WEEK 8: HEMOGLOBIN METABOLISM
HEMOGLOBIN
→ is one of the most studied proteins in the body
because of the ability to easily isolate it from red
blood cells (RBCs).
→ 95% of cytoplasmic content of RBCs
→ When released into the plasma, it is rapidly
salvaged to preserve its iron and amino acid
components; when salvage capacity is exceeded, it
is excreted by the kidneys
→ Concentration: 34 g/dL (normal MCHC)
→ Molecular weight: 64,000 Daltons
FUNCTIONS:
○ Transport oxygen to the tissues and carbon
dioxide from the tissues to the lungs
○ Contributes to acid-base balance by binding
and releasing hydrogen ions (Bohr effect)
○ Transports nitric oxide → relaxation of
vascular wall smooth muscle and vasodilation
HEMOGLOBIN STRUCTURE
→ Spherical, has four heme groups attached to four
polypeptide chains, and may carry up to four
molecules of oxygen
→ Conjugated globular protein (TETRAMER)
consisting of:
o Globin → two different pairs of polypeptide
chains (4 globin chains)
o Heme → four heme groups, with one heme
group imbedded in each of the four
polypeptide chains
o 2,3-diphosphoglycerate (2,3-DPG)
- Produced in the anaerobic glycolytic pathway
(Luebering-Rapoport pathway)
- Bonded in the center of beta chains
- Inversely related to the hemoglobin-oxygen
affinity
GLOBIN STRUCTURE
→ The four globin chains comprising each
hemoglobin molecule consist of two identical pairs
of unlike polypeptide chains (Ex. HgbA1🡪 2 alpha
and 2 beta globin chains)
→ Variations in amino acid sequences give rise to
different types of polypeptide chains
→ Each chain is designated by a Greek letter
GLOBIN BIOSYNTHESIS
→ Six structural genes code for the six globin chains ;
are on the short arms of chromosomes
o  Chromosome 16 – alpha and zeta
o  Chromosome 11 – beta, delta, gamma, and
epsilon
→ Production of globin chains takes place in the
nucleus and ribosomes of erythroid precursors from
the pronormoblast through the circulating
polychromatic erythrocyte (reticulocyte), but not in
mature erythrocytes
→  Transcription of the globin genes to messenger
ribonucleic acid (mRNA) occurs in the nucleus, and
translation of mRNA to the globin polypeptide chain
occurs on ribosomes in the cytoplasm
→  Although transcription of a-globin genes produces
more mRNA than the b-globin gene, there is less
efficient translation of the a-globin mRNA;
Therefore, a and b chains are produced in
approximately equal amounts
→ After translation is complete, chains are released
from the ribosomes in the cytoplasm
HEME STRUCTURE
→ Also called as Ferroprotoporphyrin IX
→ Heme consists of:
o Protoporphyrin IX - ring of carbon,
hydrogen, and nitrogen atom
o Central atom of divalent ferrous iron
(Fe2+) ; reduced iron
→ Each of the four heme groups is positioned
in a pocket of the polypeptide chain near
the surface ofthe hemoglobin molecule
→ The ferrous iron in each heme molecule
reversibly combines with one oxygen
molecule
→ When the ferrous irons are oxidized to the
ferric state (Fe 3+), they no longer can bind
oxygen( methemoglobin)
HEME BIOSYNTHESIS
 → Heme biosynthesis occurs in the
mitochondria and cytoplasm of bone
marrow erythroid precursors, beginning
with the pronormoblast through the
circulating polychromatic erythrocyte
(reticulocyte
→ As they lose their ribosomes and
mitochondria, mature erythrocytes can no
longer make hemoglobin
→ Porphyrias - disorders in the heme
biosynthesis pathway
IMPORTANT NOTES:
⮚ Transferrin, a plasma protein, carries iron in the
