Transcript
Page 1: HEMOGLOBIN DERIVATIVES

Haemoglobin Derivatives

Gandham. Rajeev

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• Hemoglobin derivatives are formed by the

combination of different ligands with the

heme part, or change in the oxidation state

of iron.

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Carboxy-Hemoglobin (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.

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• 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.

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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

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• 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.

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Clinical Manifestations

• Clinical symptoms manifest when carboxy-Hb

levels exceed 20%.

• Breathlessness, headache, nausea, vomiting,

& chest pain.

• At 40-60% saturation, death can result.

• Administration of O2 is the treatment.

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Methemoglobin (Met-Hb)

• When the ferrous (Fe2+ ) iron is oxidized to

ferric (Fe3+) 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 & cytochrome b5

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Methemoglobinemias

• Normal blood has only less than 1% of

methemoglobin.

• It has markedly decreased capacity for

oxygen binding and transport.

• An increase in methemoglobin in blood,

(methemoglobinemia) is manifested as

cyanosis.

• Causes may be congenital or acquired.

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Congenital Methemoglobinemia

• Presence of Hb variants like HbM can cause

congenital methemoglobinemia.

• Cytochrome b5 reductase deficiency is characterized

by cyanosis from birth.

• 10-15% of hemoglobin may exist as methemoglobin.

• 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.

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Acquired or Toxic Methemoglobinemia

• Met-hemoglobinemia may develop by intake

of water containing nitrates or due to

absorption of aniline dyes.

• Drugs which produce met-hemoglobinemia -

acetaminophen, phenacetin, sulphanilamide,

amyl nitrite, & sodium nitroprusside.

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Sulf-hemoglobinemia

• When hydrogen sulfide acts on oxy-Hb, sulf-

hemoglobin is produced.

• It occur in people taking drugs like

sulphonamides, phenacetin, acetanilide,

dapsone, etc.

• It cannot be converted back to oxy-

hemoglobin.

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Hemoglobinopathies

• Abnormal hemoglobins are the resultant of

mutations in the genes that code for α or β

chains of globin

• As many as 400 mutant hemoglobins are

known.

• About 95% of them are due to alteration in

single amino acid of globin

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Types of abnormal Hb

• Two types:

• If the mutation affects structural gene, it

results in replacement of a single amino acid

in Hb by some other amino acid resulting into

abnormal Hb.

• E.g: Hb-S, Hb-M, Hb-C, Hb-D & others.

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• If the mutation affects the regulator gene,

which affects the rate of synthesis of

peptide chains, the amino acid sequence

remains unaffected.

• E.g: Thalassaemias

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Globin synthesis

• The globin genes are organised into two gene

families or clusters

• α-Gene family:

• There are 2 genes coding for α-globin chain

present on each one of chromosome 16.

• The ζ (zeta)-gene, other member of a-gene

cluster is also found on chromosome 16 & is

active during the embryonic development

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• β-Gene family:

• The synthesis of β-globin occurs from a single

gene located on each one of chromosome 11.

• This chromosome also contains four other

genes.

• One ε-gene expressed in the early stages of

embryonic development.

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• Two γ-genes (Gγ & Aγ) synthesize γ-globin

chains of fetal hemoglobin (HbF).

• One δ-gene producing δ-globin chain found in

adults to a minor extent (HbA2).

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Sickle-cell anemia (HbS)

• Sickle-cell anemia (HbS) is the most common

form of abnormal hemoglobins.

• Erythrocytes of these patients adopt a sickle

shape (crescent like) at low oxygen

concentration

• It primarily occurs in the black population.

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Molecular basis of HbS

• The glutamic acid in the 6th position of β chain

of HbA is changed to valine in HbS.

• This single amino acid substitution leads to

polymerization of hemoglobin molecules

inside RBCs.

• This causes a distortion of cell into sickle

shape

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Normal & HbS

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• The substitution of hydrophilic glutamic acid

by hydrophobic valine causes a localized

stickiness on the surface of the molecule

• The deoxygenated HbS may be depicted with

a protrusion on one side and a cavity on the

other side, so that many molecules can

adhere and polymerize

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• The sickled cells form small plugs in

capillaries.

• Occlusion of major vessels can lead to

infarction in organs like spleen.

• Death usually occurs in the second decade of

life.

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Homozygous and heterozygous HbS

• Sickle cell anemia is said to be homozygous, if

caused by inheritance of two mutant genes

(one from each parent) that code for β-chains.

• In case of heterozygous HbS, only one gene (of

β-chain) is affected while the other is normal

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• The erythrocytes of heterozygotes contain

both HbS & HbA & the disease is referred to as

sickle cell trait.

• The individuals of sickle-cell trait lead a normal

life, & do not usually show clinical symptoms.

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Abnormalities associated with HbS

• Life-long hemolytic anemia:

• The sickled erythrocytes are fragile & their

continuous breakdown leads to life-long

anemia.

• Tissue damage and pain:

• The sickled cells block the capillaries resulting

in poor blood supply to tissues.

• This leads to extensive damage & inflammation

of certain tissues causing pain.

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• Increased susceptibility to infection :

• Hemolysis & tissue damage are accompanied

by increased susceptibility to infection &

diseases.

• Prematured eath:

• Homozygous individuals of sickle-cell anemia

die before they reach adulthood (< 20 years)

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Mechanism of sickling in sickle-cell anemia

• Glutamate is a polar amino acid & it is

replaced by a non-polar valine in sickle-cell

hemoglobin.

• This causes a marked decrease in the solubility

of HbS in deoxygenated form

• Solubility of oxygenated HbS is unaffected

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Sticky patches & formation ofdeoxyhemoglobin fibres

• The substitution of valine for glutamate

results in a sticky patch on the outer surface

of β-chains.

