Hemoglobin Structure & Types - PDF

# **Structure & Types of Hemoglobin** *Red blood cells contain several hundred thousand hemoglobin molecules, which transport oxygen.* ## Hemoglobin Molecule - Hemoglobin is a complex molecule consisting of four polypeptide chains (globins) and a heme group. - Oxygen binds to heme on the h...

# **Structure & Types of Hemoglobin** *Red blood cells contain several hundred thousand hemoglobin molecules, which transport oxygen.* ## Hemoglobin Molecule - Hemoglobin is a complex molecule consisting of four polypeptide chains (globins) and a heme group. - Oxygen binds to heme on the hemoglobin molecule. ## Structure of Hemoglobin - Hb is a spherical molecule consisting of 4 peptide subunits (globins) = Quaternary structure - Hb of adults (Hb A) is a tetramer consisting 2 α and 2 β globins --- each globin contains 1 heme group with a central Fe2+ ion (ferrous ion) - The structure of heme is shown as $H_3C \ \ N \ \ CH_2CH_2COOH$ - It is a cyclic compound 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 ## **Case Study** *Sarah, an 8-year-old female of Mediterranean descent, complains of failure to thrive in early childhood, persistent fatigue and weakness, anemia, jaundice with hepato-splenomegaly.* *Also reports a family history of anemia and an older brother being diagnosed with genetic blood disorder.* ## Clinical Features **Clinical manifestations of beta thalassemia major include:** * Failure to thrive in early childhood * Anemia * Jaundice, usually slight; gallstones * Hepatosplenomegaly, which may be massive; hypersplenism * Bone abnormalities: * Abnormal facies, prominence of malar eminences, frontal bossing, depression of bridge of the nose and exposure of upper central teeth * Skull radiographs showing hair-on-end appearance due to widening of diploic spaces * Fractures due to marrow expansion and abnormal bone structure * Generalized skeletal osteoporosis. * Growth retardation, delayed puberty, primary amenorrhea in females and other endocrine disturbances secondary to chronic anemia and iron overload * Leg ulcers * Skin bronzing. ## Structure of Heme - Heme Is a Fe-porphyrin compound - 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 ## Learning Objectives * **At the end of this lecture, students will be able to answer**: * Explain the structure of hemoglobin * Describe the types of hemoglobin * Discuss the biochemical function of hemoglobin * Discuss the clinical significance of hemoglobin ## Heme Structure (Cyclic tetra pyrrole) - Heme is a metaloporphyrine - It contains a ring of tetrapyrrole, formed by four pyrrole rings. - A pyrrole has four atoms of carbon and one atom of nitrogen arranged in the shape of a pentagon. - If the tetrapyrrole contains a metal ion between its ring structure, this ring structure is known as porphyrin. ## Structure of Hemoglobin - Conjugated globular Tetrameric protein - Non-protein component: HEME (4) - Protein component: GLOBIN (4) - Molecular weight - app. 65,000 daltons - Chromo protein (heme - red color) - Example of quaternary structure of protein - Ex of Globular protein ## Derivatives of Haemoglobin - Haemoglobin readily react with any gas, other substance **1. Oxyhaemoglobin** - An unstable and reversible compound - Iron remains in the ferrous state. **2. Reduced haemoglobin or deoxygenated haemoglobin** - Oxygen is released from the oxyhaemoglobin. - HbO2 → Hb + O2 - (Oxyhaemoglobin) -> (Reduced haemoglobin) **3. Carbamino-haemoglobin** with carbon dioxide - HbNH2 + CO2 → HbNHCOOH **4. Carboxyhaemoglobin** or carbon monoxyhaemoglobin, Hb with carbon monoxide (CO) - Hb + CO → COHb - The affinity of Hb for CO is 200-250 times its affinity for oxygen - CO poisoning **5. Methaemoglobin** - When reduced or oxygenated Hb is treated with an oxidizing agent, e.g., potassium ferricyanide, the ferrous Fe2+ is oxidized to ferric (Fe3+); the sixth bond is attached to OH to form the compound methaemoglobin. Methaemoglobin is represented as HbOH. It cannot unite