Comprehensive Study Notes on Respiratory System Biochemistry: Hemoglobin, Myoglobin, and Lung Surfactants

Respiratory System Biochemistry: Overview of Gas Transport

• The transport of oxygen ($O_2$) and carbon dioxide ($CO_2$) to and from tissues would be grossly inadequate without specialized proteins.

• Approximately 99% of the $O_2$ that dissolves in the blood combines with the $O_2$ carrying protein hemoglobin ($Hb$).

• Approximately 94.5% of the $CO_2$ that dissolves enters a series of reversible chemical reactions to be converted into other compounds.

• Significance of hemoglobin:

  • Both gases are poorly soluble in plasma.

  • Hemoglobin increases the $O_2$-carrying capacity of the blood 70-fold.

  • Chemical reactions involving $CO_2$ increase the total blood $CO_2$ content 17-fold.

• Multicellular animals depend on these proteins not just for transport, but for the storage of oxygen.

Structure of Myoglobin and Hemoglobin

• The two primary oxygen-binding proteins are Hemoglobin and Myoglobin.

• Both proteins contain iron ($Fe^{2+}$) to reversibly bind oxygen.

• Heme Structure:

  • Contains a porphyrin ring composed of four pyrrole rings.

  • The iron ($Fe^{2+}$) is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring.

  • Iron forms two additional coordination bonds:

    • One bond with the Proximal histidine (F8) of the globin chain of myoglobin or hemoglobin.

    • One bond with $O_2$, whose binding is stabilized by a Distal histidine (E7).

• Distal Histidine (E7):

  • Forms a hydrogen bond with $O_2$.

  • It is located far enough away that it does not bind directly with the heme iron.

• Chemical Substituents of Heme:

  • M = Methyl ($-CH_3$)

  • V = Vinyl ($-CH=CH_2$)

  • P = Propionic acid ($-CH_2-CH_2-COOH$)

Physiological Importance of Myoglobin

• Function: Myoglobin in tissues (especially muscle) accepts oxygen from hemoglobin in circulating arterial blood and delivers it to the mitochondria when oxygen demand is high.

• Oxygen Affinity:

  • The $p50$ (oxygen partial pressure required for 50% saturation) for myoglobin is very low (approx. $1\,mmHg$).

  • A low $p50$ signifies a very high affinity for oxygen, which is essential for extracting oxygen from hemoglobin.

• Release Mechanism: As metabolic activity increases and internal $pO_2$ falls, myoglobin releases its oxygen. It only gives away oxygen under conditions of hypoxia or in rapidly exercising muscle.

Hemoglobin-A (Adult Hemoglobin)

• Characteristics:

  • The most abundant hemoglobin in adults.

  • Found exclusively in red blood cells (RBCs).

  • Contains four oxygen-binding sites; it transports four molecules of oxygen from the lungs to the tissues.

  • Oxygen binding is regulated by allosteric effectors.

• Quaternary Structure:

  • Made of a tetramer consisting of two $\alpha$-chains and two $\beta$-chains.

  • The tetramer is composed of two identical dimers: $(\alpha\beta)_1$ and $(\alpha\beta)_2$.

  • Dimers are held together by non-covalent interactions.

  • Oxygen affinity varies between different subunits.

• Conformational States:

  • T-state (Taut/Tight): The deoxy form of hemoglobin. Dimers interact through ionic and hydrogen bonds (salt bridges), restricting movement. It has a low oxygen affinity.

  • R-state (Relaxed): The high oxygen affinity form. Binding of $O_2$ causes the rupture of some inter-dimer bonds, allowing movement.

Molecular Mechanism of Oxygenation

• Iron Movement:

  • In unoxygenated myoglobin/hemoglobin, the Proximal histidine pulls the $Fe^{2+}$ approximately $0.03\,nm$ outside the plane of the heme ring.

  • When $O_2$ binds at the sixth coordination position, it pulls the $Fe^{2+}$ into the plane of the ring (moving closer, to within $0.01\,nm$ of the plane).

