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Structure-Function Relations of Human

Hemoglobins

Alain J. Marengo-Rowe

To cite this article: Alain J. Marengo-Rowe (2006) Structure-Function Relations of

Human Hemoglobins, Baylor University Medical Center Proceedings, 19:3, 239-245, DOI:
10.1080/08998280.2006.11928171

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Structure-function relations of human hemoglobins
Alain J. Marengo-Rowe, MD

cidation of the structure of hemoglobin (1). For this endeavor

In 1949 Pauling and his associates showed that sickle cell hemoglobin he was awarded the Nobel Prize in chemistry in 1962.
(HbS) belonged to an abnormal molecular species. In 1958 Ingram, who In 1957 Ingram demonstrated that sickle cell anemia was
used a two-dimensional system of electrophoresis and chromatography to caused by the replacement of one of the 287 amino acid residues
break down the hemoglobin molecule into a mixture of smaller peptides, in the half molecule of hemoglobin (2). is finding facilitated
defined the molecular defect in HbS by showing that it differed from nor- understanding of disease at the molecular level, since for the first
mal adult hemoglobin by only a single peptide. Since then, more than 200 time a point mutation in a structural gene was shown to cause
variant and abnormal hemoglobins have been described. Furthermore, the substitution of one amino acid in the protein controlled by
the construction of an atomic model of the hemoglobin molecule based that gene. Furthermore, the accumulation of the sickle cell gene
on a high-resolution x-ray analysis by Dr. Max Perutz at Cambridge has in malarial regions of the world became a convincing illustration
permitted the study of the stereochemical part played by the amino of evolution by natural selection (3). Persons with the sickle cell
acid residues, which were replaced, deleted, or added to in each of trait (HbA/S) have a selective advantage over normal individu-
the hemoglobin variants. Some of the variants have been associated als when they contract falciparum malaria because the parasite
with clinical conditions. The demonstration of a molecular basis for a count remains low and lethal cerebral malaria is avoided.
disease was a significant turning point in medicine. A new engineered To date, well over 200 hemoglobin variants have been de-
hemoglobin derived from crocodile blood, with markedly reduced oxygen scribed. e term “variant” rather than “abnormal” is preferred
affinity and increased oxygen delivery to the tissues, points the way for because most hemoglobins are not associated with disease. e
future advances in medicine. late Professor Herman Lehmann at Cambridge University in
England and his “musketeers” in different parts of the world
have been responsible for discovering many of these variants.

H
emoglobin has played a spectacular role in the history Furthermore, as knowledge accumulated, it became evident
of biology, chemistry, and medicine. is paper, written that the structure-function relations of various hemoglobins in
primarily for the clinician, is a brief outline of the com- stereochemical terms could be related to clinical symptomatol-
plex problems associated with abnormal hemoglobins. ogy (4, 5).
e thalassemias have been intentionally omitted and will be
presented in a separate publication. STRUCTURE OF HEMOGLOBIN
Hemoglobin is a two-way respiratory carrier, transporting Hemoglobin comprises four subunits, each having one poly-
oxygen from the lungs to the tissues and facilitating the return peptide chain and one heme group (Figure 1). All hemoglobins
transport of carbon dioxide. In the arterial circulation, hemoglo- carry the same prosthetic heme group iron protoporphyrin IX
bin has a high affinity for oxygen and a low affinity for carbon associated with a polypeptide chain of 141 (alpha) and 146
dioxide, organic phosphates, and hydrogen and chloride ions. (beta) amino acid residues. e ferrous ion of the heme is
In the venous circulation, these relative affinities are reversed. linked to the N of a histidine. e porphyrin ring is wedged
To stress these remarkable properties, Jacques Monod conferred into its pocket by a phenylalanine of its polypeptide chain. e
on hemoglobin the title of “honorary enzyme.” If we call heme polypeptide chains of adult hemoglobin themselves are of two
its active site, oxygen its substrate, and hydrogen ions its in- kinds, known as alpha and beta chains, similar in length but
hibitors, then hemoglobin mimics the properties of an enzyme. differing in amino acid sequence. e alpha chain of all hu-
erefore, it became evident that unraveling the properties of man hemoglobins, embryonic and adult, is the same. e non-
hemoglobin was necessary to understanding the mechanism of
hemoglobin function as it pertains to respiratory physiology. From the Department of Pathology, Baylor University Medical Center, Dallas,
In 1937, Dr. G. S. Adair gave Dr. Max Perutz crystals of Texas.
horse hemoglobin (personal communication, Max Perutz, Corresponding author: Alain J. Marengo-Rowe, MD, Department of Pathology,
1966). is started Dr. Perutz on the path that led to the elu- Baylor University Medical Center, 3500 Gaston Avenue, Dallas, Texas 75246.