• It is present on oxy- & deoxyhemoglobin S

but absent on HbA.

• There is a site or receptor complementary to

sticky patch on deoxyHbS.

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• The sticky patch of one deoxyHbS binds with

the receptor of another deoxyHbS & this

process continuous resulting in the formation

of long aggregate molecules of deoxyHbS

• The polymerization of deoxy-HbS molecules

leads to long fibrous precipitates.

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• These stiff fibres distort the erythrocytes into

a sickle or crescent shape

• The sickled erythrocytes are highly

vulnerable to lysis.

• ln case of oxyHbS, the complementary

receptor is masked, although the sticky patch

is present.

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HbS gives protection against malaria

• HbS affords protection against Plasmodium

falciparum infection

• Hence the abnormal gene was found to offer

a biologic advantage.

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Sickle cell trait protects from malaria

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Diagnosis of sickle cell anemia

• Sickling test:

• A simple microscopic examination of blood

smear prepared by adding reducing agents

such as sodium dithionite.

• Sickled erythrocytes can be detected under

the microscope

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Electrophoresis

• Electrophoresis at alkaline pH shows a slower

moving band than HbA.

• At pH 8.6, carboxyl group of glutamic acid is

negatively charged.

• Lack of this charge on HbS makes it less negatively

charged, & decreases the electrophoretic mobility

• At acidic pH, HbS moves faster than HbA.

• In sickle cell trait, both the bands of HbA and HbS can

be noticed

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Electrophoresis at pH 8.6

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Management of sickle cell disease

• Administration of sodium cyanate inhibits

sickling of erythrocytes

• Cyanate increases the affinity of O2 to HbS &

lowers the formation of deoxyHbS

• It causes certain side effects like peripheral

nerve damage

• In severe anemia, repeated blood transfusion

is required.

• It result in iron overload & cirrhosis of liver

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Hemoglobin C disease

• Cooley's hemoglobinemia (HbC) is characterized by

substitution of glutamate by lysine in the sixth position

of β-chain.

• Due to the presence of lysine, HbC moves more slowly

on electrophoresis compared to HbA and HbS.

• HbC disease occurs only in blacks.

• Both homozygous & heterozygous individuals of HbC

disease are known.

• It is characterized by mild hemolytic anemia.

• No specific therapy is recommended.

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Hemoglobin D

• Caused by the substitution of glutamine in

place of glutamate in the 121st position of β-

chain.

• Several variants of HbD are identified from

different places indicated by the suffix.

• For instance, HbD (Punjab)

• HbD, on electrophoresis moves along with

HbS.

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Hemoglobin E

• Most common abnormal hemoglobin after HbS.

• lt is estimated that about 10% of the population in

South-East Asia (Bangladesh, Thailand, Myanmar)

suffer from HbE disease.

• In India, it is prevalent in West Bengal.

• HbE is characterized by replacement of glutamate by

lysine at 26th position of β-chain.

• The individuals of HbE (either homozygous or

heterozygous) have no clinical manifestations

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Thalassemias

• Thalassemias are a group of hereditary

hemolytic disorders characterized by

impairment/imbalance in the synthesis of globin

chains of Hb

• Thalassemias (Greek: thalassa-sea) mostly

occur in the regions surrounding the

Mediterranean sea, hence the name.

• Also prevalent in Central Africa, India.

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Molecular basis of thalassemias

• Hemoglobin contains 2α & 2β globin chains.

• The synthesis of individual chains is so

coordinated that each α-chain has a β-chain

partner & they combine to finally give

hemoglobin (α2β2).

• Thalassemias are characterized by a defect in

the production of α-or β-globin chain

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• Thalassemias occur due to a variety of

molecular defects

• Gene deletion or substitution,

• Underproduction or instability of mRNA,

• Defect in the initiation of chain synthesis,

• Premature chain termination.

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α-Thalassemiasas

• α-Thalassemias are caused by a decreased

synthesis or total absence of α-globin chain of

Hb.

• There are four copies of α-globin gene, two on

each one of the chromosome 16.

• Four types of α-thalassemias occur which

depend on the number of missing α-globin

genes

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Salient features of different α -thalassemias

• Silent carrier state is due to loss of one of the

four α -globin genes with no physical

manifestations.

• α -Thalassemia trait caused by loss of two genes

(both from the same gene pair or one from each

gene pair).

• Minor anemia is observed

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• Hemoglobin H disease, due to missing of three

genes, is associated with moderate anemia

• Hydrops fetalis is the most severe form of α-

thalassemias due to lack of all the four genes.

• The fetus usually survives until birth & then dies.

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β-thalassemias

• Decreased synthesis or total lack of the

formation of β-globin chain causes β-

thalassemias.

• The production of α-globin chain continues to

be normal, leading to the formation of a globin

tetramer (α4) that precipitate.

• This causes premature death of erythrocytes.

• There are mainly two types of β-thalassemias

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β-Thalassemia minor

• This is an heterozygous state with a defect in

only one of the two β-globin gene pairs on

chromosome 11.

• Also known as β -thalassemia trait, is usually

asymptomatic, since the individuals can make

some amount of β-globin from the affected

gene

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β-Thalassemia major

• This is a homozygous state with a defect in

both the genes responsible for β-globin

synthesis.

• The infants born with β-thalassemia major

are healthy at birth since β-globin is not

synthesized during the fetal development

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• They become severely anemic and die within

1-2 years.

• Frequent blood transfusion is required for

these children.

• This is associated with iron overload which in

turn may lead to death within 15-20 years

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References

• Text book of Biochemistry – U Satyanarayana

• Text book of Biochemistry – DM Vasudevan

• Text book of Biochemistry – MN Chatterjea

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Thank You


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