reversibly with gaseous oxygen; the O2 of the attached OH is not given off in a vacuum. **6. Glycosylated haemoglobin** - A1C (HbA1C), glucose is attached to terminal valine in the / chains - Diabetes mellitus ## Physiological Varieties of Haemoglobin - Adult haemoglobin or haemoglobin A (HbA (α2β2)] - Fetal haemoglobin or haemoglobin F (HbF (α2y2)] **Two types of adult Haemoglobin:** **(i) Haemoglobin A (HbA (α2β2)]** - The main form of normal adult Hb - Its globin part consists of two α and two β polypeptide chains - It is a spheroidal molecule with a molecular weight of 68,000 **(ii) Haemoglobin A2 (HbA2 (α2δ2)]** - Minor component (about 2.5% of the total Hb) in normal adults - Globin part consists of two α and two δ polypeptide chains. - δ chains slightly different amino acid composition (out of 146, 10 amino acids are different) as compared to B chains. ## Physiological Varieties of Haemoglobin - Adult haemoglobin or haemoglobin A (HbA (α2β2)] - Fetal haemoglobin or haemoglobin F (HbF (α2y2)] **Two types:** **(i) Haemoglobin A (HbA (α2β2)]** - The main form of normal adult Hb - Its globin part consists of two α and two β polypeptide chains - It is a spheroidal molecule with a molecular weight of 68,000 **(ii) Haemoglobin A2 (HbA2 (α2δ2)]** - Minor component (about 2.5% of the total Hb) in normal adults - Globin part consists of two α and two δ polypeptide chains. - δ chains slightly different amino acid composition (out of 146, 10 amino acids are different) as compared to B chains. ## Varieties of Haemoglobin - Physiological varieties of Hb - Haemolobinopathies ## **Which part of the cell does the Heme synthesis take place?** ## Myoglobin - Myoglobin (Mb) is monomeric O2 binding hemoprotein - Found in heart and skeletal muscle - It has single polypeptide (153 A.As) chain with heme moiety - Myoglobin (mol wt. 17,000) structurally resembles the individual subunits of hemoglobin molecule - Myoglobin functions as a reservoir for oxygen. - It serves as oxygen carrier that promotes the transport of oxygen to the rapidly respiring muscle cells ## Quaternary structure of Hb exist in two alternate conformations: - **T state (tense)** - with low affinity for O2 - deoxy state - **R state (relaxed)** - with higher affinity for O2 - oxy state - In T subunits - The binding sites are closed. - In R state - The binding sites are open. - With successive addition of O2, T shifts the equilibrium towards R state. ## Organs mainly involved in Heme synthesis  *Variable* *Constant* ## Embroynic Hb - Several Hb are found at fetal life but absent in adult life since it undergoes complex changes during development. - Hb Gover 1 – 32€2 - Hb Gover 2 - α₂E2 - Hb Portland - 3212 *By the end of the first trimester:* (and ε replaced by α and y - HbF *In the third trimester:* β subunits synthesis begins ## Binding of O2 to haemoglobin - One molecule of Hb can bind with four molecules of O2. - Myoglobin with one heme, which can bind with only one molecule of oxygen - In other words, each heme moiety can bind with one O2. ## Biomedical Importance - Transport of respiratory gases - O2 from lungs to tissues, H+, CO2 from tissues to lungs. - Major blood buffer - 36 histidine residues in Hb - Change in the structure of hemoglobin can give rise to disorders. * Sickle Cell Anemia * Thalassemia * Met-hemoglobinemia ## Differences between α & β-chains of adult normal Hb | | α-Subunit | β-Subunit | |:---------------:|:---------:|:---------:| | Molecular weight | 15126 | 15866 | | Total amino acids | 141 | 146 | | C-terminal amino acid | Arginine | Histidine | | N-terminal amino acid| Val-Leu | Val-His-Leu | | α-Helices | 7 | 8 | | Heme-pocket | Adequate for entry of one molecule O2 | Entry of O2 in heme-pocket is blocked by valine | ##  *Primary structure* *Secondary structure* *Tertiary structure* *Quaternary structure* *Amino acid residues* *Helix* *Polypeptide chain* *Assembled subunits* ## Types of adult hemoglobin | Form | Chain composition | Fraction of total hemoglobin | |---|---|---| | HbA | α2β2 | 90% | | HbF | α2γ2 | <2% | | HbA2 | α2δ2 | 2-5% | | HbA1c | α2 β2-glucose | <5% | **HBA1:** The major hemoglobin in humans. **HBA2:** First appears 12 weeks after birth, a minor component of normal adult HB increased in β-thalassemia. **HBF:** Normally synthesized only during fetal development. In fetus and newborn infants Hb F binds O2 at lower tension than Hb A. Hb F has a higher affinity to O2. After birth, Hb F is replaced by Hb A during the first few months of life. **HBA1C:** Has glucose residues attached to β-globin chains; increased amounts in DM. ## Differentiation of Hb-A from Hb-F | | Hb-A | Hb-F | |:---------------|:--------------|:--------------| | | Two α & two β chains | Two α & two γ chains | | Denatured by | Alkali | Resistant to alkali denaturation | | At pH 8.9 | Hb-A moves ahead of Hb-F | Hb-F moves behind Hb-A | | 2.3-BPG content | Is high | Is low | | Affinity of O2 | Is less | Is more | | Delivery power of O2 | More (unloading) | Is decreased | | Concentration at birth| Hb-A-85% | 15% | | | | Hb-F disappears by end of first year, persistence of Hb-F after one year is pathological | ## Types of Hemoglobin  ## Hb is an ideal respiratory pigment - Transport large quantities of O2. - Great solubility - Take up & release O2 at appropriate partial pressures. - Powerful buffer ## Hemoglobin is an Allosteric protein - Multimeric - Forms - Inactive / Tform (Deoxy-Hb) - Active/ R form (Oxy-Hb) - Regulated by allosteric modulators ## Hemoglobin is the red blood pigment (Hemoprotein) present in the RBC's. - Heme is synthesized from Reticuloendothelial cells. - Heme activates synthesis of globin in nucleated reticulocytes of bone marrow cells. - Two chains of globin are formed independently at the same rate. (15% a.a from daily protein intake) ## Normal level in blood: - 14-16 g/dl in males - 13-15 g/dl in females ## Normal Blood Haemoglobin Levels - Adult males = 15.5 g/dL (range 14–18 g/dL) - Adult females = 14 g/dL (range 12-15.5 g/dL) **The normal blood Hb concentration at different ages is:** - In fetus, just before birth (from the umbilical cord) ranges from 16.5 to 18.5 g/dL. - After birth, the Hb concentration increases rapidly and may reach up to 23 g/dL. (The transfusion of cells from the placenta to infant and haemoconcentration by reduction of plasma volume). - At the end of 3 months: After two days of birth, the Hb levels start falling and stabilize at the end of 3 months to 10.5g/dL. - At 1 year of age. The concentration then rises gradually to reach 12 g/dL at 1 year of age. ## Functions of Haemoglobin - **Transport of O2 from lungs to tissues** - In the lungs, one molecule of O2 is attached loosely and reversibly at the sixth covalent bond of each iron atom of the Hb to form oxyhaemoglobin. Hb + O2→ HbO2 - Oxygenation of first haem molecule in Hb increases affinity of second haem for oxygen which in turn increases the affinity of third haem and so on - The affinity of Hb for fourth oxygen molecule is many times that for the first molecule - The affinity of Hb for oxygen is influenced by: pH, temperature and concentration of 2,3-DPG (a product of metabolism of glucose) in the RBCs ## **Hemoproteins (heme as prosthetic gr.)** - Hemoglobin-RBC's - Myoglobin- Muscles - Cytochromes-heme gr serves as e carrier - Catalase - It catalyzes H2O2 ## Factors Controlling Haemoglobin Formation - **Role of proteins** - Provide amino acids required for the synthesis of globin part of the Hb. - A low protein intake retards Hb regeneration even in the presence of excess iron; the limiting factor being lack of globin - **Role of iron:** Necessary for formation of the haem part of haemoglobin - In addition to dietary iron, the iron released by degradation of RBCs is also reused for the synthesis of Hb. - **Role of other metals:** - Copper is essential for the Hb synthesis, as it promotes the absorption, mobilization and utilization of iron. - Cobalt increases the production of erythropoietin which in turn