Hemoglobin Synthesis

Introduction 

• Hemoglobin is a complex iron-containing protein present in red blood cells.

• It is responsible for the transport of oxygen from the lungs to body tissues.

• Hemoglobin also plays a crucial role in transporting carbon dioxide from tissues to the lungs.

• Each hemoglobin molecule consists of four globin chains and four heme groups.

• The iron present in the heme group enables reversible binding of oxygen.

• Hemoglobin plays a significant role in maintaining blood pH by acting as a buffer.

• It is synthesized in the erythroid precursor cells of the bone marrow.

• Normal hemoglobin structure and function are essential for efficient tissue oxygenation.

• Abnormalities in hemoglobin synthesis or structure result in anemia and hemoglobinopathies.

• Understanding hemoglobin is fundamental in biochemistry, hematology, and clinical diagnosis.

 

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Hemoglobin Synthesis

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Hemoglobin synthesis occurs mainly in the erythroid precursor cells of the bone marrow and involves the coordinated production of globin chains and heme, which later assemble to form a functional hemoglobin molecule.

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Haemoglobin comprises four polypeptide chains, each containing an iron-bound heme group. The synthesis of Hemoglobin involves several steps:

Hemoglobin Structure

• Hemoglobin is a tetrameric conjugated protein present inside red blood cells.

• Each hemoglobin molecule consists of four polypeptide (globin) chains.

• In adult hemoglobin (HbA), the globin chains are:

  • Two alpha (α) chains

  • Two beta (β) chains

• Each globin chain is tightly associated with a heme group, making a total of four heme groups per hemoglobin molecule.

• The heme group is a non-protein prosthetic group composed of:

  • A porphyrin ring (protoporphyrin IX)

  • A centrally placed ferrous iron (Fe²⁺) atom

• The iron atom forms reversible bonds with oxygen, allowing hemoglobin to bind and release oxygen efficiently.

• Each heme group can bind one molecule of oxygen, so one hemoglobin molecule can carry four oxygen molecules.

• The globin chains are arranged in a quaternary structure, which is essential for cooperative oxygen binding.

• The interaction between globin chains allows hemoglobin to shift between:

  • Tense (T) state – low oxygen affinity

  • Relaxed (R) state – high oxygen affinity

• This unique structural arrangement makes hemoglobin an efficient oxygen transporter under varying physiological conditions.

 

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Synthesis of Globin Chains

Chromosomal Location of Globin Genes

• Alpha (α) globin genes → Chromosome 16

• Beta (β), Gamma (γ), Delta (δ) globin genes → Chromosome 11

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Types of Globin Chains and Their Expression

• α chains: Synthesized throughout fetal and adult life

• γ chains: Predominant during fetal life → form HbF

• β chains: Predominant after birth → form HbA

• δ chains: Minor component in adults → form HbA₂

This transition from γ to β chain synthesis after birth is known as globin gene switching.

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Steps in Synthesis of Globin Chains

1. Transcription

• Occurs in the nucleus of erythroid precursor cells.

• Globin genes are transcribed into messenger RNA (mRNA).

• Regulated by transcription factors and regulatory DNA sequences.

2. mRNA Processing

• Newly formed mRNA undergoes:

  • Capping

  • Polyadenylation

  • Removal of introns (splicing)

• Mature mRNA exits the nucleus into the cytoplasm.

3. Translation

• Takes place on ribosomes in the cytoplasm.

• mRNA is translated into globin polypeptide chains.

• Each chain folds into its specific secondary and tertiary structure.

4. Post-Translational Stability

• Globin chains are unstable without heme.

• Binding of heme protects globin chains from degradation.

• Balanced synthesis ensures efficient hemoglobin assembly.

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Regulation of Globin Chain Synthesis

• Controlled by gene availability, transcription rate, and mRNA stability.

• Heme availability enhances globin chain synthesis.

• Erythropoietin indirectly stimulates globin production by increasing erythropoiesis.

• Imbalance between α and β chain synthesis leads to pathological conditions.