Proc (Bayl Univ Med Cent) 2006;19:239–245 239

 Lug worm
100 Man + H++ BPG + CO2

Hemoglobin
Scuba

Oxygen saturation (%)

50

50 100
Partial pressure of oxygen (mm Hg)
Figure 2. Diagrammatic representation of oxygen equilibrium curves of the lug
Figure 1. Model of the hemoglobin molecule. Two identical white (alpha) poly- worm, man, and hemoglobin Scuba. The effect of hydrogen ions, 2,3-bisphospho-
peptide chains and two identical black (beta) polypeptide chains form a complete glycerate, and carbon dioxide (H+ + BPG + CO2) is to promote a right shift. If man
molecule. The hemes are shown as discs. O2 marks the oxygen binding site. had the hemoglobin of the lug worm (left shift), he would die of anoxia.
Reprinted courtesy of Dr. Max Perutz.
the ferrous atom than oxygen does. Once carboxyhemoglobin is
alpha chains include the beta chain of normal adult hemoglobin formed, oxygen cannot displace carbon monoxide to any extent.
(α2β2), the gamma chain of fetal hemoglobin (α2γ2), and the is forms the molecular basis of coal gas poisoning.
delta chain of HbA2. In some variants, the gamma genes are In the body, the adequacy of the oxygen transport system
duplicated, giving rise to two kinds of gamma chains. depends on the adequacy of oxygenation of blood in the lungs,
Oxygen binds reversibly to the ferrous iron atom in each the rate and distribution of blood flow, the oxygen-carrying
heme group. e heme group that has become oxygen bound capacity of the blood (hemoglobin concentration), and the
varies with the partial pressure of oxygen. e sigmoid shape affinity of hemoglobin for oxygen so as to allow unloading
of the oxygen equilibrium curve shows that there is cooperative of oxygen in peripheral capillaries. Hence, the availability of
interaction between oxygen binding sites. Hence, as oxygen- oxygen to the body may be altered by abnormalities at any
ation proceeds, combination with further molecules of oxygen point in this physiological pathway. In this paper, only the role
is made easier. e oxygen equilibrium (or dissociation) curve of hemoglobin affinity for oxygen will be considered as variant
is not linear but S-shaped and varies according to environments forms of hemoglobin are discussed.
and species (Figure 2). At a partial pressure of oxygen of 100
mm Hg, the hemoglobin in the red cell is fully saturated with SICKLE CELL HEMOGLOBIN
oxygen. e dissociation curve is plotted as percentage of oxygen Sickle cell hemoglobin (HbS) has existed in humans for
saturation against partial pressure. thousands of years. Dr. Konotey-Ahulu, a Ghanaian physician,
e structure of hemoglobin has been extensively studied reports that among West African tribes, specific names were
by x-ray analysis (6). e arrangement of the subunits—which assigned to clinical syndromes identifiable as sickle cell anemia
is known as the quaternary structure—differs in the oxy- and (7). However, sickle cells were first described in the peripheral
deoxyhemoglobin. blood of an anemic patient from the West Indies by the Chicago
In human hemoglobin, the fit between the polypeptide physician Robert Herrick in 1910 (8). While homozygous sickle
chain is critical because the gap between two of the polypeptide cell anemia is the most common and severe form of sickle cell
chains in the hemoglobin molecule becomes narrower when ox- disease (SCD), other sickling disorders combining HbS with
ygen molecules become attached to the ferrous atoms. is has beta or alpha thalassemia, hemoglobin C, hemoglobin D, and
been likened by Max Perutz to a molecular form of paradoxical other hemoglobins share a similar pathophysiology with com-
breathing: unlike the lungs, the hemoglobin molecule contracts mon as well as distinguishing clinical features.
when oxygen enters and expands when oxygen leaves. HbS results from a single base-pair mutation in the gene
Compounds other than oxygen, such as nitric oxide and car- for the beta-globin chain of adult hemoglobin. An adenine-to-
bon monoxide, also are able to combine with the ferrous atom thymine substitution in the sixth codon replaces glutamic acid
of hemoglobin. Carbon monoxide attaches itself more firmly to with valine in the sixth amino acid position of the beta-globin