ferric (Fe3+) form to developing erythroid cells
⮚ Transferrin binds to transferrin receptors on
erythroid precursor cell membranes and the
receptors and transferrin (with bound iron) are
brought into the cell in an endosome
⮚ Acidification of the endosome releases iron from
transferrin
⮚ Iron is transported out of the endosome and into
the mitochondria, where it is reduced to the
ferrous state and is united with protoporphyrin IX
to make heme
⮚ Heme leaves the mitochondria and is joined to the
globin chains in the cytoplasm
Heme Biosynthesis Pathway
1. Succinyl coA + Glycine
2. Delta Aminolevunilic Acid (D-ALA)
3. Porphobilinogen
4. Hydroxymethylbilane
5. Uroporphyrinogen III
6. Coproporphyrinogen IIII
7. Protoporphyrinogen IX
8. Protoporphyrinogen IX
9. Heme
HEMOGLOBIN ASSEMBLY
→ After their release from ribosomes, each globin
chain binds to a heme molecule, then forms a
heterodimer
→ Two heterodimers then combine to form a
tetramer; this completes the hemoglobin molecule
→ In the native configuration of the hemoglobin
molecule, the four hemes and four polypeptide
chains are assembled in a very specific spatial
configuration
→ Each of the four chains in the molecule coils into
eight helices, forming an egg-shaped molecule
with a central cavity
→ In the process of the binding of the first heme
group to a molecule of oxygen, a change in the
overall configuration of the hemoglobin molecule
occurs
→ This altered configuration of the molecule favors
the additional binding of oxygen to the remaining
heme groups, if sufficient oxygen pressure is
present
COMPLETE HEMOGLOBIN MOLECULE
The hemoglobin molecule can be described by its
primary, secondary, tertiary, and quaternary protein
structures
 Primary refers to the amino acid sequence
of the polypeptide chains
Secondary refers to chain arrangements in
helices and non-helices
Tertiary refers to the arrangement of the
helices into a pretzel-like
configuration
Quaternary also called a tetramer, describes
the complete hemoglobin
molecule
Hb A, is composed of two a-
globin chains and two b-globin
chains.; strong a a1-b1 and a2-b2
bonds hold the dimers in a stable
form
The ai-b2 and a2-b1 bonds are
important for the stability of the
quaternary structure in the
oxygenated and deoxygenated
forms
o Are used for fractionation, presumptive
identification, quantification of normal
hemoglobin and hemoglobin variants
HEMOGLOBIN OXYGEN DISSOCIATION
CURVE
→ The function of hemoglobin is to readily bind
oxygen molecules in the lungs
o High oxygen affinity to transport
oxygen
o Low oxygen affinity to efficiently
unload oxygen to the tissues
→ During oxygenation, each of the four heme iron
atoms in a hemoglobin molecule can reversibly
bind one oxygen molecule
→ Approximately 1.34 mL of oxygen is bound by
each gram of hemoglobin (3.47 mg of iron)
→ The affinity of hemoglobin for oxygen relates to
the partial pressure of oxygen (PO2), often
defined in terms of the amount of oxygen needed
to saturate 50% of hemoglobin, called P50 value
NORMAL VALUE 27 mm/Hg
SHIFT TO THE <27 mm/Hg
LEFT

NORMAL HEMOGLOBINS SHIFT TO THE >27 mm/Hg

RIGHT
HEMOGLOB MOLECUL STAGE OF NEWB ADULT
IN AR LIFE ORN (%) → The relationship is described by the oxygen
STRUCTUR (%) dissociation curve of hemoglobin, which plots the
E
percent oxygen saturation of hemoglobin versus
PORTLAND 2 zeta; 2 Embryonic 0% 0% the PO2
gamma
→ The curve is sigmoidal, which indicates low
Gower I 2 zeta; 2 Embryonic 0% 0% hemoglobin affinity for oxygen at low oxygen