• Conformational Change:

  • Oxygenation is accompanied by the motion of the iron, the His F8 residue, and residues linked to His F8.

  • This leads to a $15^{\circ}$ rotation of one $\alpha_2\beta_2$ dimer relative to the other.

  • This movement moves the helix containing the proximal histidine, causing the loss of salt bridges.

  • Conformational changes are induced in all other subunits in a concerted manner, shifting from T to R.

• Steric hindrance: In the deoxy state, a valine residue covers the heme pocket of the $\beta$-subunit, blocking $O_2$ entry. During the T to R transition, this valine moves to open the cleft for binding.

Oxygen Dissociation Curves (ODC)

• P50 Definition: The partial pressure of oxygen needed to achieve half-saturation. It is an index of oxygen affinity.

  • Higher $p50$ = Lower oxygen affinity.

  • Lower $p50$ = Higher oxygen affinity.

• Hemoglobin ODC:

  • Sigmoidal (S-shaped) curve.

  • $p50$ is approximately $26\,mmHg$.

  • This shape serves its role in transport: fully saturated in lung alveoli ($pO_2 = 100\,mmHg$) and unloads oxygen easily in tissues.

• Myoglobin ODC:

  • Hyperbolic shape.

  • $p50$ is approximately $1\,mmHg$.

  • Indicates the absence of allosteric interactions or cooperativity.

• Cooperativity (Heme-Heme Interactions):

  • Binding of the first $O_2$ molecule increases the affinity for the second, and so on.

  • The affinity for the fourth $O_2$ molecule is approximately 300 times greater than for the first.

Carbon Dioxide Transport and the Bohr Effect

• Oxygen Transport breakdown:

  • Dissolved in plasma: 3%.

  • Bound to hemoglobin: 97%.

• CO2 Transport breakdown:

  • Dissolved gas in solution: 7% ($3.5\,ml$ of $CO_2$ per $100\,ml$ of blood).

  • As Bicarbonate ($HCO_3^-$): 70% ($44.8\,ml$). This is known as isohydric transport.

  • As Carbaminohemoglobin: 23% ($8\,ml$). $CO_2$ binds to the N-terminal amino groups of the globin chains (not the heme).

• Bohr Effect:

  • Tissues produce $CO_2$ which reacts with water via carbonic anhydrase ($ca$):

  • $CO_2 + H_2O \rightleftharpoons H_2CO_3 \rightleftharpoons H^+ + HCO_3^-$

  • High $H^+$ (low pH) and high $CO_2$ stabilize the deoxy (T) state by protonating His 146, forming ionic bonds.

  • This decreases Hb affinity for $O_2$, shifting the ODC to the right and facilitating oxygen release to tissues.

• Haldane Effect:

  • In the lungs, high $pO_2$ and high pH facilitate $O_2$ binding to Hb.

  • Binding of $O_2$ causes the release of protons ($H^+$) from Hb. These protons react with bicarbonate to form $CO_2$ for expiration.

  • Oxyhemoglobin is a stronger acid than deoxyhemoglobin.

The Chloride Shift

• During isohydric transport, $HCO_3^-$ produced in RBCs diffuses out into the plasma.

• To maintain electrical neutrality, an equal number of chloride ions ($Cl^-$) move from the plasma into the RBC.

• This exchange is mediated by Anion Exchanger 1 (AE1), also known as the Band 3 protein.

Allosteric Effectors of Hemoglobin

• 2,3-Diphosphoglycerate (2,3-BPG):

  • Derived from the glycolytic intermediate 1,3-bisphosphoglycerate via the Rapoport–Luebering shunt.

  • Binds in a central cavity of the deoxy (T) hemoglobin tetramer via salt bridges with Lys and His residues on the $\beta$-chains.

  • Stabilizes the T-state, shifting the ODC to the right to facilitate $O_2$ unloading.

  • Levels increase during chronic hypoxia and anemia.

• Carbon Monoxide (CO):

  • Binds tightly and reversibly to heme iron forming carboxyhemoglobin.