240 Baylor University Medical Center Proceedings Volume 19, Number 3

chain (9, 10). is substitution yields the electrophoretically aplastic. Damage to the red blood cell membrane gives rise to
distinct hemoglobin described by Linus Pauling in 1949 (11). reduced cell survival and chronic hemolytic anemia. If severe
In the deoxygenated form of HbS, the beta-6 valine becomes enough, this damage increases the risk of bilirubin gallstone for-
buried in a hydrophobic pocket on an adjacent beta-globin mation, stroke, and heart failure. Also, the anemia is aggravated
chain, joining the molecules together to form insoluble poly- by the mechanical impedance to blood flow caused by sickled red
mers (9). In sufficient concentration, these insoluble polymers blood cells, resulting in widespread vasoocclusive complications.
give rise to the classical sickle morphology. is process causes Interestingly, the anemia to some degree can be protective against
severe damage to the red cell membrane. Sickled red cells may vasoocclusive complications, as it moderates the increase in vis-
then aggregate and go on to cause microvascular obstruction. cosity associated with sickling in the microcirculation. Hence,
Also, these abnormal red cells adhere to endothelial cells (12) judicious exchange transfusion therapy and blood transfusion
and can interact with various cytokines (13). is indicated for the prevention of pain crises, stroke, pulmonary
e process of microthrombosis and microembolization is hypertension, and other related conditions (19).
the foundation of SCD pathology. Occlusion of the microvas- Blood transfusion not only increases the oxygen-carrying
culature by sickled erythrocytes causes painful crises, priapism, capacity of blood but also decreases the percentage of cells
pulmonary emboli, and osteonecrosis, and ultimately damages capable of sickling. It is recommended that transfusion should
every organ system including the retinae, spleen, liver, and be carried out with phenotypically matched, leuko-reduced,
kidneys. Many patients with SCD have hematocrits of 20% sickle-cell–negative blood in order to attain a posttransfusion
to 35% and chronic reticulocytosis. Clinical symptoms can be hematocrit of about 36%. (20). e complications of transfu-
precipitated by fever, infection, excessive exercise, temperature sion are well known and include allo- and autoimmunization,
changes, hypoxia, and hypertonic solutions. e clinical severity iron overload, and the transmission of infectious diseases such
of the symptoms experienced is related to the concentration of as hepatitis and HIV. Also, a considerable number of patients
HbS in the red blood cell and expression of other hemoglobins, with sickle cell anemia worldwide have undergone successful
endothelial factors, nitric oxide and other factors. Also, patients bone marrow transplantation (21). Only selected patients are
with SCD have a higher proportion of dense, dehydrated eligible for the procedure. Even then, bone marrow transplanta-
erythrocytes (14). tion was associated with a 5% to 10% mortality, mostly from
In about 11% of SCD patients under 20 years of age, stroke graft-versus-host disease.
occurs because of stenotic cranial artery lesions, demonstrable Another approach to reducing the effect of HbS polymer
by transcranial Doppler ultrasonography. A regular program of formation has been to augment the production of fetal hemo-
transfusion aimed at reducing the sickle cell population to <50% globin (HbF). rough population and clinical observation, it
prevents about 90% of stroke cases. Unfortunately, the high risk has long been recognized that higher blood HbF levels corre-
of stroke returns after transfusion is discontinued (15). late with fewer clinical manifestations of SCD. Pharmacologic
e surface of HbS consists mainly of hydrophilic amino manipulation of HbF in the therapy of sickling disorders has
acid side chains together with some smaller hydrophobic side been proposed since the mid-1950s. To date, several agents have
chains. Since adult hemoglobin is present at a very high concen- been tried, but the safest and most effective has proven to be
tration within the red cell and yet appears to remain free from hydroxyurea (22). e mechanism of increased HbF production
aggregation at all levels of saturation with oxygen, the amino by hydroxyurea is not fully understood. Also, recent studies have
acids on the surface of the molecule must be arranged so as to found that hydroxyurea contributes to the production of nitric
avoid attraction between adjacent molecules. Of the majority of acid, a potent endothelial relaxing factor (23).
hemoglobin variants with surface amino acid substitutions, only Numerous inflammatory markers associated with endo-
a minority are associated with any significant clinical abnormali- thelial surfaces and white blood cells are elevated in SCD,
ties. Except for HbS, none of those more common hemoglobins including C-reactive protein. Baseline granulocyte counts are
found in the homozygous state, such as hemoglobins C, D, and often increased. Leukocytosis itself is a risk factor for increased
E, are associated with any greater abnormality than mild anemia. mortality (24). Finally, laminin, a constituent of the endothe-
e surface of hemoglobin A is therefore able to accommodate lial matrix that binds to the Lutheran antigen on red cells, is
a variety of different amino acid changes without its structure expressed on sickled red blood cells in greater quantities than
or function being affected (16). on normal red blood cells (25).
e valine-for-glutamic acid substitution has very little ef- Almost every aspect of hemostasis tending to hypercoagu-
fect on the oxygenated form of HbS (17). However, when the lability has been described in SCD (26). However, it is not
concentration of deoxygenated HbS becomes sufficiently great, known whether the hypercoagulability is the cause or the result
its properties differ markedly from those of deoxygenated hemo- of vasoocclusion. rombocytosis is due to hyposplenia, and
globin A, causing the formation of insoluble fibers and bundles, platelet aggregation is increased (27). Antiphospholipid anti-
which distort the red blood cell into the sickle shape. bodies may be elevated, and protein C and S levels are decreased
Since the discovery of HbS, the clinical symptomatology (28). Also, high levels of von Willebrand factor and factor VIII
and associated pathophysiology of SCD have gradually been can be found (29). erapeutic trials of heparins, coumadin,
elucidated (18). SCD is characterized by anemia and four types and antiplatelet agents have been limited, yielding inconclusive
of crises: painful (vasoocclusive), sequestrative, hemolytic, and information, but they are ongoing.

