Essay on Hemoglobin: Meaning, Structure and Properties

Essay on Hemoglobin:- 1. Meaning of Hemoglobin 2. Structure of Hemoglobin 3. Properties 4. Biosynthesis 5. Transportation Provided 6. 2, 3-Biphosphoglycerate (BPG) Stabilizes the T Structure 7. De-oxy-hemoglobin S can Form Fibres that Distort Erythrocytes 8. Varieties 9. Technique for Identification.

Contents:

Essay on the Meaning of Hemoglobin
Essay on the Structure of Hemoglobin
Essay on the Properties of Hemoglobin
Essay on the Biosynthesis of Hemoglobin
Essay on the Transportation Provided by Hemoglobin
2, 3-Biphosphoglycerate (BPG) Stabilizes the T Structure of Hemoglobin
De-oxy-hemoglobin S can Form Fibres that Distort Erythrocytes
Varieties of Human Hemoglobin
Essay on the Technique for Identification of Hemoglobins

Essay # 1. Meaning of Hemoglobin:

Hemoglobin is the red colouring matter of blood which is present in the red blood cells. It is a conjugated protein consisting of heme and the protein globin. It has a molecular weight of 64,450. It can combine with oxygen and acts as the transport mechanism for oxygen within the blood. It contains 4 gram atoms of iron per mole in the ferrous (Fe⁺⁺) state.

Essay # 2. Structure of Hemoglobin:

The structure of Hemoglobin can be classified under two headings:

a. Structure of Heme, the prosthetic group.

b. Structure of Globin, the protein part— apoprotein.

a. Structure of Heme:

i. It is an iron porphyrin. The porphyrins are cyclic compounds with “tetra pyrrole” structure.

ii. Four pyrrole rings called I to IV are linked through methylene bridges or methylidene bridges.

iii. The outer carbon atoms, which are not linked with the methylidene bridges, are numbered 1 to 8.

iv. The methylidene bridges are designated as α, β, γ, δ, respectively.

v. Iron in the ferrous state is bound to the nitrogen atom of the pyrrole rings.

vi. Iron is also linked internally (5th linkage) to the nitrogen of the imidazole ring of Histidine of the polypeptide chains.

vii. The propionic acid of 6th and 7th position of heme of III and IV pyrroles are also linked to the amino acids Arg and Lys of the polypeptide chain, respectively.

The porphyrins are found in nature in which the various side chains are substituted for the 8 hydrogen atoms as numbered in the porphin nucleus. The arrangement of the A and P substituents in the uroporphyrin shown here is asymmetric (in ring IV the expected order of the acetate and propionate substituents is reversed).

This type of asymmetric substitution is classified as a type III porphyrin. A porphyrin with a completely symmetrical arrangement of the substituents is classified as a type I porphyrin. Only types I and III are found in nature and the type III series is more abundant.

b. Structure of Globin:

i. The globin of hemoglobin is a protein which is composed of 4 parallel layers of closely packed polypeptide chains.

ii. Two of the chains (α-chains) have identical amino acid composition of 141 amino acids. The two other chains may be two of the 4 polypeptide chains designated as β, γ, δ, and ɛ (epsilon). Each is having 146 amino acids.

iii. The total number of amino acids in globin is 574.

iv. α chains have Val-Leu-Ser in N terminal residues and Lys-tyr-Arg in C terminal residues.

v. β chains have Val-His-Leu in N-terminal residues and Lys-tyr-His in C-terminal residues.

vi. γ chains have Gly-His-Phe. N-terminal residues and Arg-Tyr-His in C-terminal residues.

vii. Hemoglobin molecule and its sub-units contain mostly hydrophobic amino acids internally and hydrophilic amino acids on their surfaces. So they form “‘Heme pockets”.

viii. In “heme pockets” α subunits are of size necessary for entry of O₂ molecule but the entry of O₂ molecule in β subunit is blocked by valine residue.