  • Affinity is 220 times greater than that of oxygen.

  • Shifts Hb to the R-state and shifts the ODC to the left, preventing the release of oxygen to tissues.

• Summary of ODC Shifts:

  • Right Shift (Lower Affinity): Increased $pCO_2$, increased $[H^+]$ (lower pH), increased 2,3-BPG, increased Temperature.

  • Left Shift (Higher Affinity): Decreased $pCO_2$, decreased $[H^+]$, decreased 2,3-BPG, decreased Temperature, HbF (Fetal Hemoglobin).

Hemoglobin Variants and Clinical Correlations

• Hemoglobin F (HbF):

  • Primary hemoglobin in fetuses/newborns. Consists of $\alpha_2\gamma_2$.

  • Higher affinity for $O_2$ than adult HbA because $\gamma$-chains have Serine (Ser) instead of Histidine (His 143).

  • Ser cannot form salt bridges with 2,3-BPG, resulting in weak binding of BPG and higher oxygen affinity.

• Hemoglobin A1c (Glycosylated Hemoglobin):

  • Formed by non-enzymatic glycosylation of amino terminals when blood glucose enters RBCs.

  • Normal level is approximately 5%.

  • Reflects mean blood glucose over the preceding 6–8 weeks (average RBC lifespan is 60–120 days).

• Sickle Cell Anemia (HbS):

  • Genetic disorder caused by a point mutation in the $\beta$-globin chain.

  • Charged Glutamate is replaced by nonpolar Valine at the 6th position.

  • Deoxyhemoglobin S polymerizes into insoluble fibrous polymers (filaments), distorting the RBC into a sickle shape.

Pulmonary Surfactant

• Lung Development:

  • Lung development begins at 3 weeks gestation until 8 years of age.

  • Alveolarization begins between weeks 24 and 38.

• Alveolar Cell Types:

  • Type I (AT-I): Cover 95% of alveolar surface; primary site for gas diffusion; thin with few organelles.

  • Type II (AT-II): Site of surfactant synthesis and secretion; contain lamellar bodies (LB); play a role in host defense.

• Surfactant Composition:

  • 70% to 80% Phospholipids: Primarily Dipalmitoylphosphatidylcholine (DPPC).

  • 10% Cholesterol.

  • 10% Proteins: Surfactant Proteins SP-A, B, C, and D.

• Surfactant Synthesis and Secretion:

  • Synthesized in the ER (lipids in SER, proteins in RER).

  • Transported to Golgi, then to multivesicular bodies (MVB), then to Lamellar Bodies (LB).

  • ABCA3 is the transporter that moves surfactant into LBs.

  • Secreted into the alveolar space via exocytosis induced by mechanical stretch (breathing) or chemical stimuli (Calcium, cAMP, Adrenergic).

  • Forms Tubular Myelin (TM) before reorganizing into a functional Monolayer.

Functions of Lung Surfactant

• Reduces Surface Tension (ST):

  • Surface tension at $37^{\circ}C$ for pure water is $70\,mN/m$.

  • Surfactant reduces this to $20-25\,mN/m$ (and near zero during expiration).

  • Mechanism: Hydrophilic parts bond with the water layer; hydrophobic parts interact with air, breaking intermolecular forces between water molecules.

• Increases Lung Compliance: Decreases the work required to inflate the lungs.

• Prevents Lung Collapse: Prevents atelectasis at the end of expiration.

• Keeps Alveoli Dry: Prevents pulmonary edema by reducing the force that draws fluid across the alveolar wall.

• Promote Alveolar Stability: Reduces pressure differences between alveoli of different sizes (Law of Laplace).

• Host Defense: SP-A and SP-D act as innately immune molecules; they have carbohydrate recognition domains to opsonize bacteria and viruses for macrophages.

Specific Surfactant Components

• Dipalmitoylphosphatidylcholine (DPPC): Main component; synthesis starts at 28 weeks, peaks at 36 weeks.