July 2006 Structure-function relations of human hemoglobins 241

 individuals give up less oxygen to the tissues. e
Table 1. Examples of hemoglobins with increased oxygen affinity relative anoxia increases erythropoietin production
and causes polycythemia.
Site in molecule
Hemoglobin Substitution affected p50 Reference Most of the abnormal hemoglobins with
Hb Chesapeake α92 (Arg→Leu) α1β2 contact 19.0 Charache et al, 1966 (32)
increased oxygen affinity manifest themselves by
causing polycythemia in the carrier. e increased
Hb J Capetown α92 (Arg→Gln) α1β2 contact ↓ Botha et al, 1966 (33)
oxygen affinity reduces tissue oxygen delivery, caus-
Hb Yakima β99 (Asp→His) α1β2 contact 12.0 Jones et al, 1967 (34)
ing an increase in erythropoietin secretion and in
Hb Brigham β100 (Pro→Leu) α1β2 contact 19.6 Lokich et al, 1973 (35) red cell mass. e possibility of an abnormal hemo-
Hb Rainer β145 (Tyr→Cys) C-terminal 12.9 Adamson et al, 1969 (36) globin with high oxygen affinity should be consid-
Hb Bethesda β145 (Tyr→His) C-terminal 12.8 Bunn et al, 1972 (37) ered in those atypical patients with polycythemia
Hb Syracuse β143 (His→Pro) BPG β contact 11.0 Jensen et al, 1975 (38) in which the white blood cell and platelet counts
p50 indicates 50% saturation of hemoglobin. are not elevated and splenomegaly is absent. e
importance of establishing the correct diagnosis is
mainly to protect the patient from the chemothera-
peutic treatment of polycythemia. Family members should be
Table 2. Examples of hemoglobins with reduced oxygen affinity advised that their children may be affected. e life expectancy
of affected individuals is essentially normal, and most patients
Hemoglobin Substitution p50 Reference
are symptom free. However, if such patients become symptom-
Hb Kansas β102 (Asn→Thr) 70 Reissmann et al, 1961 (39)
atic and their hematocrit rises towards 60%, then phlebotomy
Hb Yoshizuka β108 (Asn→Asp) ↑ Imamura et al, 1969 (40) may be necessary to reduce blood viscosity.
Hb Agenogi β90 (Gln→Lys) ↑ Imai et al, 1970 (41)
p50 indicates 50% saturation of hemoglobin. Reduced oxygen affinity
Only a handful of abnormal hemoglobins have been reported
in which a reduced oxygen affinity is the sole abnormality (Table
HEMOGLOBINS WITH ALTERED OXYGEN AFFINITY 2). Because of the increased oxygen delivery resulting from the
e hemoglobin loading and unloading of oxygen can be low oxygen affinity, it might be expected that the erythropoietin
expressed by an oxygen dissociation curve. e physiologic con- response would be reduced and these variants would be associ-
sequences of the abnormal hemoglobins depend on the oxygen ated with mild anemia. While this response occurs in most of
affinity, which defines the point of 50% saturation (p50). e these variants, it is not so with Hb Kansas carriers. With Hb
oxygen dissociation curve of normal hemoglobin represents the Kansas, the oxygen affinity is so low that even at normal arte-
reaction of hemoglobin with oxygen as modified by hydrogen rial oxygen tensions there is sufficient desaturation to give rise
ions (Bohr effect) and 2,3-bisphosphoglycerate (BPG) (30, 31). to clinical cyanosis. e possibility of low-affinity hemoglobins
Hemoglobin oxygen affinity increases with falling temperature should be considered in patients with low hematocrit or cyanosis
and decreases with rising pH and 2,3-BPG. Hence, red blood with no other cause apparent after evaluation. e p50 is usu-
cells containing such an abnormal hemoglobin may have an ally elevated. Despite these findings, patients usually require no
abnormal oxygen dissociation curve because of 1) an intrinsic specific treatment once the correct diagnosis is established.
abnormality of hemoglobin-oxygen dissociation, 2) an altered
interaction of hemoglobin with BPG, 3) an altered Bohr effect, THE UNSTABLE HEMOGLOBINS
or 4) a combination of any or all of the above. It is common At the molecular level, considering the three-dimensional
to speak of the oxygen-dissociation curve as being shifted to model of the hemoglobin molecule, it would appear that the
the left (increased oxygen affinity) or to the right (decreased stability of the hemoglobin tetramer is dependent on both the
oxygen affinity). internal molecular positioning of nonpolar amino acids and
the stability of the large α1β1 contacts. ese properties serve
Increased oxygen affinity to hold the four chains together. In most unstable hemoglobins
Some hemoglobins have been described in which the as- one or more of these properties have been disrupted.
sociated clinical manifestations can be ascribed to an increased Unstable hemoglobins are hemoglobins that, because of
oxygen affinity (Table 1). High-affinity hemoglobins bind oxy- the nature of the substitution, deletion, or insertion of amino
gen more readily and deliver less oxygen to tissues. acids (Table 3), tend to undergo spontaneous oxidation within
Several hemoglobins with increased oxygen affinity have the red cell and precipitate to form insoluble inclusions called
substitutions affecting the α1β2 contact of the tetramer. Oth- Heinz bodies. eir presence results in the so-called congenital
ers have substitutions involving the C-terminal residues of the Heinz body hemolytic anemia. Most patients with this condi-
beta chain or of the BPG binding sites. All these substitutions tion are found to have a nonspherocytic hemolytic anemia.
favor the oxygenated conformation and cause a left shift of the e anemia is exacerbated by infections and oxidative drugs
oxygen dissociation curve, which reflects an increased blood af- such as sulfonamides, pyridium, and antimalarials. It must be
finity for oxygen. erefore, it follows that the red cells of such remembered that the normal red cell is undergoing continual