Biosynthesis of Porphyrins:

Chlorophyll (magnesium-containing porphyrin), the photosynthetic pigment of plants and heme (the iron-containing porphyrin) of hemoglobin in animals are synthesized in living cells by a common pathway:

i. The starting materials are ‘active succinate’ (succinyl-CoA) derived from the citric acid cycle and glycine. Pyridoxal phosphate (B₆-PO₄) is necessary to activate glycine. The product of the condensation reaction is α-amino-β-ketoadipic acid which is catalyzed by the enzyme AmLev synthetase (ALA synthase).

ii. α-amino-β-ketoadipic acid is rapidly decarboxylated by the same enzyme AmLev synthetase producing δ-aminolevulinic acid (AmLev). Synthesis of aminolevulinic acid occurs in the mitochondria. The anemia has been observed in the deficiency of vitamin B₍, or pantothenic acid.

iii. 2 mols of AmLev condense to form porphobilinogen (the first precursor of pyrrole) which is catalyzed by the enzyme δ-aminolevulinase (AmLev dehydrase).

iv. 3 mols of porphobilinogen condense first to form a tripyrrylmethane which then breaks down into a di-pyrrylmethane and a monopyrrole. The dipyrryl compounds are of two types A and B. The formation of tetrapyrrole occurs by condensation of two dipyrrylmethanes. If two of the (A) components condense, a type I porphyrin results; if one (A) and one (B) condense, a type III results.

v. The uroporphyrinogens I and III are converted to coproporphyrinogens I and III by decarboxylation being catalyzed by uroporphyrinogen decarboxylase.

vi. The coproporphyrinogen III then enters the mitochondria where it is converted to protoporphyrinogen III and then to protoporphyrin III. The enzyme coproporphyrinogen oxidase catalyzes the formation of protoporphyrinogen III. The oxidation of protoporphyrinogen to protoporphyrin is catalyzed by the enzyme protoporphyrinogen oxidase.

The enzyme coproporphyrinogen oxidase is able to act on type III coproporphyrinogen only for which type I protoporphyrin has not been identified in natural materials. In mammalian liver the reaction of conversion of coproporphyrinogen to protoporphyrin requires molecular oxygen.

vii. In the final step of heme synthesis ferrous ion (Fe⁺⁺) is incorporated into protoporphyrin III which is catalyzed by heme synthetase or ferrochelatase. The reaction takes place readily in the absence of enzymes but becomes rapid in presence of enzymes.

A summary of the steps is given:

Note:

a. The porphyrinogens are the reduced porphyrins containing 6 extra hydrogen atoms. The oxidized porphyrins cannot be used for heme or chlorophyll synthesis.

b. The porphyrinogens are readily auto-oxidized to the respective porphyrins in presence of light.

Essay # 3. Properties of Hemoglobin:

i. Oxy-hemoglobin:

It forms oxy-hemoglobin in combination with oxygen. When hemoglobin is exposed to air, it takes up two atoms of oxygen for each atom of ferrous ion (Fe⁺⁺) present. Thus, hemoglobin will take up 4 molecules of oxygen. In low oxygen tension, oxy-hemoglobin gives up O₂ readily. By this way, blood carries O₂ to different parts of the body.

ii. Formation of Carhamino Compound:

It reacts with CO₂ forming carbamino compounds.

Hb-NH₂ + CO₂ → Hb-NH.COOH

iii. Reaction with Carbon Monoxide:

It forms carboxy hemoglobin after reacting with carbon monoxide (CO). Carboxy hemoglobin is stable and prevents the formation of oxy-hemoglobin. So inhalation of even small amounts of carbon monoxide is highly dangerous.

iv. Buffering Action:

One mol of hemoglobin contains 35 histidine residues. Histidine exerts its buffering action through its basic imidazole ring. Hence, hemoglobin plays an important role in regulating the acid-base balance of blood.

v. Formation of Methemoglobin:

Methemoglobin is formed as a result of the oxidation of hemoglobin by the mild oxidizing agent, potassium ferricyanide.

The ferrous ion (Fe⁺⁺) is oxidized to the ferric ion (Fe⁺⁺⁺). Methemoglobin cannot carry oxygen in blood.

It is also formed by the action of some drugs. This is found in the blood of some individuals owing to inborn errors of metabolism.

This can be reduced to hemoglobin by vitamin C which is used in the treatment of methemoglobinemia.

vi. Sulphemoglobin:

It is formed by the administration of certain drugs. It continues to remain in the blood and cannot be reconverted into hemoglobin.

vii. Cyanomethemoglobin:

It is formed by the addition of cyanide to methemoglobin. It has a bright red colour.

viii. Absorption Spectra:

The different hemoglobin derivatives can be easily identified by this characteristic absorption spectra.

(a) Oxy-hemoglobin:

Two bands—one narrow and the other wide in the green region.