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Table: Summary of Globin Chain Synthesis

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Synthesis of Heme

Cellular Location of Heme Synthesis

• Mitochondria: Initial and final steps

• Cytosol: Intermediate steps

• This compartmentalization is crucial for regulation and efficient synthesis.

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Steps of Heme Synthesis

Step 1: Formation of δ-Aminolevulinic Acid (ALA)

• Occurs in the mitochondria

• Glycine + Succinyl-CoA → δ-ALA

• Enzyme: ALA synthase (rate-limiting enzyme)

• Requires pyridoxal phosphate (vitamin B₆)

• This step is inhibited by heme (feedback inhibition)

Step 2: Formation of Porphobilinogen

• Occurs in the cytosol

• 2 ALA → Porphobilinogen

• Enzyme: ALA dehydratase

• Highly sensitive to lead inhibition

Step 3: Formation of Hydroxymethylbilane

• 4 Porphobilinogen → Hydroxymethylbilane

• Enzyme: Porphobilinogen deaminase

• Defect leads to acute intermittent porphyria

Step 4: Formation of Uroporphyrinogen III

• Hydroxymethylbilane is cyclized to uroporphyrinogen III

• Enzyme: Uroporphyrinogen III synthase

Step 5: Formation of Coproporphyrinogen III

• Occurs in cytosol

• Uroporphyrinogen III undergoes decarboxylation

• Enzyme: Uroporphyrinogen decarboxylase

Step 6: Formation of Protoporphyrin IX

• Coproporphyrinogen III re-enters mitochondria

• Converted to protoporphyrin IX

• Enzymes: Coproporphyrinogen oxidase and Protoporphyrinogen oxidase

Step 7: Formation of Heme

• Final mitochondrial step

• Protoporphyrin IX + Fe²⁺ → Heme

• Enzyme: Ferrochelatase

• Also inhibited by lead

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Table: Steps of Heme Synthesis

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Regulation of Heme Synthesis

• ALA synthase is the key regulatory enzyme.

• Heme acts as a feedback inhibitor of ALA synthase.

• Iron availability directly influences the final step of heme formation.

• In erythroid cells, synthesis is regulated by globin chain availability.

 

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Hemoglobin Assembly

1. Association of Heme with Globin Chains

• Each synthesized globin chain binds one heme molecule.

• This binding occurs mainly in the cytoplasm of erythroid cells.

• The iron (Fe²⁺) of heme forms a strong coordination bond with the globin chain.

• Proper heme insertion is essential for globin chain stability.

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2. Formation of Globin–Heme Monomers

• A single globin chain plus one heme group forms a heme–globin monomer.

• This step ensures that globin chains are protected from degradation.

• Free globin chains without heme are unstable and rapidly degraded.

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3. Dimer Formation

• One α-globin–heme complex pairs with one β-globin–heme complex to form an αβ dimer.

• In fetal life, α-globin pairs with γ-globin to form αγ dimers.

• Dimer formation is a prerequisite for tetramer assembly.

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4. Tetramer Formation

• Two αβ (or αγ) dimers associate to form a hemoglobin tetramer.

• Adult hemoglobin: α₂β₂ (HbA)

• Fetal hemoglobin: α₂γ₂ (HbF)

• The tetrameric structure is stabilized by non-covalent interactions.

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5. Development of Functional Conformation

• The tetramer exists in two interconvertible states:

  • Tense (T) state – low oxygen affinity

  • Relaxed (R) state – high oxygen affinity

• Binding of oxygen shifts hemoglobin from T-state to R-state.

• This structural transition is responsible for cooperative oxygen binding.

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Factors Influencing Hemoglobin Assembly

• Balanced globin chain synthesis is essential for effective assembly.

• Availability of iron and intact heme synthesis pathway.

• Adequate erythropoietin stimulation for erythropoiesis.

• Absence of genetic defects in globin genes.

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Table: Hemoglobin Assembly

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Hemoglobin Function

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1. Oxygen Transport 

• Hemoglobin transports oxygen (O₂) from the lungs to peripheral tissues.