242 Baylor University Medical Center Proceedings Volume 19, Number 3

 Table 3. Examples of unstable hemoglobins

Hemoglobin Substitution Reference Histidine

Hb Köln β98 (Val→Met) Carrell et al, 1966 (42)
Hb Hammersmith β42 (Phe→Ser) Dacie et al, 1967 (43) Porphyrin
Hb Bristol β67 (Val→Asp) Sakuragawa et al, 1984 (44) Fe++
Hb Gun Hill β91→95 deleted Murari et al, 1977 (45) O2 O2
β73→75 deletion,
Hb Montreal Plaseska et al, 1991 (46)
insertion

Histidine
physical stress and has to be able to deform in arterioles in order
to travel through the microcirculation. e insoluble Heinz
bodies are torn out of the red cell during passage in the micro- Figure 3. Diagrammatic representation of the heme pocket
circulation of the spleen, which is ≤3 microns across (47). In formed by amino acids. Oxygenation can occur only between the
such circumstances, Heinz bodies are pitted out of the red cell non–heme-linked histidine and iron.
along with some membrane, leading to the presence of “bite
cells” in the peripheral smear. Other disturbances such as K+
and Ca++ changes are secondary to the physical damage caused Table 4. Examples of hemoglobin M variants
by Heinz bodies.
Clinical
e first report of a child with idiopathic congenital non- Hemoglobin Substitution presentation Reference
spherocytic hemolytic anemia associated with cyanosis and Gerald and Efron,
splenomegaly is attributed to Cathie (48). e patient was a HbM Boston α58 (His→Tyr) Cyanosis at birth
1961 (50)
small boy. His spleen was removed and, several months later, Stavem et al,
the red cells were found to contain numerous Heinz bodies. In HbM Saskatoon β63 (His→Tyr) Cyanosis
1972 (51)
1966 Carrell et al described the amino acid substitution giving Hayashi et al,
HbM Iwate α87 (His→Tyr) Cyanosis at birth
rise to an unstable hemoglobin (Hb Köln) as the cause of this 1966 (52)
anemia (42). Hutt et al,
HbM Hyde Park β92 (His→Tyr) Cyanosis
e clinical findings in patients suffering from unstable 1998 (53)
hemoglobin disease include neonatal jaundice, anemia, cya- Hain et al,
HbFM Fort Ripley α92 (His→Tyr) Cyanosis at birth
nosis, pigmenturia, splenomegaly, and drug intolerance. e 1994 (54)
severity of the disease is highly dependent on the degree of
instability of the abnormal hemoglobins. e disorder is clearly
expressed in heterozygotes, and it seems likely that with most “heme pocket” to the amino acid residue histidine—the proxi-
substitutions or deletions, homozygosity would be lethal. Heinz mal histidine. Another histidine is situated on the other side of
bodies are usually not seen until the spleen has been removed; the pocket. is second histidine is not directly linked to the
they can be detected in the peripheral smear by supravital stain- ferrous atom and is called the distal histidine. Normally oxygen
ing. Unstable hemoglobins are detected by their precipitation is able to slip freely between the distal histidine and the ferrous
in isopropanol or after heating to 50°C. HbA2 and HbF may atom during oxygenation and deoxygenation (Figure 3). In the
be increased. Hemoglobin electrophoresis reveals that most normal individual there is a balance between the spontaneous
unstable hemoglobins migrate like HbA or HbS. Complete process of methemoglobin formation and a series of protective
characterization includes amino acid sequencing and gene mechanisms that reconvert the pigment back to hemoglobin.
cloning and sequencing. Methemoglobinemia may be caused by the ingestion of
Not for the first time, observations made on patients suf- nitrites and nitrobenzenes, enzyme deficiencies such as met-
fering from certain abnormal hemoglobin have provided the hemoglobin reductase or diaphorases, and certain abnormal
stimulus for basic scientific work. hemoglobins. In 1948 Hörlein and Weber (49) described a
German family in which some members had been cyanotic from
HEMOGLOBIN M AND METHEMOGLOBINEMIA birth and found that the abnormality was associated with the
For hemoglobin to combine with oxygen, its iron atoms must globin and not with the heme. Hemoglobin M has subsequently
be in the ferrous state. Should oxidation (or de-electronation) been recognized as a perfect example of a molecular abnormality.
of the hemoglobin molecule occur, the ferrous iron is converted Such abnormal hemoglobins, collectively called hemoglobin M,
to ferric iron and methemoglobin is formed. Methemoglobin is all have amino acid substitutions involving either the histidyls
valueless as a respiratory pigment. Every day about 1% of the themselves or amino acids lining the heme pocket (Table 4).
total circulating hemoglobin concentration is converted into Carriers of hemoglobin M are often cyanotic and suffer from
methemoglobin. e iron is itself attached on one side of the anemia. e anemia is more significant than the hemoglobin

