramer via a second hydrophobic patch of the -globin chain consisting of phenelalanine-85and leucine-88. The patch made up of phe-85 and leu-88 is exposed only when hemoglobin isdeoxygenated (the T state of the molecule), which explains why sickling usually occurs underconditions of low oxygen tension (such as in the organs or muscles). Both chains of a tetramerparticipate in this type of contact, and the ensuing structure is a slightly twisted double strand,with the tetramers oriented in an energetically favorable staggered arrangement (Figure 10.1).

    Formation of higher order structures

    The double strand of sickled hemoglobin then serves as a building block for higher-orderstructures. The fibers within sickled cells actually consist of seven pairs of strands, twistedinto a rope-like helical arrangement, with two central pairs surrounded by five outer pairs(see Figure 10.1). These, then, can be ordered into larger domains in which parallel align-ments of fibers form sheets, which transform the cell from its usual shape into the aberrantsickle or holly leaf forms typical of the disease (Figure 10.2).

    FIGURE 10.1. Polymerization of deoxyhemoglobin S. The left panel depicts the interactions between subunitsthat result in the formation of the double strand. The right panel depicts the 14-stranded fiber.

    ADouble strand 14-stranded fiber



    FIGURE 10.2. Smears of peripheral blood from a normal control (left) and a patient with sickle-cell disease(right). Photomicrographs courtesy of Dr. Gregory Kato, Johns Hopkins University.

  • Fiber formation is cooperative

    Fiber formation has been found to be highly cooperativealthough the initiation of a fiberis difficult, once a nucleus of 10 or so tetramers has formed the fiber will rapidly con-tinue to grow. Thus, polymerization is exquisitely sensitive to HbS concentration, whichexplains why heterozygotes, with half as much HbS as homozygotes, are usually asymp-tomatic. Decreasing the concentration of HbS by half increases the time for fiber forma-tion 1000-fold. By this time a red blood cell will have traversed the microcapillaries wheresickling is most likely to occur and reached the relative safety of the larger veins leadingto the lungs.

    Episodes of Vaso-Occlusion Are the Most Troublesome Feature of Sickle-Cell Disease

    The episodes are extremely painful

    The vaso-occlusive events common in those with sickle-cell disease cause painful crises,the most distressing symptom of the illness. Painful crises can be triggered by a minor ill-ness, overexertion, minor trauma, or cold weather, but they often appear unpredictably. Notethat the patient was admitted for a painful crisis following a hiking excursion, and thus thecrisis was likely triggered by over exertion. During exercise the rapidly metabolizing musclecells tend to deplete hemoglobin of its oxygen, and this together with dehydration can trig-ger a crisis. Crises vary greatly in severity and frequency. They can occur from less than onceto 15 or 20 times per year, and can last for hours to weeks. Treatment for these crises islargely symptomatic but can also include some measures to minimize sickling. Note thatupon admission the patient in the case was treated with an analgesic to control the pain andoxygen therapy to reduce sickling.

    In addition to causing severe pain, vaso-occlusive crises, in blocking food flow and de-priving bodily tissues of nutrients and oxygen, can cause damage to vital organs, which overtime can compromise their function. Kidney damage is common, and the spleen is commonlyaffected as well, resulting in increased susceptibility to infections, especially those of bacter-ial origin. Prior to a landmark study in 1986 that showed marked improvement in prognosiswith long-term treatment with penicillin, pneumococcal infections were a leading cause ofdeath in children with sickle-cell disease. Hence the long-term treatment of the patient dis-cussed here with penicillin throughout his early childhood period.

    Sickling damages red blood cells and causes anemia

    The anemia associated with the disease is a result of the increased fragility of red blood cellsin affected individuals. Sickling damages the cell membrane and renders cells more suscep-tible to lysis, with the average half-life of an erythrocyte being 1020 days in a patient withsickle-cell disease, rather than the usual 120. This shortened life span results in a chronicshortage of red blood cells (anemia) and an increased turnover of the hemoglobin withinthem. Bilirubin is a breakdown product of hemoglobin, explaining the elevated levels of thecompound observed in the patient.

    Acute Chest Syndrome

    Acute chest syndrome (ACS) is a life-threatening complication of sickle-cell disease, which oc-curs in approximately 50% of patients and is fatal in 1020% of cases. It is likely caused by anocclusion in the lung vasculature, and can lead to respiratory failure and death if not treated ag-gressively. It is often accompanied by an infection, and, in fact, infection may be the very causefor the episode. Infection can deplete a tissue of oxygen, thus triggering the sickling that leads to


  • 6 CHAPTER 10

    the pulmonary occlusion. Fortunately, the patient in the case was rapidly diagnosed and treatedwith antibiotics, oxygen, and blood transfusion. He was, in fact, treated with an exchange trans-fusion rather than a simple transfusion. In substituting his HbS erythrocytes with those from ahealthy donor, the diseased cells were greatly diluted, reducing sickling and the risk of furthervaso-occlusion.

