STRUCTURE AND

FUNCTIONS OF HEME
• Red blood cells (RBC) are biconcave discs, with a
diameter of about 7 microns.
• RBCs live for about 120 days in peripheral circulation
• Mature RBC is non-nucleated; have no mitochondria
and does not contain TCA cycle enzymes.
• However, the glycolytic pathway is active which
provides energy and 2,3-bisphospho glycerate (2,3-BPG).
• The HMP shunt pathway provides the NADPH.
• RBC formation in the bone marrow requires amino acids,
iron, copper, folic acid, vitamin B12, vitamin C, pyridoxal
phosphate, and pantothenic acid; they are used as
hematinics in clinical practice.
Structure of Hemoglobin
• Hemoglobin is a conjugated protein having heme as
the
prosthetic group and the protein, the globin.
• It is a tetrameric protein with 4 subunits, each subunit
having a prosthetic heme group and the globin
polypeptide.
• The polypeptide chains are usually two alpha and two
beta chains.
• Hemoglobin has a molecular weight of about 67,000
Daltons.
• Each gram of Hb contains 3.4 mg of iron.
• Heme is present in
a. Hemoglobin
b. Myoglobin
c. Cytochromes
d. Peroxidase
e. Catalase
f. Tryptophan pyrrolase
g. Nitric oxide synthase
• Heme is produced by the combination of iron with a
porphyrin ring.
• Chlorophyll, the photosynthetic green pigment in plants is
magnesium-porphyrin complex.
Structure of Heme
• Heme is a derivative of the porphyrin.
• Porphyrins are cyclic compounds formed by fusion of
4 pyrrole rings linked by methenyl (=CH-) bridges.
• Since an atom of iron is present, heme is a
ferroprotoporphyrin.
• The pyrrole rings are named as I, II, III, IV and the bridges
as alpha, beta, gamma and delta.

Pyrrole ring
Porphyrin Ring

Porphyrin ring. The pyrrole rings are numbered I to IV; the bridges are named as alpha to
delta. The possible sites of substitutions are denoted from 1 to 8. For brevity, the bridges
and double bonds are sometimes omitted, as shown on the right.
• When the substituent groups have a symmetrical
arrangement (1,3,5,7 and 2,4,6,8) they are called the I
series.
• The III series have an asymmetrical distribution of
substituent groups (1,3,5,8 and 2,4,6,7).
• Type III is the most predominant in biological systems.
• It is also called series 9, because Fischer, the pioneer in
porphyrin chemistry has placed it as the 9th in a series
of 15 possible isomers.
• The usual substitutions are:
a. propionyl (–CH2–CH2–COOH) group
b. acetyl (–CH2–COOH) group
c. methyl (–CH3) group
d. vinyl (–CH=CH2) group.
Porphyrins of Biological Importance
Name of Porphyrin Order of substituents from 1st
to 8th positions

