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

FUNCTION:
 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:
 Globin  two different pairs of polypeptide chains (4 globin chains)
 Heme  four heme groups, with one heme group imbedded in each of the four
polypeptide chains
 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. Hgb A1  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
 Chromosome 16 – alpha and zeta
 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:
 Protoporphyrin IX  ring of carbon, hydrogen,
and nitrogen atom
 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 of
the 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 (Fe3+), 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

 IMPORANT 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
HEME BIOSYNTHESIS
Delta Aminolevulinic
Succinyl CoA + Glycine Porphobilinogen
Acid (D-ALA)

Coproporphyrinogen IIII Uroporphyrinogen III Hydroxymethylbilane

Protoporphyrinogen IX Protoporphyrin IX 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 confi
guration
 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 a1-b1 and a2-b2 bonds
hold the dimers in a stable form
 The a1-b2 and a2-b1 bonds are important for the stability of the quaternary structure in the
oxygenated and deoxygenated forms
NORMAL HEMOGLOBINS
MOLECULAR
HEMOGLOBIN STAGE OF LIFE NEWBORN (%) ADULTS (%)
STRUCTURE
Portland 2 zeta; 2 gamma Embryonic 0% 0%
Gower I 2 zeta; 2 epsilon Embryonic 0% 0%
Gower II 2 alpha; 2 epsilon Embryonic 0% 0%
Fetal 2 alpha; 2 gamma Newborn; Adult 60-90% 1-2%
A1 2 alpha; 2 beta Newborn; Adult 10-40% >95%
A2 2 alpha; 2 delta Newborn; Adult <0.5% 3.5%

 Embryonic hemoglobin  present up to the 3rd month of fetal life

 Fetal hemoglobin  predominant during second and third trimester of fetal life and at birth (up to 6 months)
 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
 Are used for fractionation, presumptive identification, and quantification of normal hemoglobins and
hemoglobin variants
 The function of hemoglobin is to readily bind oxygen molecules in the lungs
 High oxygen affinity to transport oxygen
 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 left: <27 mm/Hg
 Shift to the right: >27 mm/Hg
 The relationship is described by the oxygen dissociation curve of hemoglobin, which plots the percent oxygen
saturation of hemoglobin versus the PO2
 The curve is sigmoidal, which indicates low hemoglobin affinity for oxygen at low oxygen tension and high affinity for
oxygen at high oxygen tension
 SHIFT TO THE LEFT  Increased oxygen affinity to hemoglobin ; OXYGEN IS NOT DELIVERED TO THE TISSUES
 SHIFT TO THE RIGHT  Decreased oxygen affinity to hemoglobin ; OXYGEN IS DELIVERED TO THE TISSUES
FACTORS AFFECTING THE HGB-O2 DISSOCIATION CURVE
2, 3-DPG
Body Temperature
Blood pH  Bohr effect
Carbon dioxide  Haldane effect
Fetal hemoglobin  Causes left shift
Abnormal hemoglobin variants  causes left shift
FACTORS AFFECTING THE HGB-O2 DISSOCIATION CURVE
FACTORS AFFECTING SHIFT TO THE LEFT (Low except pH) SHIFT TO THE RIGHT (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
 Blood transfusions  Conditions that produce
 Increased carboxyhemoglobin hypoxia (high altitude,
ASSOCIATED CONDITIONS  Methemoglobinemia pulmonary insufficiency,
 Presence of Hgb F congestive heart failure,
 Some Hb variants with high severe anemia)
affinity for oxygen  Some Hb variants with low
affinity for oxygen
THE ROLE OF 2,3-DPG
 OXYHEMOGLOBIN
 Oxygenated or Relaxed state
 As hemoglobin binds oxygen molecules, a change in conformation of the hemoglobin tetramer
occurs with a change in hydrophobic interactions at the a1b2 contact point, a disruption of the salt
bridges, and release of 2,3-BPG
 A 15-degree rotation of the a1b1dimer, relative to the a2b2 dimer, occurs along the a1b2 contact
point
 OXYGEN IS NOT RELEASED TO THE TISSUES