• In the lungs, where partial pressure of oxygen (pO₂) is high, oxygen binds to hemoglobin forming oxyhemoglobin.

• Each hemoglobin molecule can carry four oxygen molecules, one per heme group.

• In tissues with low pO₂, hemoglobin releases oxygen for cellular metabolism.

• This process is facilitated by the sigmoidal oxygen–hemoglobin dissociation curve, indicating cooperative binding.

Physiological Advantages

• Ensures efficient oxygen loading in lungs.

• Enables maximal oxygen unloading in metabolically active tissues.

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2. Carbon Dioxide Transport

• Hemoglobin helps transport carbon dioxide (CO₂) from tissues to the lungs.

• CO₂ binds to the globin portion of hemoglobin forming carbaminohemoglobin.

• Approximately 20–30% of CO₂ is transported in this form.

• Deoxygenated hemoglobin binds CO₂ more readily (Haldane effect).

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3. Acid–Base Buffering Action

• Hemoglobin acts as a powerful buffer system in blood.

• It binds hydrogen ions (H⁺) released during tissue metabolism.

• This buffering capacity helps maintain normal blood pH (≈7.4).

• Hemoglobin works in coordination with the bicarbonate buffer system.

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4. Transport of Nitric Oxide (NO)

• Hemoglobin modulates nitric oxide availability, influencing vascular tone.

• NO binding contributes to vasodilation, improving tissue perfusion.

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5. Regulation of Oxygen Delivery (Bohr Effect)

• Increased CO₂, H⁺ concentration, and temperature reduce hemoglobin’s oxygen affinity.

• This promotes oxygen release in actively metabolizing tissues.

• Known as the Bohr effect, it enhances tissue oxygenation.

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Table: Functions of Hemoglobin

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Clinical Importance

• Reduced hemoglobin concentration leads to anemia and tissue hypoxia.

• Structural abnormalities cause hemoglobinopathies (e.g., sickle cell disease).

• Altered oxygen affinity affects oxygen delivery in lung and cardiac diseases.

 

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Hemoglobin Degradation

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1. Lifespan and Destruction of Red Blood Cells

• Red blood cells have an average lifespan of ~120 days.

• Senescent or damaged RBCs lose membrane flexibility.

• These cells are removed by macrophages of the reticuloendothelial system (RES), mainly in:

  • Spleen (primary site)

  • Liver

  • Bone marrow

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2. Breakdown of Hemoglobin

Inside macrophages, hemoglobin is split into two major components:

A. Globin Part

• Globin chains are hydrolyzed into amino acids.

• Amino acids are released into circulation.

• Reused for new protein synthesis.

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B. Heme Part

Heme undergoes a series of enzymatic conversions:

Step 1: Conversion of Heme to Biliverdin

• Enzyme: Heme oxygenase

• Heme is opened to form biliverdin

• Iron (Fe³⁺) is released

• Carbon monoxide (CO) is produced as a by-product

Step 2: Conversion of Biliverdin to Bilirubin

• Enzyme: Biliverdin reductase

• Biliverdin is reduced to bilirubin

• Bilirubin at this stage is unconjugated, lipid-soluble, and toxic

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3. Transport of Unconjugated Bilirubin

• Unconjugated bilirubin is transported in blood bound to albumin.

• Albumin prevents bilirubin from crossing the blood–brain barrier.

• Delivered to the liver for further processing.

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4. Hepatic Conjugation of Bilirubin

• In hepatocytes, bilirubin undergoes conjugation.

• Enzyme: UDP-glucuronosyltransferase

• Bilirubin is converted into bilirubin diglucuronide (conjugated bilirubin).

• Conjugation makes bilirubin water-soluble and non-toxic.

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5. Excretion and Intestinal Metabolism

• Conjugated bilirubin is excreted into bile.

• Bile enters the intestine.

• Intestinal bacteria convert bilirubin into urobilinogen:

  • Majority → Stercobilin (excreted in feces; gives brown color)

  • Small amount → Reabsorbed → excreted as urobilin in urine (yellow color)

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6. Iron Recycling

• Iron released from heme is oxidized to Fe³⁺.

