Hemoglobin and Myoglobin

Haemoglobin

• One of the first proteins to have its molecular mass accurately determined.
• First protein to be characterized by ultracentrifugation and associated with the physiological function of oxygen transport.
• First protein in which a point mutation was proven to cause a single amino acid change (sickle-cell anemia).
• A sophisticated oxygen delivery system that delivers the proper amount of oxygen to tissues across varying circumstances.
• Transports oxygen from the lungs, gills, or skin to capillaries for use in respiration.
• Required by larger organisms because O2 diffusion rate through tissue thicker than 1 mm is too slow to support life.

Myoglobin

• Originally assumed to store oxygen, but this is significant only in aquatic mammals which have Mb concentrations 10-30 times greater than land mammals.
• In terrestrial mammals, it facilitates oxygen transport in rapidly respiring muscle.
• Increases effective solubility of O2 in muscle.
• Functions as a kind of molecular bucket brigade to facilitate O2 diffusion.
• Myoglobin is the oxygen-carrying protein of muscle tissue.
• Abundant in diving mammals, allowing them to use oxygen underwater for extended periods.
• Functions include the detoxification of nitric oxide (NO) to nitrate ion (NO_3) under normal conditions and synthesis from nitrite ion (NO_2) under hypoxic conditions.

Physical and Chemical Properties of Haemoglobin and Myoglobin

• Myoglobin and each of the four subunits of hemoglobin noncovalently bind a single heme group.
• Heme is responsible for the characteristic red color of blood and is the site at which each globin monomer binds one molecule of O2.
• The heterocyclic ring system of heme is a porphyrin derivative consisting of four pyrrole rings linked by methene bridges.
• The porphyrin in heme, with its particular arrangement of four methyl, two propionate, and two vinyl substituents, is known as protoporphyrin IX. Heme is protoporphyrin IX with a centrally bound iron atom.
• The iron atom normally remains in the Fe (II) (ferrous) oxidation state whether or not the heme is oxygenated.
• The Fe atom in deoxygenated Hb and Mb is 5-coordinated by a square pyramid of N atoms: four from the porphyrin and one from a His side chain of the protein.
• On oxygenation, the O2 binds to the Fe(II) on the opposite side of the porphyrin ring from the His ligand, so that the Fe(II) is octahedrally coordinated.
• Certain small molecules, such as CO, NO, CN, and H2S, coordinate to the sixth liganding position of the Fe(II) in Hb and Mb with much greater affinity than oxygen.
• The Fe(II) of Hb and Mb can be oxidized to Fe(III) to form methemoglobin (metHb) and metmyoglobin (metMb).
• MetHb and metMb do not bind O2; their Fe(III) is already octahedrally coordinated with an H2O molecule in the sixth liganding position.
• Erythrocytes contain methemoglobin reductase, which converts the small amount of metHb that spontaneously forms back to the Fe(II) form.

Myoglobin's Role in Nitric Oxide Metabolism

• Nitric oxide (NO) induces vasodilation in tissues.
• Eliminated rapidly to prevent its interference with subsequent NO signals.
• NO is detoxified in muscle under normal O2 concentrations through its reaction with oxygenated myoglobin (oxyMb) to yield nitrate ion and metmyoglobin
• Metmyoglobin is subsequently reduced to Mb via an intracellular metmyoglobin reductase.
• Oxygenated hemoglobin (oxyHb) detoxifies NO present in blood.

Heme and Globin Chain Attachment

• There are 4 heme residues per Hb molecule, one for each subunit in Hb, which accounts for about 4% of the whole mass of Hb.
• Heme is located in a hydrophobic cleft of the globin chain.
• The iron atom of heme occupies the central position of the porphyrin ring; the reduced state is called ferrous (Fe++) and the oxidized state is ferric (Fe+++).
• Ferrous iron has 6 valencies, and ferric has 5 valencies; in hemoglobin, iron remains in the ferrous state.
• Iron is linked to pyrrole nitrogen by 4 coordinate valency bonds and a 5th to the imidazole nitrogen of the proximal histidine.
• In oxy-Hb, the 6th valency of iron binds the O2; oxygen atom directly binds to Fe and forms a hydrogen bond with an imidazole nitrogen of the distal histidine.
• In deoxy-Hb, a water molecule is present between the iron and distal histidine
• As the porphyrin molecule is in resonance, the central iron atom is linked by a coordinate bond; distal histidine lies on the side of the heme ring.