epsilon tension and high affinity for oxygen at high
GOWER II 2 alpha; 2 Embryonic 0% 0% oxygen tension
epsilon
SHIFT TO THE LEFT SHIFT TO THE
Fetal 2 alpha; 2 Newborn; 60- 1-2%
gamma adult 90% RIGHT
A1 2 alpha; 2 Newborn; 10- >95% Increased oxygen Decreased oxygen
beta Adult 40%
affinity to hemoglobin affinity to hemoglobin
A2 2 alpha; 2 Newborn; <0.5% 3.5%
delta Adult OXYGEN IS NOT OXYGEN IS
DELIVERED TO THE DELIVERED TO THE
● Embryonic hemoglobin – present up to the 3 rd TISSUES TISSUES
month of fetal life
● Fetal hemoglobin – predominant during second
and third trimester of fetal life and at birth (up to
6 month)
● Adult hemoglobin (HgbA1) – predominant by 6
months of age until adulthood with small amounts
of HgbA2
● Hemoglobin variants – genetic abnormalities in
the hemoglobin molecule (350 hemoglobin
variants)
● Hemoglobin electrophoresis and HPLC
 FACTORS AFFECTING THE HGB-O2 → Abnormal hemoglobin variants - causes left
DISSOCIATION CURVE shift
→ 2, 3-DPG
→ Body Temperature
→ Blood pH - Bohr effect
→ Carbon dioxide - Haldane effect
→ Fetal hemoglobin - Causes left shift

FACTORS SHIFT TO THE LEFT (Low except SHIFT TO THE RIGHT

AFFECTING pH) (Rise except pH)
PH INCREASED (BASIC) DECREASED (ACIDIC)
Temperature Decreased Increased
Carbon dioxide Decreased (tissues) Increased (Lungs)
2,3-DPG Decreased Increased
→ Lowered Body temperature → High Body temperature
→ Alkalosis → Acidosis
ASSOCIATED
→ Blood transfusions → Conditions that produce
CONDITIONS
→ Increased carboxyhemoglobin → hypoxia (high altitude,
→ Methemoglobinemia pulmonary insufficiency,
→ Presence of Hgb F congestive heart failure,
severe anemia)
→ Some Hb variants with high affinity
→ Some Hb variants with low
for oxygen
affinity for oxygen
THE ROLE OF 2,3-DPG
DYSHEMOGLOBINS
→ OXYHEMOGLOBIN
○ Oxygenated or Relaxed state → Dysfunctional hemoglobin that are unable to
- As hemoglobin binds oxygen molecules, change transport oxygen
in conformation of the hemoglobin tetramer occurs → Form and may accumulate to toxic levels, after
with a change in hydrophobic interactions at the
a1b1 contact point, a disruption of the salt bridges, exposure to certain drugs or environmental
and release of 2,3 -BPG chemicals or gasses., the offending agent modifies
- A 15-degree rotation of the a1b1 dimer, relative to the structure of the hemoglobin molecule,
the a2b2 dimer, occurs along the a1b2 contact preventing it from binding oxygen
point → EXAMPLES:
- OXYGEN IS NOT RELEASED TO THE
TISSUES - METHEMOGLOBIN
- SULFHEMOGLOBIN
→ DEOXYHEMOGLOBIN
Deoxygenated or Tense state - CARBOXYHEMOGLOBIN
- Binding of 2,3-BPG between the beta-globin METHEMOGLOBIN (MetHb) or Hi
chains; the formation of salt bridges between
the phosphates of 2,3-BPG and positively → Is formed by the reversible oxidation of heme
charged groups on the globin chains further iron to the ferric state (Fe3+)
stabilizes the tetramer in the T conformation → A small amount of methemoglobin is
- The binding of 2,3-BPG shifts the oxygen continuously formed by oxidation of iron during
dissociation curve to the right, OXYGEN IS normal oxygenation and deoxygenation of
RELEASED TO THE TISSUES hemoglobin (1% of total hemoglobin)
→ COLOR OF BLOOD:CHOCOLATE BROWN
→ Cannot carry oxygen because oxidized ferric
iron cannot bind it; an increase in
methemoglobin level results in decreased
delivery of oxygen to the tissues
→ TOXIC LEVELS:
 o <25% - asymptomatic → Ineffective for oxygen transport, and patients with