July 2006 Structure-function relations of human hemoglobins 243

level suggests because some 25% of circulating hemoglobin is 8. Herrick JB. Peculiar elongated and sickle-shaped red corpuscles in a case
in the ferric form and therefore is not functional. No effective of severe anemia. Arch Intern Med 1910;6:517.
9. Bunn HF. Pathogenesis and treatment of sickle cell disease. N Engl J Med
treatment exists for the cyanosis that is present in patients with 1997;337(11):762–769.
hemoglobin M. 10. Raphael RI. Pathophysiology and treatment of sickle cell disease. Clin
Adv Hematol Oncol 2005;3(6):492–505.
POSSIBLE CLINICAL DEVELOPMENTS IN HEMOGLOBIN 11. Pauling L, Itano HA, Singer SJ, Wells IC. Sickle cell anemia, a molecular
RESEARCH disease. Science 1949;110:543–548.
12. Nagel RL, Platt OS. General pathophysiology of sickle cell anemia. In
It is well known that crocodiles kill their prey by drowning Steinberg MH, Forget BG, Higgs DR, eds. Disorders of Hemoglobin.
them. Crocodiles are capable of remaining under water without Cambridge: Cambridge University Press, 2001:494–526.
surfacing to breathe for more than an hour. It has been shown 13. Pathare A, Kindi SA, Daar S, Dennison D. Cytokines in sickle cell disease.
that when crocodiles hold their breath, bicarbonate ions, the fi- Hematology 2003;8(5):329–337.
nal product of respiration, accumulate and markedly reduce the 14. Hebbel RP, Mohandas N. Cell adhesion and microrheology in sickle-
cell disease. In Steinberg MH, Forget BG, Higgs DR, eds. Disorders of
oxygen affinity of their hemoglobin. is releases a large fraction
Hemoglobin. Cambridge: Cambridge University Press, 2001:527–549.
of hemoglobin-bound oxygen to the tissues (55). Hence, the 15. Adams RJ, Brambilla D; Optimizing Primary Stroke Prevention in Sickle
oxygen affinity of crocodile hemoglobin is markedly reduced by Cell Anemia (STOP 2) Trial Investigators. Discontinuing prophylactic
the physiologic concentration of carbon dioxide. e bicarbon- transfusions used to prevent stroke in sickle cell disease. N Engl J Med
ate ions thus formed bind to deoxyhemoglobin and facilitate 2005;353(26):2769–2778.
16. Marengo-Rowe AJ, Beale D, Lehmann H. New human haemoglobin vari-
the giving up of oxygen (Figure 2).
ant from southern Arabia: G-Audhali (alpha-23B4 glutamic acid→valine)
Amino acid sequence identity between crocodile and hu- and the variability of B4 in human haemoglobin. Nature 1968;219(159):
man hemoglobin is 68% for the alpha subunit and 51% for the 1164–1166.
beta subunit. In crocodile hemoglobin, the amino acid residues 17. Perutz RR, Ligouri AM, Eirich F. X-ray and solubility studies of the
involved in bicarbonate ion binding are located at the α1β2 haemoglobin of sickle-cell anaemia patients. Nature 1951;167(4258):
contact. is junction acts as a flexible joint during hemoglobin 929–931.
18. Ballas SK, Smith ED. Red blood cell changes during the evolution of the
respiration. sickle cell painful crisis. Blood 1992;79(8):2154–2163.
In collaboration with Osaka University in Japan, Jeremy 19. Vichinsky EP. Comprehensive care in sickle cell disease: its impact on
Tame at the MRC Laboratory of Molecular Biology in Cam- morbidity and mortality. Semin Hematol 1991;28(3):220–226.
bridge, England, was able to transplant this unique allosteric 20. National Heart, Lung, and Blood Institute, National Institutes of Health.
effect from the Nile crocodile (Crocodylus niloticus) into human e Management of Sickle Cell Disease (NIH Publication No. 02-2117).