• Transported in plasma by transferrin.

• Utilized for:

  • New hemoglobin synthesis in bone marrow

  • Storage as ferritin or hemosiderin in liver and spleen

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Table: Hemoglobin Degradation

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Clinical Importance of Heamoglobin

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1. Role in Oxygen Delivery and Tissue Hypoxia

• Hemoglobin determines the oxygen-carrying capacity of blood.

• Reduced hemoglobin levels lead to tissue hypoxia, even when lung function is normal.

• Clinical manifestations include:

  • Fatigue

  • Dyspnea

  • Palpitations

  • Poor exercise tolerance

• Severe hypoxia can cause organ dysfunction, particularly in the brain and heart.

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2. Anemia and Its Clinical Significance

• Anemia is defined as a reduction in hemoglobin concentration below normal limits.

• Causes include:

  • Iron deficiency

  • Vitamin B₁₂ or folate deficiency

  • Chronic disease

  • Bone marrow failure

• Chronic anemia leads to:

  • Compensatory tachycardia

  • Cardiac hypertrophy

  • High-output heart failure (in severe cases)

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3. Hemoglobinopathies (Structural Abnormalities)

• Genetic defects affecting hemoglobin structure lead to hemoglobinopathies.

• Example:

  • Sickle cell disease – abnormal β-globin structure causes RBC sickling under low oxygen tension.

• Clinical effects:

  • Chronic hemolysis

  • Vaso-occlusive crises

  • Increased susceptibility to infections

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4. Thalassemia (Disorders of Globin Chain Synthesis)

• Caused by reduced or absent synthesis of globin chains.

• Types:

  • α-thalassemia

  • β-thalassemia

• Consequences:

  • Ineffective erythropoiesis

  • Severe anemia

  • Bone marrow expansion

  • Iron overload due to repeated transfusions

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5. Disorders of Heme Synthesis (Porphyrias)

• Enzyme defects in heme synthesis cause porphyrias.

• Clinical features depend on the accumulated intermediate:

  • Abdominal pain

  • Neurological symptoms

  • Photosensitivity

• Lead poisoning inhibits heme synthesis enzymes, causing:

  • Microcytic anemia

  • Neuropathy

  • Abdominal pain

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6. Jaundice and Bilirubin Metabolism Disorders

• Excess hemoglobin breakdown increases bilirubin production.

• Types of jaundice:

  • Hemolytic jaundice – excess unconjugated bilirubin

  • Hepatic jaundice – impaired conjugation

  • Obstructive jaundice – blocked bile flow

• Neonatal jaundice occurs due to immature liver conjugating enzymes.

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7. Iron Metabolism and Storage Disorders

• Hemoglobin degradation is the major source of body iron.

• Disorders include:

  • Iron deficiency anemia

  • Iron overload (hemochromatosis, transfusion-related)

• Abnormal iron metabolism affects:

  • Hemoglobin synthesis

  • Erythropoiesis

  • Organ function (liver, heart, pancreas)

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8. Role in Acid–Base Balance

• Hemoglobin is a major non-bicarbonate buffer of blood.

• It binds hydrogen ions generated during metabolism.

• Impaired hemoglobin buffering worsens:

  • Metabolic acidosis

  • Respiratory acidosis

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9. Diagnostic and Laboratory Importance

• Hemoglobin estimation is used for:

  • Screening anemia

  • Monitoring therapy response

  • Assessing nutritional status

• Abnormal hemoglobin variants are detected by:

  • Electrophoresis

  • HPLC

  • Genetic testing

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10. Cardiopulmonary and Critical Care Relevance

• Low hemoglobin worsens outcomes in:

  • Cardiac disease

  • Respiratory failure

  • Sepsis

• Hemoglobin levels guide:

  • Blood transfusion decisions

  • Oxygen therapy

  • ICU management

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Table: Clinical Conditions Related to Hemoglobin

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MCQs

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1. Hemoglobin synthesis occurs mainly in:

A. Liver
B. Kidney
C. Bone marrow
D. Spleen

✅ Answer: C

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2. The protein part of hemoglobin is called:

A. Heme
B. Globin
C. Porphyrin
D. Ferritin

✅ Answer: B

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3. Alpha globin genes are located on chromosome:

A. 11
B. 12
C. 16
D. 18

✅ Answer: C

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4. Beta globin genes are located on chromosome:

A. 16
B. 11
C. 13
D. 21

✅ Answer: B

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5. Adult hemoglobin (HbA) consists of:

A. α₂γ₂
B. α₂δ₂
C. α₂β₂
D. α₄

✅ Answer: C

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6. Fetal hemoglobin (HbF) consists of:

A. α₂β₂
B. α₂γ₂
C. β₄
D. α₂δ₂

✅ Answer: B

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7. Globin chain synthesis occurs in:

A. Mitochondria
B. Nucleus only
C. Cytoplasm
D. Lysosomes

✅ Answer: C

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8. Transcription of globin genes occurs in:

A. Cytoplasm
B. Ribosomes
C. Mitochondria
D. Nucleus

✅ Answer: D

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9. Translation of globin mRNA occurs on:

A. Nucleus
B. Ribosomes
C. Golgi apparatus
D. Lysosomes

✅ Answer: B

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10. Heme synthesis occurs in:

A. Cytosol only
B. Mitochondria only
C. Both mitochondria and cytosol
D. Nucleus

✅ Answer: C

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11. First step of heme synthesis occurs in:

A. Cytosol
B. Mitochondria
C. Nucleus
D. Golgi

✅ Answer: B

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12. Rate-limiting enzyme of heme synthesis is:

A. Ferrochelatase
B. ALA dehydratase
C. ALA synthase
D. Porphobilinogen deaminase

✅ Answer: C

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13. ALA synthase requires which vitamin as cofactor?

A. Vitamin B₁
B. Vitamin B₂
C. Vitamin B₆
D. Vitamin B₁₂

✅ Answer: C

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14. Substrates for ALA synthase are:

A. Glycine + Acetyl-CoA
B. Glycine + Succinyl-CoA
C. Alanine + Succinyl-CoA
D. Glycine + Propionyl-CoA

✅ Answer: B

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15. ALA dehydratase is inhibited by:

A. Iron
B. Copper
C. Lead
D. Zinc

✅ Answer: C

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16. Final step of heme synthesis is insertion of:

A. Zinc
B. Copper
C. Magnesium
D. Iron

✅ Answer: D

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17. Enzyme inserting iron into protoporphyrin IX is:

A. ALA synthase
B. Ferrochelatase
C. Heme oxygenase
D. Biliverdin reductase

✅ Answer: B

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18. Ferrochelatase is inhibited by:

A. Mercury
B. Arsenic
C. Lead
D. Cadmium

✅ Answer: C

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19. Heme is synthesized mainly in:

A. Hepatocytes only
B. Erythroid cells only
C. Erythroid cells and liver
D. Kidney cells

✅ Answer: C

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20. Protoporphyrin IX is formed in:

A. Cytosol
B. Mitochondria
C. Nucleus
D. Golgi

✅ Answer: B

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21. Balanced synthesis of globin chains is essential to prevent:

A. Porphyria
B. Jaundice
C. Thalassemia
D. Hemochromatosis

✅ Answer: C

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22. Excess unmatched globin chains cause:

A. RBC stabilization
B. Increased lifespan
C. RBC damage
D. Improved oxygen binding

✅ Answer: C

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23. Globin gene switching refers to:

A. α → β chain change
B. β → γ chain change
C. γ → β chain change
D. δ → β chain change

✅ Answer: C

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24. HbA₂ contains which globin chains?

A. α₂β₂
B. α₂γ₂
C. α₂δ₂
D. β₄

✅ Answer: C

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25. HbA₂ percentage is increased in:

A. Iron deficiency anemia
B. β-thalassemia
C. Sickle cell disease
D. Aplastic anemia

✅ Answer: B

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26. Each hemoglobin molecule contains how many heme groups?

