HARAMAYA UNIVERSITY

COLLEGE OF HEALTH AND

MEDICAL SCIENCES
SCHOOL OF MEDICINE

Module Title: Hematology System

Course Title: Biochemistry
Topics to be covered: Hemoglobin
Target students: Preclerkship-I
Instructor: Teka Obsa (Asst. Prof.
of Medical Biochemistry, MSc, BSc in MLT))
Haemoglobin structure,
function & metabolism
 Outline
• Hemoproteins
• Structure of Hemoglobin
• Functions of Hemoglobin
• Forms of Hemoglobin
• Oxygen Dissociation Curve
• Heme metabolism
• Heme is iron containing porphyrin.
• Porphyrin is cyclic compound formed by fusion of
4 pyrrole rings linked by methine bridges (=CH-).
• Heme is the prosthetic group of several proteins
and enzymes such as:
• Hemoglobin (O2 transport)
• Myoglobin (O2 storage)
• Cytochrome (ETC component)
• Cytochrome P450 (microsomal
hydroxylation)
• Catalase (acts on H2O2)
• Peroxidase (acts on H2O2)
• Hemoglobin: Heme + Globin
• Red colour of blood is due to Hb in RBCs
• Each gram of hemoglobin can carry 1.34 mL of oxygen.
• This means the O2 content in 15g/dL of hemoglobin is
approximately 20ml/100ml.
• Hemoglobin has 4 binding sites for oxygen.
• Under normal conditions, the hemoglobin is 97% to 98% saturated.
 Structure of Hemoglobin

• 4 polypeptide (2α and 2β) chains

• α-chain is common for all variants of

hemoglobin and contains 141 aas.
• Beta (β), gamma (γ), delta (δ) or epsilon (ε) varies as
per the variants of hemoglobin, contains 146 aas.
• Normal HbA1 (α2β2): 2 X 141 + 2 X 146 = 574 aas

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• Heme is iron protoporphyrin
• Protoporphyrin–four pyrrole rings
linked by methene bridge (=CH) to
form porphyrin
• 4 Methyl (CH3), 2 vinyl (-CH=CH2),
2 propionate (CH2-CH2-COOH) side
chain groups are attached to the
Porphyrin ring (protoporphyrin IX).

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Iron of heme can form 6 cordinate bonds.
4 bonds are formed between the iron
and nitrogen atoms of porphyrin

5th bond is formed between nitrogen

atom of histidine residue of the globin
6th bond is formed with O2.

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What helps hemoglobin maintain
its quaternary structure?
1. Salt bridges
2. Vander Waals forces
3. Ionic bonds
4. Hydrophobic Interactions
5. Hydrogen bonds

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 Functions of Hemoglobin
• Transport of O2 from lungs to tissues
• Transport of CO2 from tissues to lungs
• Transport of H from tissues to lungs and kidney
• Act as an intracellular buffer

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• Oxygen is carried in blood in 2 forms:
1) 97% bound to hemoglobin.
2) 3% dissolved in the water of the plasma and
blood cells.
• Hemoglobin increases the O₂ carrying capacity of
blood to 30 to 100 fold.
Hemoglobin in RBCs account for
• 33% of the RBC volume (1/3)
• 90-95% of the dry weight of RBC is Hb.
Normal level of Hb
• Newborn: 14-24 gm/dL
• 6 months - 6 year: 10-14 g/dL
• Adult male: 13.5 - 17.5 gm/dL
• Adult female: 12 - 15.5 gm/dL
• Pregnant female: 11 - 14 gm/dL

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Functions of globin
Forming a protective hydrophobic
pocket for haem in order to protect
the reduced form of iron (Fe2+).

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 Normal Hemoglobin Variants

Type Composition % of Total

and Symbol Hemoglobin

HbA1 α2β2 97%

HbA2 α2δ2 2%
HbF α2γ2 <1% (at birth
80%)
HbA1c α2β2-glucose <6%

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Terminologies

Carbaminohemoglobin
Carboxyhemoglobin
Deoxyhemoglobin
Methemoglobin
Oxyhemoglobin
Forms of Hemoglobin
T form (tense/taut/) – deoxyhemoglobin
R form (relaxed) – oxyhemoglobin
 T Form of Hb R Form Of Hb
Deoxy Hb is in T form binds with Oxy Hb is in R form binds only with
CO2,H+ and 2,3BPG Oxygen

T form has 8 salt bridges linked in Salt bridges are broken in

between the dimer subunits between the dimer subunits
during oxygenation of Hb.