• Phosphatidyl glycerol (PG): Last phospholipid produced (appears at 36 weeks); reliable marker for lung maturity.

• SP-A & SP-D: Hydrophilic; host defense; promote tubular myelin formation; secreted in LB-independent manner.

• SP-B & SP-C: Hydrophobic; positively charged; help with lipid adsorption onto the monolayer; localized within LBs.

Clinical Correlation: Respiratory Disorders

• Respiratory Distress Syndrome (RDS):

  • Affects premature infants due to surfactant deficiency.

  • Leads to high surface tension, decreased compliance, and alveolar collapse (atelectasis).

  • Diagnosis: Fetal Lung Maturity (FLM) testing.

    • L/S Ratio: Lecithin (DPPC) to Sphingomyelin ratio. L/S $\ge 2/1$ at 35 weeks indicates maturity.

    • Risk: L/S 1.5–1.9 (40% risk), L/S < 1.5 (75% risk).

    • PG: Presence of PG ($> 2\%$) is a definitive marker of maturity.

  • Treatment: Exogenous surfactant; maternal antenatal corticosteroids (Glucocorticoids) to stimulate production.

• Pulmonary Alveolar Proteinosis (PAP):

  • Excessive accumulation of surfactant in alveoli.

  • Often caused by hypo-responsiveness to Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), which is needed for alveolar macrophages to degrade surfactant.

  • Treatment: Alveolar wash (physical removal) or systemic GM-CSF.

Factors Increasing Surfactant Synthesis

• Glucocorticoids: Increase activity of synthesis enzymes (given to mothers at 30–32 weeks if preterm labor is imminent).

• Labor: Enhances phospholipid synthesis and secretion.

• Estrogen: Increases lung maturation enzyme activity.

• Thyroid Hormones: Accelerate lipid translocation into lamellar bodies.

• The transport of oxygen ($O_2$) and carbon dioxide ($CO_2$) to and from tissues would be grossly inadequate without specialized proteins.

• Approximately 99% of the $O_2$ that dissolves in the blood combines with the $O_2$ carrying protein hemoglobin ($Hb$).

• Approximately 94.5% of the $CO_2$ that dissolves enters a series of reversible chemical reactions to be converted into other compounds.

• Significance of hemoglobin:

  • Both gases are poorly soluble in plasma.

  • Hemoglobin increases the $O_2$-carrying capacity of the blood 70-fold.

  • Chemical reactions involving $CO_2$ increase the total blood $CO_2$ content 17-fold.

• Multicellular animals depend on these proteins not just for transport, but for the storage of oxygen.

Structure of Myoglobin and Hemoglobin

• The two primary oxygen-binding proteins are Hemoglobin and Myoglobin.

• Both proteins contain iron ($Fe^{2+}$) to reversibly bind oxygen.

• Heme Structure:

  • Contains a porphyrin ring composed of four pyrrole rings.

  • The iron ($Fe^{2+}$) is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring.

  • Iron forms two additional coordination bonds:

  • One bond with the Proximal histidine (F8) of the globin chain of myoglobin or hemoglobin.

  • One bond with $O_2$, whose binding is stabilized by a Distal histidine (E7).

• Distal Histidine (E7):

  • Forms a hydrogen bond with $O_2$.

  • It is located far enough away that it does not bind directly with the heme iron.

• Chemical Substituents of Heme:

  • M = Methyl ($-CH_3$)

  • V = Vinyl ($-CH=CH_2$)

  • P = Propionic acid ($-CH_2-CH_2-COOH$)

Physiological Importance of Myoglobin

• Function: Myoglobin in tissues (especially muscle) accepts oxygen from hemoglobin in circulating arterial blood and delivers it to the mitochondria when oxygen demand is high.

• Oxygen Affinity:

  • The $p50$ (oxygen partial pressure required for 50% saturation) for myoglobin is very low (approx. $1 \, mmHg$).

  • A low $p50$ signifies a very high affinity for oxygen, which is essential for extracting oxygen from hemoglobin.