 DEOXYHEMOGLOBIN
 Deoxygenated or Tense state
 Binding of 2,3-BPG between the beta-globin chains; the formation of salt bridges between the
phosphates of 2,3-BPG and positively charged groups on the globin chains further stabilizes the
tetramer in the T conformation
 The binding of 2,3-BPG shifts the oxygen dissociation curve to the right, OXYGEN IS RELEASED TO
THE TISSUES
DYSHEMOGLOBINS
Dysfunctional hemoglobins that are unable to transport oxygen
Form and may accumulate to toxic levels, after exposure to certain drugs or
environmental chemicals or gasses., the offending agent modifies the structure of the
hemoglobin molecule, preventing it from binding oxygen
EXAMPLES:
 METHEMOGLOBIN
 SULFHEMOGLOBIN
 CARBOXYHEMOGLOBIN
METHEMOGLOBIN (MetHb or Hi)
 Is formed by the reversible oxidation of heme iron to the ferric state (Fe3+)
 A small amount of methemoglobin is continuously formed by oxidation of iron during normal oxygenation
and deoxygenation of 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:
 <25% - asymptomatic
 >30% - cyanosis and symptoms of hypoxia
 >50% - coma or death
 is assayed by spectral absorption analysis instruments such as the CO-oximeter (absorption
peak/wavelength at 630 nm)
METHEMOGLOBIN (Hi)
 METHEMOGLOBINEMIA
 Increased in methemoglobin ; can be acquired or hereditary
 Acquired
 Aka toxic methemoglobinemia ; occurs in normal individuals after exposure to an
exogenous oxidant, such as nitrites, primaquine, dapsone, or benzocaine
 Treatment  removal of offending oxidant ; >30% is treated with methylene blue or
exchange transfusion
 Hereditary
 Mutations in the gene (CYB5R3)  required for NADH-cytochrome b5 reductase 3
o Autosomal dominant
o Hemoglobin is called as Hemoglobin M  30-50% of total hemoglobin
o No effective treatment
 Cytochrome b5 reductase deficiency
o Autosomal recessive
o <50% of total hemoglobin
SULFHEMOGLOBIN (HgbS)
 Formed by the addition of a sulfur atom to the pyrrole ring of heme; In vitro and in the presence of
oxygen, hemoglobin reacts with hydrogen sulfide
 Produces an IRREVERSIBLE CHANGE in the polypeptide chains of the hemoglobin molecule due to oxidant
stress, and further change can result in denaturation and the precipitation of hemoglobin as Heinz bodies

 Can combine with carbon monoxide to form carboxysulfhemoglobin

 Sulfhemoglobin has a similar peak (630 nm) to methemoglobin on a spectral absorption instrument ;
cannot be converted to cyanmethemoglobin

 COLOR OF BLOOD: GREENISH PIGMENT (MAUVE-LAVENDER in sulfhemoglobinemia)

SULFHEMOGLOBIN (HgbS)
 CAUSES:
 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

 Ineffective for oxygen transport, and patients with elevated levels present with cyanosis
 Cannot be converted to normal Hb A; it persists for the life of the cell
 Treatment consists of prevention by avoidance of the offending agent
CARBOXYHEMOGLOBIN (HgbCO)

 Results from the combination of carbon monoxide (CO) with heme iron
 Carbon monoxide has 210-240 times more affinity for hemoglobin compared to oxygen
 CO shifts the hemoglobin-oxygen dissociation curve to the left (shift to the left) further increasing its
affinity and severely impairing release of oxygen to the tissues
 Some carboxyhemoglobin is produced endogenously, but it normally comprises less than 2% of total
hemoglobin
 Has been termed the silent killer because it is an odorless and colorless gas, and victims may quickly
become hypoxic
 May be detected by spectral absorption instruments at 540 nm
 COLOR OF BLOOD: CHERRY RED
CARBOXYHEMOGLOBIN (HgbCO)
 TOXIC LEVELS:
 20-30% - headache, dizziness, disorientation
 >40% - coma, seizure, hypotension, cardiac arrhythmias, pulmonary edema, and death

 CAUSES:
 Exhaust of automobiles
 Tobacco smoke – in smokers, carboxyhemoglobin levels may be as high as 15% (higher hematocrit and
polycythemia to compensate for hypoxia)
 Industrial pollutants (coal, coal gas, charcoal burning)

 Diagnosis of carbon monoxide poisoning is made if the level is >3% in nonsmokers and >10% in smokers
 Treatment:
 Removing the carbon monoxide source and administration of 100% oxygen
 Use of hyperbaric oxygen therapy is controversial; it is primarily used to prevent neurologic and
cognitive impairment after acute carbon monoxide exposure in patients whose level exceeds 25%
END