Uroporphyrin I A,P, A,P, A,P, A,P

Uroporphyrin III A,P, A,P, A,P, P,A
Coproporphyrin I M,P, M,P, M,P, M,P
Coproporphyrin III M,P, M,P, M,P, P,M
Protoporphyrin III M,V, M,V, M,P, P,M
(A = acetyl; P = propionyl; M =
methyl; V = vinyl)
Structure of Heme
Normal Hemoglobin
• Normal level of Hemoglobin (Hb) in blood in males is
14-16 g/dl and in females, 13-15 g/dl.
• Hb is globular in shape.
• The adult Hb (HbA) has 2 alpha chains and 2 beta chains.
• Molecular weight of HbA is 67,000 Daltons.
• Hb F (Fetal Hb) is made up of 2 alpha and 2 gamma
chains.
• Hb A2 has 2 alpha and 2 delta chains.
• Normal adult blood contains 97% HbA, about 2% HbA2
and about 1% HbF.
• Each alpha chain has 141 amino acids.
• The beta, gamma and delta chains have 146 amino acids.
• There are 36 histidine residues in Hb molecule; these are
important in buffering action.
• The 58th residue in alpha chain is called distal histidine,
because it is far away from the iron atom.
• The 87th residue in alpha chain is called proximal
histidine, because it lies near to the iron atom.
Fetal Hemoglobin (HbF)
• HbF has 2 alpha chains and 2 gamma chains. Gamma
chain has 146 amino acids.
• The differences in physicochemical properties when
compared with HbA are:
o a. Increased solubility of deoxy HbF
o b. Slower electrophoretic mobility for HbF
o c. Increased resistance of HbF to alkali denaturation
o d. HbF has decreased interaction with 2,3-BPG.
• The synthesis of HbF starts by 7th week of gestation; it
becomes the predominant Hb by 28th week.
• At birth, 80% of Hb is HbF.
• During the first 6 months of life, it decreases to about 5%
of total.
• HbF level may remain elevated in children with anemia
and beta thalassemia, as a compensatory measure.
Hemoglobin A2
• It is a normal adult Hb; it is about 2% of total Hb.
• It has 2 alpha chains and 2 delta chains.
• The delta chain has sequence homology with beta chain.
• In beta thalassemia, as a compensation, HbA2 is
increased.
• The iso-electric pH of HbA2 is 7.4, while HbA has the pI
value of 6.85.
• HbA2 is slower moving on electrophoresis.
Hemoglobin molecule is made up of two alpha and two beta chains.
Oxygenation and Oxidation
• When hemoglobin carries oxygen, the Hb is
oxygenated.
• The iron atom in Hb is still in the ferrous state.
• Oxidised Hemoglobin is called Met-Hb; then iron is in
ferric state and the oxygen carrying capacity is lost.
• The alpha and beta subunits are connected by relatively
weak non-covalent bonds like van der Waals forces,
hydrogen bonds and electrostatic forces.
• There are 4 heme residues per Hb molecule, one for each
subunit in Hb.
• The 4 heme groups account for about 4% of the whole
mass of Hb.
• The heme is located in a hydrophobic cleft of globin
chain.
Linkage of Heme with Globin

Pink circle represents the globin chain. Blue rectangle

represents the protoporphyrin ring
• The iron atom of heme occupies the central position of
the porphyrin ring.
• The reduced state is called ferrous (Fe++) and the
oxidized state is ferric (Fe+++).
• The ferrous iron has 6 valencies and ferric has 5
valencies.
• In hemoglobin, iron remains in the ferrous state.
Iron Carries Oxygen
• The iron is linked to the pyrrole nitrogen by 4 co-
ordinate
valency bonds and a fifth one to the imidazole nitrogen
of the proximal histidine.
• In oxy-Hb, the 6th valency of iron binds the O2.
• The oxygen atom directly binds to Fe, and forms a
hydrogen bond with an imidazole nitrogen of the distal
histidine.
• In deoxy-Hb, a water molecule is present between the
iron and distal histidine.
• As the porphyrin molecule is in resonance, central iron
atom is linked by coordinate bond.
• The distal histidine lies on the side of the heme ring.
Transport of Oxygen by Hemoglobin
• Hemoglobin is an ideal oxygen carrying pigment because
–
o a. It can transport large quantities of oxygen
o b. It has great solubility
o c. It can take up and release oxygen at appropriate partial
pressures
o d. It is a powerful buffer.
Transport of Oxygen by Hemoglobin

In oxy-Hb, the 6th valency of iron binds the O2. The oxygen atom
directly binds to Fe. In deoxy-Hb, oxygen is removed and a water
molecule is present between the iron and distal histidine.
Subunit Interaction in Hemoglobin