• Release Mechanism: As metabolic activity increases and internal $pO_2$ falls, myoglobin releases its oxygen. It only gives away oxygen under conditions of hypoxia or in rapidly exercising muscle.

Hemoglobin-A (Adult Hemoglobin)

• Characteristics:

  • The most abundant hemoglobin in adults.

  • Found exclusively in red blood cells (RBCs).

  • Contains four oxygen-binding sites; it transports four molecules of oxygen from the lungs to the tissues.

  • Oxygen binding is regulated by allosteric effectors.

• Quaternary Structure:

  • Made of a tetramer consisting of two $\beta$-chains and two $\beta$-chains.

  • The tetramer is composed of two identical dimers: $( ext{αβ})<em>1$ and $( ext{αβ})</em>2$.

  • Dimers are held together by non-covalent interactions.

  • Oxygen affinity varies between different subunits.

• Conformational States:

  • T-state (Taut/Tight): The deoxy form of hemoglobin. Dimers interact through ionic and hydrogen bonds (salt bridges), restricting movement. It has a low oxygen affinity.

  • R-state (Relaxed): The high oxygen affinity form. Binding of $O_2$ causes the rupture of some inter-dimer bonds, allowing movement.

Molecular Mechanism of Oxygenation

• Iron Movement: In unoxygenated myoglobin/hemoglobin, the Proximal histidine pulls the $Fe^{2+}$ approximately $0.03 \, nm$ outside the plane of the heme ring.

• When $O_2$ binds at the sixth coordination position, it pulls the $Fe^{2+}$ into the plane of the ring (moving closer, to within $0.01 \, nm$ of the plane).

• Conformational Change:

  • Oxygenation is accompanied by the motion of the iron, the His F8 residue, and residues linked to His F8.

  • This leads to a $15^{ ext{o}}$ rotation of one $ext{α}<em>2 ext{β}</em>2$ dimer relative to the other.

  • This movement moves the helix containing the proximal histidine, causing the loss of salt bridges.

  • Conformational changes are induced in all other subunits in a concerted manner, shifting from T to R.

• Steric hindrance: In the deoxy state, a valine residue covers the heme pocket of the $\beta$-subunit, blocking $O_2$ entry. During the T to R transition, this valine moves to open the cleft for binding.

Oxygen Dissociation Curves (ODC)

• P50 Definition: The partial pressure of oxygen needed to achieve half-saturation. It is an index of oxygen affinity.

  • Higher $p50$ = Lower oxygen affinity.

  • Lower $p50$ = Higher oxygen affinity.

• Hemoglobin ODC:

  • Sigmoidal (S-shaped) curve.

  • $p50$ is approximately $26 \, mmHg$.

  • This shape serves its role in transport: fully saturated in lung alveoli ($pO_2 = 100 \, mmHg$) and unloads oxygen easily in tissues.

• Myoglobin ODC:

  • Hyperbolic shape.

  • $p50$ is approximately $1 \, mmHg$.

  • Indicates the absence of allosteric interactions or cooperativity.

• Cooperativity (Heme-Heme Interactions):

  • Binding of the first $O_2$ molecule increases the affinity for the second, and so on.

  • The affinity for the fourth $O_2$ molecule is approximately 300 times greater than for the first.

• The transport of oxygen ($O_2$) and carbon dioxide ($CO_2$) to and from tissues would be grossly inadequate without specialized proteins.

• Approximately 99% of the $O_2$ that dissolves in the blood combines with the $O_2$ carrying protein hemoglobin ($Hb$).

• Approximately 94.5% of the $CO_2$ that dissolves enters a series of reversible chemical reactions to be converted into other compounds.

• Significance of hemoglobin:

  • Both gases are poorly soluble in plasma.

  • Hemoglobin increases the $O_2$-carrying capacity of the blood 70-fold.

  • Chemical reactions involving $CO_2$ increase the total blood $CO_2$ content 17-fold.

• Multicellular animals depend on these proteins not just for transport, but for the storage of oxygen.