More constrained form Less constrained form

T form has low affinity for O2 R form has higher affinity for O2

T form of Hb predominates in low R form of Hb predominates at high

pO2 pO2
 Reversible Combination of Oxygen with Hemoglobin

• Oxygen molecule combines loosely and reversibly

with the heme portion of hemoglobin.
• When PO₂ is high, as in the pulmonary capillaries,
oxygen binds with the hemoglobin,
• When PO₂ is low, as in the tissue capillaries, oxygen is
released from the hemoglobin.
• When fully saturated, each gram of hemoglobin
binds 1.34 ml of oxygen.
• The degree of saturation is related to the oxygen
tension (pO2), which normally ranges from 100 mm
Hg in arterial blood to about 35 mmHg in veins.
• The relation between O2 tension and hemoglobin-
oxygen saturation is described by the oxygen-
dissociation curve (ODC).
 Oxygen Dissociation Curve

 A graph with oxygen partial pressure (pO2) along the

horizontal axis and oxygen saturation on the vertical axis
When O2 binds to the first subunit of deoxyhemoglobin it
increases the affinity of the remaining subunits for O2.
 O2 dissociation curves of myoglobin: Hyperbolic
 O2 dissociation curves of hemoglobin: Sigmoidal
 At 100 torr of pO2, Hb is fully saturated with O2.
 The “P50”

• A common point of reference on the oxygen

dissociation curve is the P50.
• The P50 represents the partial pressure at
which the hemoglobin is 50% saturated with
oxygen, typically 26.6 mmHg in adults.
• The P50 is a conventional measure of
hemoglobin affinity for oxygen.
 Shifts in the P50

• The presence of diseases or other conditions:

• Shift ODC to the right or left.
• An increased P50 causes a rightward shift and
indicates lower affinity
• A decreased P50 causes a leftward shift and
indicates a higher affinity.
 Factors Affecting O2 Dissociation Curve
The effectiveness of hemoglobin-oxygen binding can be
affected by several factors, including:
1. Temperature
2. pH
3. 2, 3-BPG
4. Carbon monoxide (CO)
5. Variants/types of hemoglobin
 Heme synthesis
• Substrates mainly include succinyl-CoA,
glycine and Fe2+
• Heme can be synthesized by almost all the
tissues
• Major sites of synthesis is bone marrow and liver
• Subcellular location: Mitochondria and
cytosol

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Haem synthesis pathway

Ferrochelatase

δ-ALA synthase Protoporphyrinogen oxidase

Coproporpyrinogen oxidase

δ-aminolevulinic acid

δ-ALA dehydratase

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Porphyrinogen Uroporphyrinogen
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deaminase decarboxylase
 1. ALA synthase
Inhibited by:
a. Heme: end-product inhibition
b. Hematin: allosteric inhibition
• Inhibits translocation of ALA synthase from cytoplasm
to mitochondria
c. Lack of Vit. B6

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Heme-synthesis Disorders:
1. Porphyrias
Porphyrias are rare genetic diseases in which
activity of one of the enzymes involved in heme
synthesis is deficient.
Symptoms vary depending on
★the enzyme
★the severity of the deficiency
★whether heme synthesis is affected primarily in liver or
in developing erythrocytes.

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 Porphyrias
can be grouped into erythropoietic porphyria and
hepatic porphyria
- hepatic can be acute or chronic
caused by hereditary or acquired defects in heme
synthesis
— hereditary : the enzymes of heme synthesis
— acquired :Liver dysfunction, lead posioning
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Biochemical causes of major sign and symptoms4of porphyria
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Abnormal Hemoglobin synthesis
/Hemoglobinopathy
• Mutations in the genes that code for globin
chains can affect their formation and
biological function of hemoglobin
• Biological function is altered due to mutation
in hemoglobin
• Quantitative and qualitative:
 Quantitative:
• ↓ synthesis of globin chain
• Chains are structurally normal
• αThalassemia
• βThalassemia
Qualitative: altered sequence of amino acids
• Sickle cell disease
• Hb C disease
• Hb M disease
 Thalassaemia
• Genetically transmitted disorders of hemoglobin
synthesis
• ↓synthesis of α or β chain
↓ production of normal hemoglobin
• The synthesis of 1 globin chain reduced, there is a
relative excess synthesis of the other globin chains
• Hemolysis of cell resulting in hypochromic anaemia
Types of -Thalassemia – Four types

Type Missing Symptoms

gene
(a) Silent carrier 1 No

(a)  Thalassemia 2 Mild anaemia

(a) Hemoglobin H 3 Moderate

Disease anaemia
(a) Hydrofetalis 4 Severe form
Types of  thalassemia – two types
(a)  Thalassemia Minor ( Thalassemia Trait)
Heterozygous state – defect in only one 
globulin gene
Usually asymptomatic.
(b)  Thalassemia Major
Homozygous state (both gene)
At birth baby is healthy
After birth severe anemic & die 1-2 yrs.
 Methemoglobin

Oxidized hemoglobin
Ferrous iron(Fe2+) oxidized to ferric state(Fe3+)
Normally, MetHb formed in the RBCs reduced back
to Fe2+ by MetHb reductase enzyme system
NADH cyt b5 – 75%
NADPH dependent system – 20%
Glutathione dependent MetHb reductase
accounts for the rest 5% activity.
 HbM Diseases:
Mutation in either proximal or distal histidine
residue of either α or β chains, which bound with
the iron in the haem molecule.
Glu residue is substituted with Lys resdue.
Histidine replaced by tyrosine.
Iron is oxidized to Fe3+ form
Sickle cell disease/sickle cell anemia
• HbS
• Due to mutation in globin genes
• Sickle cell disease: Symptomatic-anemia
• Homozygous
• Due to defective genes from each parents
• Sickle cell trait:
• Asymtomatic
• Heterozygous
• defective gene from only one parent
 HbS