Bethesda, MD: NIH, 2002. Available at http://www.nhlbi.nih.gov/
hemoglobin by replacing a total of 12 amino acids at critical health/prof/blood/sickle/; accessed February 13, 2006.
positions in the alpha and beta chains. is new engineered 21. Vermylen C, Cornu G. Hematopoietic stem cell transplantation for sickle
hemoglobin was named Hb Scuba (56). e clinical implication cell anemia. Curr Opin Hematol 1997;4(6):377–380.
of this work for transfusion medicine is mind-boggling! 22. Steinberg MH, Barton F, Castro O, Pegelow CH, Ballas SK, Kutlar A,
Orringer E, Bellevue R, Olivieri N, Eckman J, Varma M, Ramirez G, Adler
B, Smith W, Carlos T, Ataga K, DeCastro L, Bigelow C, Saunthararajah
Acknowledgment
Y, Telfer M, Vichinsky E, Claster S, Shurin S, Bridges K, Waclawiw M,
I am deeply grateful to the late Dr. Max Perutz and Pro- Bonds D, Terrin M. Effect of hydroxyurea on mortality and morbidity
fessor Herman Lehmann, who first stimulated my interest in in adult sickle cell anemia: risks and benefits up to 9 years of treatment.
hemoglobinopathies, and various commanders in the Royal JAMA 2003;289(13):1645–1651.
Air Force and Special Air Service for much assistance. Also, I 23. Cokic VP, Smith RD, Beleslin-Cokic BB, Njoroge JM, Miller JL, Glad-
win MT, Schechter AN. Hydroxyurea induces fetal hemoglobin by the
would like to thank Kathy Cypert (née Martin) for her patient
nitric oxide–dependent activation of soluble guanylyl cyclase. J Clin Invest
and determined secretarial fortitude. 2003;111(2):231–239.
24. Platt OS, Brambilla DJ, Rosse WF, Milner PF, Castro O, Steinberg MH,
1. Perutz MF, Rossmann MG, Cullis MG, Muirhead H, Will G, North ACT. Klug PP. Mortality in sickle cell disease. Life expectancy and risk factors
Structure of haemoglobin. A three-dimensional Fourier synthesis at 5.5Å for early death. N Engl J Med 1994;330(23):1639–1644.
resolution, obtained by X-ray analysis. Nature 1960;185:416–422. 25. Udani M, Zen Q, Cottman M, Leonard N, Jefferson S, Daymont C,
2. Ingram VM. Gene mutations in human haemoglobin: the chemical dif- Truskey G. Basal cell adhesion molecule/Lutheran protein. e receptor
ference between normal and sickle cell haemoglobin. Nature 1957;180: critical for sickle cell adhesion to laminin. J Clin Invest 1998;101(11):
326–328. 2550–2558.
3. Allison AC. Protection afforded by sickle-cell trait against subtertian 26. Ataga KI, Orringer EP. Hypercoagulability in sickle cell disease: a curious
malarial infection. Br Med J 1954;1:290–294. paradox. Am J Med 2003;115(9):721–728.
4. Perutz MF, Lehmann H. Molecular pathology of human haemoglobin. 27. Westwick J, Watson-Williams EJ, Krishnamurthi S, Marks G, Ellis V,
Nature 1968;219(157):902–909. Scully MF, White JM, Kakkar VV. Platelet activation during steady state
5. Marengo-Rowe A. Haemoglobinopathies. Br J Hosp Med 1971;6:617– sickle cell disease. J Med 1983;14(l):17–36.
630. 28. Westerman MP, Green D, Gilman-Sachs A, Beaman K, Freels S, Boggio
6. Perutz MF. Proteins and Nucleic Acids: Structure and Function. Amsterdam: L, Allen S, Zuckerman L, Schlegel R, Williamson P. Antiphospholipid
Elsevier, 1962:35–48. antibodies, proteins C and S, and coagulation changes in sickle cell disease.
7. Konotey-Ahulu FID. Hereditary qualitative and quantitative erythrocyte J Lab Clin Med 1999;134(4):352–362.
defects in Ghana. An historical and geographical survey. Ghana Med J
1968;7:118–119.