Structure of Myoglobin and Hemoglobin

• The two primary oxygen-binding proteins are Hemoglobin and Myoglobin.

• Both proteins contain iron ($Fe^{2+}$) to reversibly bind oxygen.

• Heme Structure:

  • Contains a porphyrin ring composed of four pyrrole rings.

  • The iron ($Fe^{2+}$) is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring.

  • Iron forms two additional coordination bonds:

  • One bond with the Proximal histidine (F8) of the globin chain of myoglobin or hemoglobin.

  • One bond with $O_2$, whose binding is stabilized by a Distal histidine (E7).

• Distal Histidine (E7):

  • Forms a hydrogen bond with $O_2$.

  • It is located far enough away that it does not bind directly with the heme iron.

• Chemical Substituents of Heme:

  • M = Methyl ($-CH_3$)

  • V = Vinyl ($-CH=CH_2$)

  • P = Propionic acid ($-CH_2-CH_2-COOH$)

Physiological Importance of Myoglobin

• Function: Myoglobin in tissues (especially muscle) accepts oxygen from hemoglobin in circulating arterial blood and delivers it to the mitochondria when oxygen demand is high.

• Oxygen Affinity:

  • The $p50$ (oxygen partial pressure required for 50% saturation) for myoglobin is very low (approx. $1 \, mmHg$).

  • A low $p50$ signifies a very high affinity for oxygen, which is essential for extracting oxygen from hemoglobin.

• Release Mechanism: As metabolic activity increases and internal $pO_2$ falls, myoglobin releases its oxygen. It only gives away oxygen under conditions of hypoxia or in rapidly exercising muscle.

Hemoglobin-A (Adult Hemoglobin)

• Characteristics:

  • The most abundant hemoglobin in adults.

  • Found exclusively in red blood cells (RBCs).

  • Contains four oxygen-binding sites; it transports four molecules of oxygen from the lungs to the tissues.

  • Oxygen binding is regulated by allosteric effectors.

• Quaternary Structure:

  • Made of a tetramer consisting of two $\alpha$-chains and two $\beta$-chains.

  • The tetramer is composed of two identical dimers: $(\alpha\beta)<em>1$ and $(\alpha\beta)</em>2$.

  • Dimers are held together by non-covalent interactions.

  • Oxygen affinity varies between different subunits.

• Conformational States:

  • T-state (Taut/Tight): The deoxy form of hemoglobin. Dimers interact through ionic and hydrogen bonds (salt bridges), restricting movement. It has a low oxygen affinity.

  • R-state (Relaxed): The high oxygen affinity form. Binding of $O_2$ causes the rupture of some inter-dimer bonds, allowing movement.

Molecular Mechanism of Oxygenation

• Iron Movement: In unoxygenated myoglobin/hemoglobin, the Proximal histidine pulls the $Fe^{2+}$ approximately $0.03 \, nm$ outside the plane of the heme ring.

• When $O_2$ binds at the sixth coordination position, it pulls the $Fe^{2+}$ into the plane of the ring (moving closer, to within $0.01 \, nm$ of the plane).

• Conformational Change:

  • Oxygenation is accompanied by the motion of the iron, the His F8 residue, and residues linked to His F8.

  • This leads to a $15^{\text{o}}$ rotation of one $\alpha<em>2\beta</em>2$ dimer relative to the other.

  • This movement moves the helix containing the proximal histidine, causing the loss of salt bridges.

  • Conformational changes are induced in all other subunits in a concerted manner, shifting from T to R.

• Steric hindrance: In the deoxy state, a valine residue covers the heme pocket of the $\beta$-subunit, blocking $O_2$ entry. During the T to R transition, this valine moves to open the cleft for binding.

Oxygen Dissociation Curves (ODC)

• P50 Definition: The partial pressure of oxygen needed to achieve half-saturation. It is an index of oxygen affinity.

  • Higher $p50$ = Lower oxygen affinity.

  • Lower $p50$ = Higher oxygen affinity.