    Rapid diagnosis and treatment is one of the most important prognostic factors for ACS,yet diagnosis of the syndrome is notoriously difficult. The symptoms are variable and oftenmimic those of an infection such as pneumonia. Common symptoms are fever, cough, wheez-ing, chest pain, and a new pulmonary infiltrate often appears on a chest x-ray. The Boy Scoutthus exhibited classic symptoms. ACS often occurs in the course of a painful crisis, and ismore common though usually less severe in children than adults. Improved prognosis is as-sociated with limited pulmonary infiltrates and mild hypoxemia (low blood oxygen). However,ACS tends to recur and repeated episodes are associated with poor long-term prognosis.


    1. Hydroxyurea, a drug first used to treat cancer, was later found to be effective in the treat-ment of sickle-cell disease. In cancer patients the drug works by inhibiting DNA repli-cation and cell division, whereas in the treatment of sickle-cell disease, it acts bystimulating the expression of fetal hemoglobin (HbF). HbF, like the adult form, is atetramer consisting of the same two a chains as the adult form, but rather than two bchains, the HbF tetramer consists of two -globin chains. The non-copolymerization ofHbS and HbF interferes with fiber formation and hence sickling. The boy described inthe case study was not treated with hydroxyurea. Can you think of a reason why?

    2. If you were asked to design a drug for the treatment of sickle-cell disease, which patho-physiological features of the disease might you target?

    3. When a malaria parasite (plasmodium species) invades a red blood cell, its metabolicwaste products result in acidification of the interior of the cell, which favors the T formof hemoglobin and can trigger sickling in carriers of sickle-cell disease (those with onedisease gene copy and one normal copy). Sickling weakens the cell and makes it less hos-pitable to the parasite, rendering these individuals somewhat resistant to the disease. Ifinfection with the malaria parasite had favored the R form of hemoglobin rather than theT form, do you think that the HbS mutation would have become as prevalent as it has?

    4. Explain the therapeutic effect of supplemental oxygen in sickle-cell patients withpainful crises.

    5. Why do you think those with sickle-cell disease often have enlarged hearts?

    6. Those homozygous for the HbS allele were previously referred to as having sickle-cellanemia. The disease was renamed sickle-cell disease to include other illnesses causedby hemoglobin mutations, and because the term sickle-cell anemia was somewhat ofa misnomer. Although HbS homozygotes are anemic due to the shortened life-spans oftheir erythrocytes, anemia is not the most troubling feature of the disease. In fact, it mayeven be somewhat of an advantage in terms of minimizing the severity of vaso-occlusiveevents. Can you explain why?

    7. The patient in the case study was treated with an exchange transfusion when he devel-oped ACS. A transfusion helps ameliorate the situation by diluting the patients HbS ery-throcytes with normal HbA erythrocytes. In addition, transfusion temporarily inhibitserythropoiesis (red blood cell production). Would you view this as an advantage or dis-advantage in this situation?

  • 8. The most widely used test for diagnosing sickle-cell disease is hemoglobin elec-trophoresis. The test is based on the different rates of migration of HbA and HbS inan electric field. Figure 10.3 illustrates a gel electrophoresis pattern in which lane Arepresents HbA and lane B represents HbS. Lane C represents the hemoglobin froma patient who may have sickle-cell disease. Does the patient have sickle-cell disease?How do you explain the difference in migration pattern between HbA and HbS?

    9. A young couple, each known to carry a single copy of the HbS hemoglobin variant,comes to you, a genetic counselor, for advice regarding their wish to have children. Whatare their chances of having a child with sickle-cell disease?

    10. ACS is often treated with blood transfusion, and once an individual has been struck withACS, it typically recurs. Can you think of a problem that might occur with repeatedtransfusions?

    11. 2,3-diphosphoglycerate (2,3-DPG) is the predominant phosphorylated compound inred blood cells, accounting for about 2/3 of red-blood-cell phosphorus. 2,3-DPG sta-blizes the T form of hemoglobin and is increased in situations associated with hypox-emia (low levels of oxygen in the blood). Can you predict what effect 2,3-DPG has onthe hemoglobin-oxygen dissociation curve and tissue delivery of oxygen?

    12. Why have prophylactic antibiotics proven useful in extending the life of sickle-cellpatients?


    1. Bunn, H. Franklin Pathogenesis and treatment of sickle-cell disease. New England Journalof Medicine 337(11)(1997):762769.

    2. Edelstein, S. J. The sickled cell: From myths to molecules. Harvard University Press (1986).


    FIGURE 10.3. Gel electrophoresis pattern of hemoglobin from a normal individual (lane A), a person withsickle-cell anemia (lane B), and from a test patient (lane C).










  • 8 CHAPTER 10

    3. Herrick, J. B. Peculiar elongated and sickle-shaped red blood corpuscles in a case of se-vere disease. Archives of Internal Medicine 6(1910):517521. (This is the first descriptionof a case of sickle-cell disease.)