Oxygen Binding: Hemoglobin vs. Myoglobin

• Oxygen diffuses from the alveoli of the lungs into the capillaries of the bloodstream and then into red blood cells, where it binds to hemoglobin.
• Hemoglobin is virtually 100% saturated in the lungs, but the oxygen pressure decreases to about 25 mm Hg as hemoglobin circulates to working muscles.
• At lower oxygen levels, hemoglobin is about 50% saturated; the released oxygen moves into the muscles, where myoglobin is found.
• At 25 mm Hg, myoglobin is almost fully saturated.
• The amount of oxygen in the mitochondria (1 or 2 mm Hg) allows myoglobin to release most of its oxygen where it is most needed.
• Hemoglobin picks up oxygen in the lungs, circulates through the bloodstream, and drops off oxygen in the muscles and other tissues.
• Myoglobin picks up the oxygen and delivers it to the mitochondria, where it is used to oxidize fuel molecules.
• Hemoglobin's oxygen binding curve is sigmoidal, meaning that it can deliver a significant amount of oxygen over a fairly narrow range of pressures.
• Myoglobin's oxygen binding curve is hyperbolic, meaning that it holds onto oxygen much tighter.
• Hemoglobin is well suited for oxygen binding in the lungs, transport in the bloodstream, and delivery to the tissues.
• Myoglobin is well suited for oxygen storage in the muscles and delivery to mitochondria when needed.

Hemoglobin: Structure and O2 Delivery

• Hemoglobin has four subunits that communicate, contributing to its success as an oxygen delivery molecule.
• This is evidenced by the cooperativity in oxygen binding.
• To achieve 25% saturation (1 O2 molecule per hemoglobin), O2 amount needs to be about 18 mm Hg.
• To increase saturation to 50% (2 O2 molecules per hemoglobin), O2 amount needs to be about 26 mm Hg.
• It is easier to bind the second molecule of O2 than the first.
• Max Perutz determined that hemoglobin exists in two forms: oxy-hemoglobin and deoxy-hemoglobin.
• Deoxy-hemoglobin has a relatively low affinity for oxygen, but when one molecule of oxygen binds to a heme group, the structure changes to the oxygenated form, which has a greater attraction for oxygen.
• The second molecule of O2 binds more easily, and the third and fourth even more easily.
• Oxygen affinity of oxy-hemoglobin is many times greater than that of deoxy-hemoglobin.

Equilibrium

• The two forms of hemoglobin are in equilibrium with one another because oxygen binding is reversible.
• Under certain conditions, the deoxy form is favored and vice versa.
• Adding more O2 would shift the reaction to the right, producing more oxy-hemoglobin.
• Myoglobin has only one form, regardless of whether oxygen is present.
• Differing forms of hemoglobin occur due to its four subunits, accounting for its cooperative oxygen binding.
• Myoglobin, with its single chain, does not exhibit cooperative oxygen binding.
• When O2 binds to a subunit of deoxyhemoglobin, it causes subtle changes in the structure of the protein.
• This alters the way that the four subunits fit together, making it easier for a subsequent molecule of oxygen to bind to the next subunit.

Factors Affecting Hemoglobin's Equilibrium

• Other substances can alter the binding of oxygen to hemoglobin, called "allosteric effectors".
• Cooperative interactions occur when the binding of one ligand is influenced by the binding of another ligand (effector or modulator) at a different (allosteric) site on the protein.
• An identical allosteric effect is called homotropic, whereas if they are allosteric binding to different sites it is called heterotropic.
• These effects are termed positive or negative depending on whether the effector increases or decreases the protein's ligand-binding affinity.
• Hemoglobin exhibits both homotropic and heterotropic effects.
• The binding of O2 to Hb results in a positive homotropic effect since it increases the O2 affinity.
• BPG, CO2, H+, and Cl- are negative heterotropic effectors of O2 binding to Hb because they decrease its affinity for O2 and are chemically different from O2.
• Allosteric effects result from interactions among subunits of oligomeric proteins.
• Hydrogen ions (protons), CO2, and 2,3-bisphosphoglycerate (BPG) all promote the release of oxygen by shifting the equilibrium towards the deoxygenated form of hemoglobin.