244 Baylor University Medical Center Proceedings Volume 19, Number 3

29. Francis RB Jr. Platelets, coagulation, and fibrinolysis in sickle cell dis- 43. Dacie JV, Shinton NK, Gaffney PJ Jr, Lehmann H. Haemoglobin Ham-
ease: their possible role in vascular occlusion. Blood Coagul Fibrinolysis mersmith (beta-42 (CDI) Phe replaced by Ser). Nature 1967;216(5116):
1991;2(2):341–353. 663–665.
30. Perutz MF. Stereochemistry of cooperative effects in haemoglobin. Nature 44. Sakuragawa M, Ohba Y, Miyaji T, Yamamoto K, Miwa S. A Japanese
1970;228(5273):726–739. boy with hemolytic anemia due to an unstable hemoglobin (Hb Bristol).
31. Perutz MF, Wilkinson AJ, Paoli M, Dodson GG. e stereochemical Nippon Ketsueki Gakkai Zasshi 1984;47(4):896–902.
mechanism of the cooperative effects in hemoglobin revisited. Annu Rev 45. Murari J, Smith LL, Wilson JB, Schneider RG, Huisman TH. Some
Biophys Biomol Struct 1998;27:1–34. properties of hemoglobin Gun Hill. Hemoglobin 1977;1(3):267–282.
32. Charache S, Weatherall DJ, Clegg JB. Polycythemia associated with a 46. Plaseska D, Dimovski AJ, Wilson JB, Webber BB, Hume HA, Huisman
hemoglobinopathy. J Clin Invest 1966;45(6):813–822. TH. Hemoglobin Montreal: a new variant with an extended beta chain due
33. Botha MC, Beale D, Isaacs WA, Lehmann H. Hemoglobin J Cape Town- to a deletion of Asp, Gly, Leu at positions 73, 74, and 75, and an insertion
alpha-2 92 arginine replaced by glutamine beta-2. Nature 1966;212(64): of Ala, Arg, Cys, Gln at the same location. Blood 1991;77(1):178–181.
792–795. 47. Winterbourn CC, Carrell RW. Studies of hemoglobin denaturation
34. Jones RT, Osgood EE, Brimhall B, Koler RD. Hemoglobin Yakina. I. Clin- and Heinz body formation in the unstable hemoglobins. J Clin Invest
ical and biochemical studies. J Clin Invest 1967;46(11):1840–1847. 1974;54(3):678–689.
35. Lokich JJ, Moloney WC, Bunn HF, Bruckheimer SM, Ranney HM. 48. Cathie IAB. Apparent idiopathic Heinz body anaemia. Great Ormond
Hemoglobin Brigham (α2Aβ2100 Pro→Leu). Hemoglobin variant associated Street J 1952;2:43–48.
with familial erythrocytosis. J Clin Invest 1973;52(8):2060–2067. 49. Hörlein H, Weber G. Über Chronishce Familiare Metthämoglobinamie
36. Adamson JW, Parer JT, Stamatoyannopoulos G. Erythrocytosis associated und Eine Modificazation des Methämoglobins. Dtsch Med Wochenschr
with hemoglobin Rainier: oxygen equilibria and marrow regulation. J Clin 1948;73:476.
Invest 1969;48(8):1376–1386. 50. Gerald PS, Efron ML. Chemical studies of several varieties of Hb M. Proc
37. Bunn HF, Bradley TB, Davis WE, Drysdale JW, Burke JF, Beck WS, Natl Acad Sci U S A 1961;47:1758–1767.
Layer MB. Structural and functional studies on hemoglobin Bethesda 51. Stavem P, Stromme J, Lorkin PA, Lehmann H. Haemoglobin M Saskatoon
(α2Aβ2l45His), a variant associated with compensatory erythrocytosis. J with slight constant haemolysis, markedly increased by sulphonamides.
Clin Invest 1972;51(9):2299–2309. Scand J Haematol 1972;9(6):566–571.
38. Jensen M, Oski FA, Nathan DG, Bunn HF. Hemoglobin Syracuse A 52. Hayashi N, Motokawa Y, Kikuchi G. Studies on relationships between
(α2Aβ2143(H21)His→Pro), a new high-affinity variant detected by special structure and function of hemoglobin M-Iwate. J Biol Chem 1966;241(l):
electrophoretic methods. Observations on the auto-oxidation of normal 79–84.
and variant hemoglobins. J Clin Invest 1975;55(3):469–477. 53. Hutt PJ, Pisciotta AV, Fairbanks VF, ibodeau SN, Green MM. DNA
39. Reissmann KR, Ruth WE, Nomura T. A human hemoglobin with low- sequence analysis proves Hb M-Milwaukee-2 is due to beta-globin gene
ered oxygen affinity and impaired heme-heme interactions. J Clin Invest codon 92 (CAC→TAC), the presumed mutation of Hb M-Hyde Park
1961;40:1826–1833. and Hb M-Akita. Hemoglobin 1998;22(1):1–10.
40. Imamura T, Fujita S, Ohta Y, Hanada M, Yanase T. Hemoglobin Yo- 54. Ham RD, Chitayat D, Cooper R, Bandler E, Eng B, Chui DH, Waye JS,
shizuka (G10(108)β asparagine→aspartic acid): a new variant with a Freedman MH. Hb FM-Fort Ripley: confirmation of autosomal dominant
reduced oxygen affinity from a Japanese family. J Clin Invest 1969;48(12): inheritance and diagnosis by PCR and direct nucleotide sequencing. Hum
2341–2348. Mutat 1994;3(3):239–242.
41. Imai K, Morimoto H, Kotani M, Shibata S, Miyaji T. Studies on the 55. Bauer C, Forster M, Gros G, Mosca A, Perrella M, Rollema HS, Vogel D.
function of abnormal hemoglobins. II. Oxygen equilibrium of abnormal Analysis of bicarbonate binding to crocodilian hemoglobin. J Biol Chem
hemoglobins: Shimonoseki, Ube II, Hikari, Gifu, and Agenogi. Biochim 1981;256(16):8429–8435.
Biophys Acta 1970;200(2):197–202. 56. Komiyama NH, Miyazaki G, Tame J, Nagai K. Transplanting a unique
42. Carrell RW, Lehmann H, Hutchinson HE. Hemoglobin Koln (β-98 va- allosteric effect from crocodile into human haemoglobin. Nature 1995;
line→methionine): an unstable protein causing inclusion-body anaemia. 373(6511):244–246.
Nature 1966;210(39):915–916.

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