• Hemoglobin ODC:

  • Sigmoidal (S-shaped) curve.

  • $p50$ is approximately $26 \, mmHg$.

  • This shape serves its role in transport: fully saturated in lung alveoli ($pO_2 = 100 \, mmHg$) and unloads oxygen easily in tissues.

• Myoglobin ODC:

  • Hyperbolic shape.

  • $p50$ is approximately $1 \, mmHg$.

  • Indicates the absence of allosteric interactions or cooperativity.

• Cooperativity (Heme-Heme Interactions):

  • Binding of the first $O_2$ molecule increases the affinity for the second, and so on.

  • The affinity for the fourth $O_2$ molecule is approximately 300 times greater than for the first.

Carbon Dioxide Transport and the Bohr Effect

• Oxygen Transport breakdown:

  • Dissolved in plasma: 3%.

  • Bound to hemoglobin: 97%.

• CO2 Transport breakdown:

  • Dissolved gas in solution: 7% ($3.5 \, ml$ of $CO_2$ per $100 \, ml$ of blood).

  • As Bicarbonate ($HCO_3^-$): 70% ($44.8 \, ml$). This is known as isohydric transport.

  • As Carbaminohemoglobin: 23% ($8 \, ml$). $CO_2$ binds to the N-terminal amino groups of the globin chains (not the heme).

• Bohr Effect:

  • Tissues produce $CO_2$ which reacts with water via carbonic anhydrase ($ca$):
    $CO_2 + H_2O \rightleftharpoons H_2CO_3 \rightleftharpoons H^+ + HCO_3^-$

  • High $H^+$ (low pH) and high $CO_2$ stabilize the deoxy (T) state by protonating His 146, forming ionic bonds.

  • This decreases Hb affinity for $O_2$, shifting the ODC to the right and facilitating oxygen release to tissues.

• Haldane Effect:

  • In the lungs, high $pO_2$ and high pH facilitate $O_2$ binding to Hb.

  • Binding of $O_2$ causes the release of protons ($H^+$) from Hb. These protons react with bicarbonate to form $CO_2$ for expiration.

  • Oxyhemoglobin is a stronger acid than deoxyhemoglobin.

The Chloride Shift

• During isohydric transport, $HCO_3^-$ produced in RBCs diffuses out into the plasma.

• To maintain electrical neutrality, an equal number of chloride ions ($Cl^-$) move from the plasma into the RBC.

• This exchange is mediated by Anion Exchanger 1 (AE1), also known as the Band 3 protein.

Allosteric Effectors of Hemoglobin

• 2,3-Diphosphoglycerate (2,3-BPG):

  • Derived from the glycolytic intermediate 1,3-bisphosphoglycerate via the Rapoport–Luebering shunt.

  • Binds in a central cavity of the deoxy (T) hemoglobin tetramer via salt bridges with Lys and His residues on the $\beta$-chains.

  • Stabilizes the T-state, shifting the ODC to the right to facilitate $O_2$ unloading.

  • Levels increase during chronic hypoxia and anemia.

• Carbon Monoxide (CO):

  • Binds tightly and reversibly to heme iron forming carboxyhemoglobin.

  • Affinity is 220 times greater than that of oxygen.

  • Shifts Hb to the R-state and shifts the ODC to the left, preventing the release of oxygen to tissues.

• Summary of ODC Shifts:

  • Right Shift (Lower Affinity): Increased $pCO_2$, increased $[H^+]$ (lower pH), increased 2,3-BPG, increased Temperature.

  • Left Shift (Higher Affinity): Decreased $pCO_2$, decreased $[H^+]$, decreased 2,3-BPG, decreased Temperature, HbF (Fetal Hemoglobin).

Hemoglobin Variants and Clinical Correlations

• Hemoglobin F (HbF):

  • Primary hemoglobin in fetuses/newborns. Consists of $\alpha<em>2\gamma</em>2$.

  • Higher affinity for $O_2$ than adult HbA because $\gamma$-chains have Serine (Ser) instead of Histidine (His 143).