stimulates RBC formation. - Calcium reported to help indirectly by conserving iron and its subsequent utilization. ## Attachment of Haem to Globin - One molecule of Hb contains four units of haem - Each attached to one of the four polypeptide chains constituting globin - Four iron atoms in one molecule of Hb which can carry four molecules (eight atoms) of oxygen ## Fate of Hemoglobin - Haemoglobin - Choleglobin (Tetrapyrrole straight chain containing both globin and iron) * Globin - Amino acids * Reutilized in the bone marrow for haemoglobin synthesis * Iron - Biliverdin (Tetrapyrrolo straight chain free of globin and iron) * Biliverdin reductase * Bilirubin * Taken up by the liver - Stored as ferritin in other tissues ## The Iron - Iron in the haem is in ferrous (Fe2+) form - Attached to the nitrogen atom of each pyrrole ring - On the iron (Fe2+) a bond is available for loose union, where: - In oxyhaemoglobin, O2 is attached - In carboxyhaemoglobin, CO is attached ## Synthesis of Globin - Globin, the protein part of the Hb, is synthesized in the ribosomes. -  ## Steps in globin chain synthesis: - Globin and α-globin gene families contain three exons (coding regions) separated by two noncoding introns. 1. **Transcription** 2. **Modification of mRNA precursor by splicing** 3. **Translation by ribosomes & further modifications** (i.e., glycosylation) -  ## Structure of Globin - Made of four polypeptide chains - Haemoglobin A (HbA) - Two α chains, each containing 141 amino acid residues - Two β chains, each containing 146 amino acid residues. - Normal adult haemoglobin A is written as HbA (α2β2) ## Fate of RBCs - The tissue macrophage system (reticuloendothelial system), includes the following phagocytic cells: - In the bone marrow these cells form part of the lining of the blood sinuses (littoral cells) - In the liver they lie at intervals along the vascular capillaries (Kupffer cells) - In the spleen they are found in the pulp - In the lymph nodes they line the lymphatic paths. ## Fate of RBCs - The cell membrane of old RBCs (after about 120 days) becomes more fragile due to decreased NADPH activity. - The destruction of red cells occurs mostly in the capillaries of spleen because they have very thin lumen - Spleen is also called the graveyard of RBCs - The haemoglobin released after the haemolysis of red cells is taken up by the tissue macrophages. ## Basic chemical steps in the formation of hemoglobin. -  - First, succinyl-CoA, formed in the krebs metabolic cycle, binds with glycine to form a pyrrole molecule. - In turn, four pyrroles combines, forming protoporphyrin IX, which then combines with iron to form the heme molecule. - Finally, each heme molecule combines with a long polypeptide chain, a globin synthesized by ribosomes, forming a subunit of hemoglobin called hemoglobin chain. ## Porphyrin ring -  - The pyrrole rings are numbered I to IV; the bridges named as alpha to delta and 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). ## Life Span of RBCs - THE AVERAGE LIFE SPAN OF RBCS IS 120 DAYS **Causes of reduction in the life span of RBCs:** - **Defects in RBCs (Corpuscular defects)** * Hereditary spherocytosis * Sickle cell anaemia * Thalassaemias * Deficiency of red cell enzymes. * Glucose 6-phosphate-dehydrogenase deficiency * Pyruvate kinase deficiency - **Extracorpuscular defects** * Transfusion of mismatched blood * Autoimmune haemolytic disorders and Hypersplenism. ## Hemoglobin - The normal Hb becomes 100% saturated when blood is equilibrated with 100% oxygen (PO2, 760mmHg) - One gram of Hb when fully saturated combines with 1.34 mL oxygen. **Normal values of oxygen carrying capacity** - males is 1.34 x 15.5 = about 21 mL% - females is 1.34 x 14 = about 18.5 mL% **Clinically, irrespective of the age, a level of 14.8 g/dL is considered as 100% Hb.