Factors That Affect Haemoglobin Equilibrium

• Oxygen binding
• 2,3-biphosphoglycerate (BPG)
• CO2
• H+
• Cl-

Oxygen Dissociation Curve (ODC)

• The strength by which oxygen binds to hemoglobin is affected by several factors and can be represented as a shift to the left or right in the oxygen dissociation curve
• A curve shift to the right indicates that hemoglobin has a decreased affinity of oxygen, thus, oxygen actively unloads.

Bohr Effect

• High concentrations of hydrogen ions and carbon dioxide are present around actively metabolizing tissues.
• The binding of these allosteric effectors in the capillaries prompts the release of oxygen from hemoglobin, which is taken up by myoglobin and delivered to the mitochondria.
• The specific reaction of hydrogen ions and carbon dioxide with hemoglobin causing oxygen release is the Bohr effect.
• When deoxygenated hemoglobin returns to the lungs, the concentrations of H+ and CO2 are low, causing these compounds to be released from hemoglobin.
• Carbon dioxide is expelled from the body through expired air.

Effect of pH and pCO2

• When pCO2 is elevated, H+ concentration increases, and pH falls.
• pCO2 is high/pH is low in the tissues due to the formation of metabolic acids like lactate.
• The affinity of hemoglobin for O2 is decreased (the ODC is shifted to the right), which allows more O2 to be released to the tissues.
• Binding of CO2 forces the release of O2.
• When pCO2 is high, CO2 diffuses into the red blood cells, where the carbonic anhydrase forms carbonic acid (H2CO3).
• When carbonic acid ionizes, the intracellular pH decreases with decreased affinity of Hb for O2, and O2 is unloaded to the tissues.
• In the lungs, the opposite reaction occurs, pCO2 is low, pH is high, and pO2 is significantly elevated, increases binding of O2 to hemoglobin and the ODC shifts to the left.

The Chloride Shift

• When CO2 is taken up, the HCO3- concentration within the cell increases and diffuses out into the plasma.
• Simultaneously, chloride ions from the plasma enter in the cell to establish electrical neutrality (chloride shift or Hamburger effect).
• From this side, RBCs are slightly bulged due to the higher chloride ion concentration.
• When the blood reaches the lungs, the reverse reaction takes place.
• Deoxyhemoglobin liberates protons that combine with HCO3 to form H2CO3, which is dissociated to CO2 and H2O by the carbonic anhydrase, and the CO2 is expelled
• As HCO3 – binds H+, more HCO3- from plasma enters the cell, and Cl- exits (reversal of chloride shift).

Effect of Temperature

• The term p50 means the pO2 at which Hb is half saturated (50%) with O2.
• The p50 of normal Hb at 37oC is 26 mm Hg.
• Elevation of temperature from 20 to 37oC causes 88% increase in p50.
• Shift in ODC to left at low temperature results in release of less O2 to the tissues and relative hypothermia.
• However, under febrile conditions, the increased needs of O2 are met by a shift in ODC to right.

Effect of 2,3-BPG

• 2,3-BPG is produced from 1,3-BPG, an intermediate of glycolytic pathway
• Preferentially binding to deoxy-Hb, it stabilizes the T conformation.
• BPG interacts with deoxygenated hemoglobin beta subunits and decreases the affinity for oxygen and allosterically promotes the release of remaining oxygen molecules
• When the T form reverts to the R conformation during oxygenation, BPG is released.
• High oxygen affinity of fetal blood (HbF) is due to the inability of gamma chains to bind 2,3-BPG.

Haemoglobinopathies

• Encompasses all genetic diseases of haemoglobin.
• There are two main groups: hemoglobin variants and thalassemias.
• Hemoglobin variants are caused by mutations in the hemoglobin gene.
• Thalassemias are caused by an underproduction of otherwise normal haemoglobin molecules.

Hemoglobin variants

• Hemoglobin S
• Hemoglobin E
• Hemoglobin C
• Hemoglobin D
• Hemoglobin M

Hemoglobin S (HbS) (Sickle Cell Hemoglobin)

• It constitutes the most common variant globally.
• The single amino acid substitution of glutamic acid to valine in the 6th position of the beta chain of HbA leads to polymerization of hemoglobin molecules inside RBCs, distorting the cell into a sickle shape.
• The substitution of glutamic acid causes a localized stickiness on the surface of the molecule, and the deoxygenated HbS may be depicted with a protrusion on one side and cavity on the other side.
• HbS can bind and transport oxygen; the sickling occurs under a deoxygenated state; the sickled cells form small plugs in capillaries; Occlusion of major vessels can lead to infarction in organs like the spleen.