  • Ser cannot form salt bridges with 2,3-BPG, resulting in weak binding of BPG and higher oxygen affinity.

• Hemoglobin A1c (Glycosylated Hemoglobin):

  • Formed by non-enzymatic glycosylation of amino terminals when blood glucose enters RBCs.

  • Normal level is approximately 5%.

  • Reflects mean blood glucose over the preceding 6–8 weeks (average RBC lifespan is 60–120 days).

• Sickle Cell Anemia (HbS):

  • Genetic disorder caused by a point mutation in the $\beta$-globin chain.

  • Charged Glutamate is replaced by nonpolar Valine at the 6th position.

  • Deoxyhemoglobin S polymerizes into insoluble fibrous polymers (filaments), distorting the RBC into a sickle shape.

Pulmonary Surfactant

• Lung Development:

  • Lung development begins at 3 weeks gestation until 8 years of age.

  • Alveolarization begins between weeks 24 and 38.

• Alveolar Cell Types:

  • Type I (AT-I): Cover 95% of alveolar surface; primary site for gas diffusion; thin with few organelles.

  • Type II (AT-II): Site of surfactant synthesis and secretion; contain lamellar bodies (LB); play a role in host defense.

• Surfactant Composition:

  • 70% to 80% Phospholipids: Primarily Dipalmitoylphosphatidylcholine (DPPC).

  • 10% Cholesterol.

  • 10% Proteins: Surfactant Proteins SP-A, B, C, and D.

• Surfactant Synthesis and Secretion:

  • Synthesized in the ER (lipids in SER, proteins in RER).

  • Transported to Golgi, then to multivesicular bodies (MVB), then to Lamellar Bodies (LB).

  • ABCA3 is the transporter that moves surfactant into LBs.

  • Secreted into the alveolar space via exocytosis induced by mechanical stretch (breathing) or chemical stimuli (Calcium, cAMP, Adrenergic).

  • Forms Tubular Myelin (TM) before reorganizing into a functional Monolayer.

Functions of Lung Surfactant

• Reduces Surface Tension (ST):

  • Surface tension at $37^{\circ}C$ for pure water is $70 \, mN/m$.

  • Surfactant reduces this to $20-25 \, mN/m$ (and near zero during expiration).

  • Mechanism: Hydrophilic parts bond with the water layer; hydrophobic parts interact with air, breaking intermolecular forces between water molecules.

• Increases Lung Compliance: Decreases the work required to inflate the lungs.

• Prevents Lung Collapse: Prevents atelectasis at the end of expiration.

• Keeps Alveoli Dry: Prevents pulmonary edema by reducing the force that draws fluid across the alveolar wall.

• Promote Alveolar Stability: Reduces pressure differences between alveoli of different sizes (Law of Laplace).

• Host Defense: SP-A and SP-D act as innately immune molecules; they have carbohydrate recognition domains to opsonize bacteria and viruses for macrophages.

Specific Surfactant Components

• Dipalmitoylphosphatidylcholine (DPPC): Main component; synthesis starts at 28 weeks, peaks at 36 weeks.

• Phosphatidyl glycerol (PG): Last phospholipid produced (appears at 36 weeks); reliable marker for lung maturity.

• SP-A & SP-D: Hydrophilic; host defense; promote tubular myelin formation; secreted in LB-independent manner.

• SP-B & SP-C: Hydrophobic; positively charged; help with lipid adsorption onto the monolayer; localized within LBs.

Clinical Correlation: Respiratory Disorders

• Respiratory Distress Syndrome (RDS):

  • Affects premature infants due to surfactant deficiency.

  • Leads to high surface tension, decreased compliance, and alveolar collapse (atelectasis).

  • Diagnosis: Fetal Lung Maturity (FLM) testing.

• L/S Ratio: Lecithin (DPPC) to Sphingomyelin ratio. L/S $\ge 2/1$ at 35 weeks indicates maturity.

• Risk: L/S 1.5–1.9 (40% risk).