o >30% - cyanosis and symptoms of elevated levels present with cyanosis
hypoxia → Cannot be converted to normal Hb A; it persists for
o >50% - coma or death the life of the cell
→ is assayed by spectral absorption analysis → Treatment consists of prevention by avoidance of
instruments such as the CO-oximeter (absorption the offending agent
peak/wavelength at 630 nm)
CARBOXYHEMOGLOBINS (HgBCO)
METHEMOGLOBIN (Hi)
→ Results from the combination of carbon monoxide
→ METHEMOGLOBINEMIA (CO) with heme iron
o Increased in methemoglobin ; can be acquired or → Carbon monoxide has 210-240 times more affinity
hereditary for hemoglobin com
o Acquired → CO shifts the hemoglobin-oxygen dissociation
▪ Aka toxic methemoglobinemia ; occurs in curve to the left (shift to the left) further increasing
normal individuals after exposure to its affinity and severely impairing release of oxygen
anexogenous oxidant, such as nitrites, to the tissues
primaquine, dapsone, or benzocaine → Some carboxyhemoglobin is produced
▪ Treatment - removal of offending oxidant ; endogenously, but it normally comprises less than
>30% is treated with methylene blue or 2% of total hemoglobin
exchange transfusion → Has been termed the silent killer because it is an
o Hereditary odorless and colorless gas, and victims may quickly
o Mutations in the gene (CYB5R3)  required become hypoxic
for NADH-cytochrome b5 reductase 3 → May be detected by spectral absorption instruments
▪ Autosomal dominant at 540 nm
▪ Hemoglobin is called as Hemoglobin M → COLOR OF BLOOD: CHERRY RED
 30-50% of total hemoglobin → TOXIC LEVELS
▪ No effective treatment o 20-30% - headache, dizziness,
o Cytochrome b5 reductase deficiency disorientation
▪ Autosomal recessive o >40% - coma, seizure, hypotension, cardiac
▪ <50% of total hemoglobin arrhythmias, pulmonary edema, and death
→ CAUSES:
SULFHEMOGLOBIN (HgbS)
o Exhaust of automobiles
→ Formed by the addition of a sulfur atom to the o Tobacco smoke – in smokers,
pyrrole ring of heme; In vitro and in the presence of carboxyhemoglobin levels may be as high
oxygen, hemoglobin reacts with hydrogen sulfide as 15% (higher hematocrit and
→ Produces an IRREVERSIBLE CHANGE in the polycythemia to compensate for hypoxia)
polypeptide chains of the hemoglobin molecule due o Industrial pollutants (coal, coal gas,
to oxidant stress, and further change can result in charcoal burning)
denaturation and the precipitation of hemoglobin as → Diagnosis of carbon monoxide poisoning is made if
Heinz bodies the level is >3% in nonsmokers and >10% in
→ Can combine with carbon monoxide to form smokers
carboxysulfhemoglobin → Treatment:
→ Sulfhemoglobin has a similar peak (630 nm) to o Removing the carbon monoxide source and
methemoglobin on a spectral absorption administration of 100% oxygen
instrument ; cannot be converted to o Use of hyperbaric oxygen therapy is
cyanmethemoglobin controversial; it is primarily used to prevent
→ COLOR OF BLOOD: GREENISH PIGMENT neurologic and cognitive impairment after
(MAUVE-LAVENDER in sulfhemoglobinemia) acute carbon monoxide exposure in patients
→ CAUSES whose level exceeds 25%
- Drugs (such as sulfanilamides, phenacetin,
nitrites, and phenylhydrazine)
- Exposure to sulfur chemicals in industrial or
environmental settings
Patients with severe constipation, in cases of
bacteremia due to Clostridium perfringens and
Clostridium welchii, and in a condition known
enterogenous cyanosis