Diagrammatic representation of the subunit interaction in

hemoglobin. Rectangles represent T state Hb monomers
and circles represent R state Hb monomers. As oxygen is
added, salt bridges are successively broken.
Simultaneously the T (taught) confirmation of deoxy-Hb is
changed into R (relaxed) confirmation of oxy-Hb.
Oxygen dissociation curve (ODC). A = Theoretical curve as per mass
action. B = Sigmoid curve, due to heme-heme interaction (Hill effect). C =
Further shift to right due to carbon dioxide (Bohr effect) and PG. This
curve represents the pattern under normal conditions. D = Further shift to
right when temperature is increased to 42oC.
Factors affecting oxygen dissociation curve.
In Tissues Oxy Hb Releases O2
Clinical Applications of Oxygen Dissociation
1. In all hypoxic states the O2 affinity is decreased with a shift in ODC to the
right and an increase in 2,3-BPG inside RBC. The adaptation to the high
altitude where pCO2 is low, includes increased pulmonary ventilation,
polycythemia, and an increase in 2,3-BPG level with a shift in ODC to the
right.
2. In anemia where the total concentration of Hb is reduced, increased
oxygen unloading alone will ensure proper oxygenation of tissues.
3. in many cases, the 2,3-BPG level varies inversely as the Hb
concentration.
4. in chronic pulmonary diseases and cyanotic cardiac disorders also, the
2,3-BPG level is increased, ensuring maximum unloading of O2 to the
tissues.
5. The red cell 2,3-BPG level is decreased in acidosis and increased in
alkalosis. Hence, the expected shift in ODC to the right or left is not
observed.
6. Transfusion of large volumes of stored blood, which has a low level of
2,3-BPG can lead to sudden hypoxia, since it can cause a left-shifted ODC.
Chloride Shift in Tissues
Chloride Shift, in Lungs
Transport of Carbon Dioxide
1. Dissolved Form
About 10% of CO2 is transported as dissolved form.
CO2 + H2O → H2CO3 → HCO3– + H+
The hydrogen ions thus generated, are buffered by the buffer systems of
plasma.

2. Isohydric Transport of Carbon Dioxide

Isohydric transport constitutes about 75% of CO 2. It means that there is
a minimum change in pH during the transport. The H+ ions are
buffered by the deoxy-Hb and this is called the Haldane effect.

3. Carriage as Carbamino Hemoglobin

The rest 15% of CO2 is carried as carbamino-hemoglobin, without much
change in pH. A fraction of CO2 that enters into the red cell is bound to Hb
as a carbamino complex.
R–NH2 + CO2 -------→-- R–NH–COOH
Transport of Carbon Dioxide
Embryonal, Fetal and Adult Hemoglobins
Name of hemoglobin Globin chains present Period of life
Hb Gower I 2 Zeta, 2 epsilon Embryonal hemoglobins.
Hb Gower2 2 alpha, 2 epsilon Upto the end of first
Hb Portland 2 gamma, 2 delta trimester of pregnancy