Sickle Cell Trait

• In heterozygous (AS) condition, 50% of Hb in the RBC is normal, the sickle cell trait does not produce clinical symptoms, allowing persons to have a normal lifespan.
• At higher altitudes, hypoxia may cause manifestation of the disease and those with chronic lung disorders may also produce hypoxia-induced sickling in HbS trait.
• HbS can be detected with normal Hb in persons by electrophoresis.
• HbS gives protection against malaria:
• A high incidence of the sickle cell gene in population coincides with the area endemic for malaria, HbS affords said protection against Plasmodium falciparum infection.

Important Haemoglobinopathies

• HbS: Point mutation at Beta 6, Amino acid substitution of Glu --> Val, Codon and base substitution of GAG --> GUG
• HbC: Point mutation at Beta 6, Amino acid substitution of Glu --> Lys, Codon and base substitution of GAG --> AAG
• HbE: Point mutation at Beta 26, Amino acid substitution of Glu --> Lys, Codon and base substitution of GAG --> AAG
• HbD (Punjab): Point mutation at Beta 121, Amino acid substitution of Glu --> Gln, Codon and base substitution of GAG --> CAG
• HbM: Point mutation at Proximal or distal histidine in alpha or beta chains, Amino acid substitution of His --> Tyr, Codon and base substitution of CAC --> UAC

Hemoglobin E

• The second most prevalent hemoglobin variant caused by the replacement of beta 26 glutamic acid by Lysine.
• Primarily seen in the orientals of South-East Asia (Thailand, Myanmar, Bangladesh, etc).
• Very prevalent in West Bengal, India; heterozygotes are completely asymptomatic.
• HbE has similar mobility to A2 by electrophoresis.

Hemoglobin C

• The 6th amino acid in the beta chain is glutamic acid, but it is replaced by lysine in HbC.
• AC heterozygotes do not show any clinical manifestations.
• Double heterozygotes for HbS and HbC (SC) have a moderate disease and homozygotes (CC) have a mild to moderate hemolytic anemia.
• HbC is slower moving than HbA on electrophoresis at alkaline pH.

Hemoglobin D

• HbD Punjab does not produce sickling from the replacement of beta 121 glutamic acid by glutamine.

M-Hemoglobins (Hb M)

• A group of variants, where the substitution occurs in the proximal or distal histidine residues of alpha or beta chains.
• Alpha 58 His →Tyr
• Beta 92 His →Tyr

Thalassemia

• Thalassemia may be defined as the normal globin chains in abnormal proportions, gene function is abnormal, but there is no abnormality in the polypeptide chains.
• Reduction in alpha chain synthesis is called alpha thalassemia and deficient beta chain synthesis is beta thalassemia.

Structural Proteins: Collagen and Elastin

• The major structural protein found in connective tissue is collagen, which comprises 25-30% of the total body weight.
• Collagen holds together the cells in the tissues.
• A major fibrous element of tissues like bone, teeth, tendons, cartilage, and blood vessels.

Intracellular Alterations During Post-Translational Processing of Collagen:

• Hydroxylation of proline and some lysine residues.
• Glycosylation of some of the hydroxylysine residues.
• Formation of intrachain and interchain disulphide bonds, mainly in the carboxy and amino terminal ends.
• Formation of triple helix.

Extracellular Alterations During Post-Translational Processing of Collagen:

• Cleavage of 25-35 kDa portions at both carboxy and amino terminal ends
• Formation of quarter-staggered alignment
• Oxidative deamination of epsilon amino groups of lysine and hydroxylysine residues
• Formation of intra- and interchain crosslinks

Abnormalities in Collagen:

• Osteogenesis Imperfecta
• Ehlers-Danlos Syndrome (EDS)
• Alport Syndrome
• Epidermolysis bullosa
• Marfan's Syndrome
• Menke's Disease
• Deficiency of Ascorbic Acid
• Homocystinuria

Elastin

• Found in connective tissue and is the major component of elastic fibers.
• These fibers can stretch and then resume their original length, with high tensile strength.

Disease Associated with Abnormalities in Elastin:

• Williams-Beuren syndrome
• Pseudoxanthoma elasticum
• Copper deficiency