    4. Steinberg M. H. Management of sickle-cell disease. New England Journal of Medicine340(13)(1999):10211030.

    For further information, see the following web sites:

    The Sickle-Cell Disease Association of America: www.sicklecelldisease.org

    National Institutes of HealthMedlinePlus: www.nlm.nih.gov/medlineplus/sicklecelldisease.html

    National Center for Biotechnology Information. Online Mendelian Inheritance in Man(OMIM): www3.ncbi.nlm.nih.gov:80/htbin-post/Omim/dispmim?603903


    1. Although used at lower dosages in treating sickle-cell disease than in treating cancer, itis not yet known how a drug that inhibits cell proliferation will affect the growth anddevelopment of children. Thus, hydroxyurea is rarely used in children.

    2. (a) A drug that increases the affinity of hemoglobin for oxygen would tend to keep he-moglobin in the R form, which does not form fibers.

    (b) A drug that inhibits the adhesion molecules on the surface of sickled cells might re-duce sticking to the walls of blood vessels (certain adhesion molecules are expressedat unusually high levels in HbS erythrocytes).

    (c) A drug that targets the intermolecular interactions between hemoglobin moleculeseither within a fiber or between fibers. (Choosing this route will be a challenge giventhe very large amounts of hemoglobin in erythrocytes. Large amounts of drug maybe necessary, which increases the chance of toxicity.)

    (d) A drug that increases the expression of fetal hemoglobin would inhibit fiber formation.

    (e) A drug that reduces red cell dehydration would reduce the intracellular hemoglobinconcentration and might minimize fiber formation.

    (f) A drug that reduces -globin expression (lowered -globin expression [-thalassemia]is associated with reduced severity of symptoms in those with sickle-cell disease, per-haps due to the lower levels of hemoglobin associated with -thalassemia).

    3. The R form of hemoglobin does not form fibers, which are what ultimately result in lim-iting the progression of the infection in heterozygous individuals. Thus, if the parasitewere to have induced the R form, the prevalence of the HbS mutation would probablynever have become so nearly so wide.

    4. The mutation in HbS facilitates a hydrophobic interaction between -globin chains thatoccurs when the molecule is in the T form (deoxygenated). Supplemental oxygen willincrease occupancy and favor the R form, preventing -globin chain interaction and sick-ling, with consequent vaso-occlusion.

    5. Damage to the lungs caused by repeated vaso-occlusive events results in poor oxygena-tion of the blood. The heart then tries to compensate by pumping more blood, whichresults in enlargement. However, hypoxemia and vaso-occlusive events in the lungs neednot be present in sickle-cell patients with enlarged hearts (cardiomegaly). Anemia is alsoa cause of cardiac enlargement because it imposes a chronic volume load on the heart.The heart must pump more blood to deliver the same amount of oxygen to the tissues.

    6. Most of the pathophysiology of the disease is caused by sickling of red blood cells, withthe resultant blockage of a blood vessel. The fewer the circulating red blood cells, theless likely a blockage is to occur; should one occur, the more likely it is to dissolve be-fore it becomes a more severe obstruction leading to a painful crisis.



    7. It is an advantage. Inhibition of erythropoiesis in a patient experiencing ACS will inhibitthe production of the patients HbS erythrocytes. It will thus keep the percentage of cir-culating HbS erythrocytes low for a longer period of time, thus improving the prognosis.

    8. The patient expresses both HbA and HbS and so has sickle-cell trait rather than sickle-cell disease. The substitution of a negatively charged residue (glutamic acid) with anuncharged residue (valine) in HbS causes the protein to migrate more slowly than wildtype toward the positive pole in an electric field, thus explaining the pattern depicted inthe figure.

    9. The disease is inherited in an autosomal recessive manner, and, thus, with both parentscarrying a copy of the disease gene, each of their children has a 25% chance of havingthe disease. Prenatal testing of a fetus and pre-implantation genetic testing of an embryocan be done to test for the disease. In the United States, those of African descent are mostlikely to be carriers of the disease, with 8% carrying the HbS allele.

    10. In the United States sickle-cell disease is most common among those of African descent,while the blood used for transfusion is derived from individuals of all backgrounds, es-pecially those of European ancestry. It is thus possible for the recipient of a blood trans-fusion to elicit an immune reaction to minor antigens present on donor red blood cells.This can result in a deadly reaction to subsequent transfusions unless donor and recip-ient are carefully matched.

    11. 2,3-DPG shifts the Hb-oxygen dissociation curve to the right, decreasing the oxygenaffinity of hemoglobin (an effect similar to that of low pH). The consequence of this shiftis that at any given oxygen tension hemoglobin is less saturated (has lower affinity) andmore easily releases bound oxygen in the tissues. Acute and chronic hypoxemia, such asthat associated with high altitude and heart or lung disease, is associated with higher2,3-DPG levels, enhancing tissue delivery of oxygen.

    12. The spleen of patients with sickle-cell disease is hypofunctional, possibly the result ofrecurrent vaso-occlusion and infarction (destruction due to the absence of blood flow).The spleen is a central location for clearance of microorganisms, particularly encapsu-lated bacteria. Antibiotic therapy, in part, compensates for reduced function of the spleenin patients with sickle-cell disease.