Genetic defect: β chain

HbA:
1 2 3 4 5 6 7 8
Val –His –leu –Thr –Pro –Glu–Glu –Lys

HbS
Val –His –leu –Thr –Pro –Val–Glu-Lys
 HbS: Replacement of Glu by Val
• Loss of -ve charge in each of 2 βchains
• Polar Glu replaced by non-polar Val produces
sticky patch at 6th position of βchain (insoluble).
• Bind to another deoxygenated HbS
(polymerization of deoxy-HbS)
• Deforming the RBCs to Sickle shape
HbS
 Characteristic Features

Sickled RBCs lose water and become fragile

• Shorter life span (17 days) lysis of RBC anaemia
• Small blood capillaries blocked by abnormal RBCs which
interrupts oxygen supply and leads to anoxia
• People with sickle cell trait show resistant for Plasmodium
falciparum
 Fate of Red Blood Cells

The average life span of RBC in blood stream is 120 days

 RBCs are phagocytosed and/or lysed
Normally, lysis occurs extravascularly in the
RES subsequent to RBC phagocytosis.
 Lysis can also occur intravascularly.

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Catabolism of Heme: Formation of Bilirubin
1. The end product of heme catabolism is bile
pigment (bilirubin)
• Bilirubin has no function in the body and is excreted.
2. The globin chains are separated - amino acids -
amino acid pool.
3. The porphyrin ring is broken down in the
reticuloendothelial ( RE) cells of liver, spleen & bone
marrow to bile pigments, mainly bilirubin

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4. Heme is degraded primarily by microsomal enzyme:
heme oxygenase.

5. The ferrous ( Fe+2) librated is oxidized to Ferric ( Fe+3) and

taken up by transferrin.

6. The linear tetrapyrrol formed is bilivedrin, which is green

in color.
• In mammals it is further reduced to bilirubin, a red-
yellow pigment.

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(Liver, Bone marrow,
& Spleen)

Phagocytosis & Lysis

Hemoglobin

Globin Heme Bilirubin

Amino acids Fe2+

Amino acid pool Excreted

 Blood Cells Stercobilin
excreted in feces Urobilin
excreted in urine
Hemoglobin

Globin
Urobilinogen
Heme formed by bacteria KIDNEY
O2 reabsorbed
INTESTINE into blood
Heme oxygenase
CO
via bile duct to intestines
Biliverdin IX
NADPH
Bilirubin diglucuronide
Biliverdin
(water-soluble)
reductase
UDP glucuronyl tranferase
NADP+ 2 UDP-glucuronic acid
Bilirubin Bilirubin
(water-insoluble) (water-insoluble) LIVER
via blood to
the liver
 Hyperbilirubinemia
 Elevated bilirubin levels in the blood (>10 mg/l)
 bilirubin may diffuse into peripheral tissues (jaundice)
Types of jaundice:
1. Pre-hepatic: excessive formation of bilirubin by
increased degradation of erythrocytes (icterus neonatus,
hemolytic anemia).
2.Hepatic: insufficient processing of bilirubin as a result of
liver defects (hepatitis, liver toxic damage, cirrhosis,
hepatic failure).
3. Post-hepatic: by impaired excretion of gall
(obstructive jaundice due to gallstones, inflammation
of biliary tract)
 Neonatal Jaundice
• Common, particularly in premature infants
• Transient (resolves in the first 10 days)
• Due to immaturity of the enzymes involved in
bilirubin conjugation (UDP glucuronyl
transferase)
• High levels of unconjugated bilirubin are toxic to
the newborn
• Causes mental retardation (kernicterus)
If bilirubin levels are judged to be too high
• Phototherapy with UV light
• converts bilirubin into water soluble, non-toxic form
• Blood transfusion
• removes excess bilirubin
• Pharmacotherapy (phenobarbital)
• Crosses placenta induces the synthesis of UDP glucuronyl
transferase
S.No Hemoglobin (Hb) Myoglobin (Mb)

1. Hb is Oxygen transport protein in Mb is Oxygen storing protein in

Differences of Hb And Mb
RBCs of blood. muscles.

2. Tetrameric has four Heme and Monomeric has one Heme and binds
binds with 4O2 with 1 O2.

3. Oxygenated at Lungs Oxygenated at Muscle Cell Cytosol.

4. HbO2 unloads oxygen at tissues MbO2 unloads oxygen at cell cytosol

when pO2 is at 40 mmHg. when pO2 is at 5 mmHg. to rapidly
respiring cells
P50 for HbA1 is 27 torr.
P50 for Mb is 2 torr.

5. ODC is sigmoid shaped ODC is hyperbolic shaped.

6. Hb has 574 amino acids. Mb has 153 amino acids.

Mol .wt-67,000 Daltons. Mol wt-17,200 Daltons.