** ## Factors Controlling Haemoglobin Formation - **Role of proteins** * Provide amino acids required for the synthesis of globin part of the Hb. * A low protein intake retards Hb regeneration even in the presence of excess iron; the limiting factor being lack of globin. - **Role of iron:** Necessary for formation of the haem part of haemoglobin * In addition to dietary iron, the iron released by degradation of RBCs is also reused for the synthesis of Hb - **Role of other metals:** * Copper is essential for the Hb synthesis, as it promotes the absorption, mobilization and utilization of iron. * Cobalt increases the production of erythropoietin which in turn stimulates RBC formation. * Calcium reported to help indirectly by conserving iron and its subsequent utilization ## Attachment of Haem to Globin - One molecule of Hb contains four units of haem. - Each attached to one of the four polypeptide chains constituting globin. - Four iron atoms in one molecule of Hb which can carry four molecules (eight atoms) of oxygen ## Fate of Hemoglobin - Haemoglobin - Choleglobin (Tetrapyrrole straight chain containing both globin and iron) * Globin * Amino acids * Reutilized in the bone marrow for haemoglobin synthesis * Iron * Biliverdin (Tetrapyrrolo straight chain free of globin and iron) * Biliverdin reductase * Bilirubin * Taken up by the liver : * Stored as ferritin in other tissues ## The Iron - Iron in the haem is in ferrous (Fe2+) form - Attached to the nitrogen atom of each pyrrole ring - On the iron (Fe2+) a bond is available for loose union, where: - In oxyhaemoglobin, O2 is attached - In carboxyhaemoglobin, CO is attached. ## Synthesis of Globin - Globin, the protein part of the Hb, is synthesized in the ribosomes. -  ## Steps in globin chain synthesis: - Globin and α-globin gene families contain three exons (coding regions) separated by two noncoding introns. 1. **Transcription** 2. **Modification of mRNA precursor by splicing** 3. ** Translation by ribosomes & further modifications** (i.e. glycosylation). -  ## Structure of Globin - Made of four polypeptide chains - Haemoglobin A (HbA) - Two α chains, each containing 141 amino acid residues - Two β chains, each containing 146 amino acid residues. - Normal adult haemoglobin A is written as HbA (α2β2) ## Fate of RBCs - The tissue macrophage system (reticuloendothelial system) includes the following phagocytic cells: - In the bone marrow these cells form part of the lining of the blood sinuses (littoral cells). - In the liver they lie at intervals along the vascular capillaries (Kupffer cells). - In the spleen they are found in the pulp. - In the lymph nodes they line the lymphatic paths. ## Fate of RBCs - The cell membrane of old RBCs (after about 120 days) becomes more fragile due to decreased NADPH activity. - The destruction of red cells occurs mostly in the capillaries of spleen because they have very thin lumen. - Spleen is also called the graveyard of RBCs. - The haemoglobin released after the haemolysis of red cells is taken up by the tissue macrophages. ## Basic chemical steps in the formation of hemoglobin. -  - First, succinyl-CoA, formed in the Krebs metabolic cycle, binds with glycine to form a pyrrole molecule. - In turn, four pyrroles combines, forming protoporphyrin IX, which then combines with iron to form the heme molecule. - Finally, each heme molecule combines with a long polypeptide chain, a globin synthesized by ribosomes, forming a subunit of hemoglobin called hemoglobin chain. ## Porphyrin ring -  - The pyrrole rings are numbered I to IV, the bridges named as alpha to delta and 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). ## Life Span of RBCs - THE AVERAGE LIFE SPAN OF RBCS IS 120 DAYS **Causes of reduction in the life span of RBCs:** - **Defects in RBCs (Corpuscular defects)** * Hereditary spherocytosis * Sickle cell anaemia * Thalassaemias * Deficiency of red cell enzymes. * Glucose 6-phosphate-dehydrogenase deficiency * Pyruvate kinase deficiency - **Extracorpuscular defects** * Transfusion of mismatched blood * Autoimmune haemolytic disorders and Hypersplenism. ## Structure of heme -  - M = Methyl - V = Vinyl - P = Propionyl