A. One
B. Two
C. Three
D. Four

✅ Answer: D

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27. Each heme group binds:

A. One oxygen molecule
B. Two oxygen molecules
C. Four oxygen molecules
D. One carbon dioxide

✅ Answer: A

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28. Globin chains without heme are:

A. Stable
B. Stored
C. Rapidly degraded
D. Excreted

✅ Answer: C

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29. Assembly of hemoglobin occurs in:

A. Nucleus
B. Cytoplasm
C. Golgi
D. Lysosome

✅ Answer: B

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30. Normal adult hemoglobin percentage is highest for:

A. HbF
B. HbA
C. HbA₂
D. HbS

✅ Answer: B

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31. Defect in globin synthesis leads to:

A. Porphyrias
B. Thalassemias
C. Jaundice
D. Hemochromatosis

✅ Answer: B

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32. Defect in heme synthesis leads to:

A. Thalassemia
B. Porphyrias
C. Sickle cell disease
D. Leukemia

✅ Answer: B

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33. Iron is incorporated into heme in which form?

A. Fe³⁺
B. Fe²⁺
C. Ferritin
D. Hemosiderin

✅ Answer: B

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34. Which is NOT involved in hemoglobin synthesis?

A. Iron
B. Glycine
C. Succinyl-CoA
D. Albumin

✅ Answer: D

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35. Heme regulates its own synthesis by inhibiting:

A. Ferrochelatase
B. ALA dehydratase
C. ALA synthase
D. Biliverdin reductase

✅ Answer: C

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36. Primary site of hemoglobin synthesis in adults is:

A. Liver
B. Spleen
C. Bone marrow
D. Kidney

✅ Answer: C

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37. Alpha globin chains are synthesized:

A. Only in fetal life
B. Only in adult life
C. Throughout life
D. After birth only

✅ Answer: C

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38. Gamma globin chains are predominant in:

A. Adult
B. Neonatal period
C. Fetal life
D. Old age

✅ Answer: C

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39. Normal hemoglobin synthesis requires all EXCEPT:

A. Iron
B. Vitamin B₆
C. Globin genes
D. Vitamin C

✅ Answer: D

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40. Lead poisoning causes anemia mainly due to inhibition of:

A. Globin transcription
B. Iron absorption
C. Heme synthesis enzymes
D. RBC membrane proteins

✅ Answer: C

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41. First stable porphyrin formed is:

A. Protoporphyrin IX
B. Uroporphyrinogen III
C. Coproporphyrinogen III
D. Biliverdin

✅ Answer: B

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42. Heme synthesis intermediate accumulated in AIP is:

A. ALA and PBG
B. Bilirubin
C. Protoporphyrin
D. Heme

✅ Answer: A

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43. Which enzyme defect causes porphyria cutanea tarda?

A. ALA synthase
B. ALA dehydratase
C. Uroporphyrinogen decarboxylase
D. Ferrochelatase

✅ Answer: C

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44. Each globin chain binds heme via:

A. Covalent bond
B. Ionic bond
C. Coordination bond
D. Hydrogen bond

✅ Answer: C

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45. Hemoglobin assembly requires formation of:

A. Monomers only
B. Dimers only
C. Tetramers
D. Trimers

✅ Answer: C

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46. Cooperative oxygen binding is due to:

A. Heme only
B. Globin only
C. Tetrameric structure
D. Iron oxidation

✅ Answer: C

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47. Site of porphobilinogen formation is:

A. Mitochondria
B. Cytosol
C. Nucleus
D. Golgi

✅ Answer: B

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48. Normal hemoglobin synthesis is essential for:

A. RBC shape
B. Oxygen transport
C. CO₂ solubility
D. Plasma volume

✅ Answer: B

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49. Excess iron from hemoglobin synthesis is stored as:

A. Transferrin
B. Hemosiderin
C. Ferritin
D. Bilirubin

✅ Answer: C

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50. Hemoglobin synthesis defects are commonly inherited as:

A. Autosomal recessive disorders
B. Autosomal dominant disorders
C. X-linked disorders
D. Mitochondrial disorders

✅ Answer: A