Fetal Hb or Hb F 2 alpha 2 gamma Second trimester to

early post natal life, upto
2 years

Hb A (adult Hb) 2 alpha ,2 Beta Immediately after birth

to end of life span

Hb A2 (adult) 2 alpha ,2 delta Adult life

Hemoglobin Derivatives
• Hemoglobin derivatives are formed by the combination
of different ligands with the heme part, or change in the
oxidation state of iron.
• Oxy-Hb is dark red, deoxy-Hb is purple, met-Hb is dark
brown, COHb is cherry red and sulph-Hb is green in
color.
• Normally concentration of deoxy-Hb is less than 5% of
the total Hb.
• If the level increases cyanosis occurs.
Carboxy-Hemoglobin (Carbon Monoxy Hb) (CO-Hb)
• Hemoglobin binds with carbon monoxide (CO) to form
carboxy-Hb.
• The affinity of CO to Hb is 200 times more than that of
oxygen.
• It is then unsuitable for oxygen transport.
• When one molecule of CO binds to one monomer of the
hemoglobin molecule, it increases the affinity of others
to O2; so that the O2 bound to these monomers are not
released.
• This would further decrease the availability of oxygen to
the tissues.
Carbon Monoxide Poisoning
• CO is a colorless, odorless, tasteless gas generated by
incomplete combustion.
• CO poisoning is a major occupational hazard for workers
in mines.
• Breathing the automobile exhaust in closed space is the
commonest cause for CO poisoning.
• The carboxy-Hb level in normal people is 0.16%.
• An average smoker has an additional 4% of CO-Hb.
• One cigarette liberates 10–20 ml carbon monoxide into
the lungs.
• Clinical symptoms manifest when carboxy-Hb levels
exceed 20%.
• Symptoms are breathlessness, headache, nausea,
vomiting, and pain in chest.
• At 40-60% saturation, death can result.
• Administration of O2 is the treatment.
• In severe cases, oxygen under high pressure (hyperbaric
oxygen) is helpful.
• The absorption bands of carboxy-Hb is similar to that of
oxy-Hb.
• But on adding a reducing agent like sodium dithionite, it
fails to convert carboxy-Hb to deoxy-Hb; whereas oxy-Hb
is converted.
Met-hemoglobin (Met-Hb)
• When the ferrous (Fe++) iron is oxidized to ferric (Fe+++)
state, met-Hb is formed.
• Small quantities of met-Hb formed in the RBCs are
readily reduced back to the ferrous state by met-Hb
reductase enzyme systems.
• About 75% of the reducing activity is due to enzyme
system using NADH and cytochrome b5.
 Methemoglobin Reductase System
Another 20% of the reducing activity is due to NADPH dependent
system.
Glutathione dependent Met-Hb-reductase accounts for the rest
5% activity.
Difference between OxyHb and MetHb.
Met-hemoglobinemias
• Normal blood has only less than 1% of met-hemoglobin.
• It has markedly decreased capacity for oxygen binding
and transport.
• An increase in methemoglobin in blood, (met-
hemoglobinemia) is manifested as cyanosis.
• Causes may be congenital or acquired.
Congenital Met-hemoglobinemia
• Cytochrome b5 reductase deficiency is characterized by
cyanosis from birth.
• 10-15% of hemoglobin may exist as met-hemoglobin.
• Oral administration of methylene blue, 100-300 mg/day
or ascorbic acid 200-500 mg/day decreases met-Hb level
to 5-10% and reverses the cyanosis.
Acquired or Toxic Met-hemoglobinemia
• Met-hemoglobinemia may develop by intake of water
containing nitrates or due to absorption of aniline dyes.
• Aniline dye workers have been known to develop met-
hemoglobinemia.
• Drugs which produce met-hemoglobinemia are:
acetaminophen, phenacetin, sulphanilamide, amyl
nitrite, and sodium nitroprusside.
Glucose-6-phosphate Dehydrogenase Deficiency
• In persons with this enzyme deficiency, the condition
may be manifested even with small doses of drugs.
• In such persons, NADPH is not available in the RBC.
• In such individuals, disease is manifested easily.
• In such patients, intravenous leukomethylene blue 2
mg/kg is effective, which will substitute for the NADPH.
Glucose-6-phosphate Dehydrogenase Deficiency
Laboratory Analysis
• Ferricyanide can oxidize oxy- or deoxy-Hb to metHb.
• The color changes to dark brown and absorption spectra
show a band in the red with its center at 633 nm, while
the bands for
Oxy-Hb persist.
• Sodium hydrosulfite or dithionite reconverts met-Hb to
oxy-Hb.
Hemin Crystals
• When iron is oxidized to Fe+++, it has a net positive
charge.
• It can combine with negatively charged chloride, to form
hemin or hematin chloride.
• Hemin crystals can be prepared from even very old
blood stains in medicolegal cases.
• Blood or eluted blood stains are heated with Nippe's
fluid (1% solution of KCI, KBr and Kl in glacial acetic acid)
over a glass slide, when dark brown rhombic crystals are
seen under the microscope.
• The test is sensitive, but is answered by the heme part of
blood of all species.
Sulf-hemoglobinemia
• When hydrogen sulfide acts on oxy-hemoglobin, sulf-
hemoglobin is produced.
• It can occur in people taking drugs like sulphonamides,
phenacetin, acetanilide, dapsone, etc.
• It cannot be converted back to oxy-hemoglobin.
• It is seen as basophilic stippling of RBC, throughout its
lifespan.
Nitric Oxide
• Hemoglobin binds nitric oxide (NO) with high affinity
similar to binding of carbon monoxide (CO).
• The NO is delivered at its site of action, i.e. capillary
endothelium.
• The positive co-operative effect and effect of H+ also
play a role in the binding and delivery of NO.
• Binding of NO by hemoglobin increases its half-life.
• The heme iron preferentially binds NO in the T
conformation.
• When hemoglobin acquires the R conformation, the NO is
transferred to a cysteinyl SH group on the beta chain and
then to the SH group of a small molecule like GSH, when
R form reverts to T state.
• The X-S-NO complex is biologically very potent and even
under low oxygen tension delivers NO to tissue
capillaries.
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