(b) Reduced hemoglobin:

One single broad band in the green region.

(c) Carboxy hemoglobin:

Two bands in the green region.

(d) Methemoglobin:

Three bands – one in red and two in the green regions.

(e) Sulphemoglobin:

Three bands similar to methemoglobin.

Essay # 4. Biosynthesis of Hemoglobin:

i. The biosynthesis of hemoglobin takes place in the bone marrow in the erythroid cell during its development to erythrocyte.

ii. It starts appearing at stage II (early normoblast) and the synthesis is complete when the cell reaches stage IV (late normoblast).

iii. Iron in the ferrous state is incorporated into protoporphyrin to form heme.

iv. The heme gets attached to the newly synthesized globin to form hemoglobin.

v. The iron of heme is coordinated to 2 imidazole nitrogen of histidine at position 38 and 87 in α-chains and 63 & 92 in β-chains.

In nature, the other metal loporphyrins which are compounds of importance in biologic processes are mentioned:

A. Erythrocruorins:

(a) They are iron porphyrinoproteins occurring in blood and tissue fluids of some invertebrates.

(b) Their function is corresponding to hemoglobin.

B. Myoglobins:

(a) They are the respiratory pigments occurring in the muscle cells of vertebrates and invertebrates.

(b) The purified one has a molecular weight of about 17,000.

C. Catalases:

(a) They are iron poiphyrin enzymes.

(b) They have been obtained in crystalline form.

(d) They contain 4 gram atoms of iron per mol.

(e) In plants, their activity is minimal.

D. Tryptophan Pyrrolase:

(a) It is an iron porphyrin protein.

(b) It catalyzes the oxidation of tryptophan to formyl kynurenine.

E. Cytochromes:

(a) Cytochromes means the cellular pigments because these pigments are widely distributed not only in the tissues of higher animals and plants but also in yeast and bacteria.

(b) At first, cytochromes a, b and c were identified and they had been shown to exist in oxidized and reduced forms and their fundamental role is in cellular respiration. At present, some thirty cytochromes are known to exist and according to original cytochromes they are designated as a₁, a₂, a₃, c₁, c₂, c₃, c₄, c₅, b₂, b₃, b₄ etc.

(d) The important example is cytochrome C which has been obtained in the purified form.

(e) Cytochrome C has a molecular weight of about 13,000 and contains 0.43% iron.

(f) The iron porphyrin group of cytochrome C is attached to protein more firmly than in the hemoglobin.

(g) Cytochrome C is quite stable to heat and acids.

(h) The reduced form of cytochrome C is not auto-oxidizable.

(i) At physiological pH Ferro cytochrome C does not combine with O₂ or CO as does hemoglobin.

(j) The peptide chain of human heart cytochrome C contains 104 amino acids, Acetyl glycine is the N-terminal amino acid and glutamic acid the C-terminal amino acid. The two cysteine residues are located at positions 14 and 17 in the peptide chain.

The linkage of iron in heme occurs through the imidazole nitrogen of a histidine residue at position 18 in the peptide chain.

(k) The degree of difference in primary structure among the 13 cytochrome C might be related to the degree of phytogenetic relationship between the species Eg. The cytochrome C of man as compared to that of rhesus monkey differs by only one amino acid of the 104 amino acids. Human cytochrome C differs from that of the dog in 11 amino acid residues, from that of the horse in 12.

(l) The enzymes that catalyse the reactions of molecular oxygen are known as oxidases. Cytochrome a₃, which is found in heart muscle and other animal tissues is called cytochrome oxidase. These oxidases catalyse many reactions in addition to terminal oxidation at the electron transport chain. They can carry three general types of reactions e.g., oxygen transfer, mixed function oxidation electron transfer.

Essay # 5. Transportation Provided by Hemoglobin:

Hemoglobin Transports CO₂ and Protons to the Lungs after releasing O₂ to the Tissues:

i. Hemoglobin can bind CO, directly when oxygen is released and CO, reacts with the amino terminal a-amino groups of the hemoglobin forming a carbamate and releasing protons.

The amino terminal is converted from a positive to a negative charge favouring salt bridge formation between the a and P chains.

ii. At the lungs, hemoglobin is oxygenated, being accompanied by expulsion and subsequent expiration of CO₂. CO₂ is absorbed in blood and the carbonic anhydrase in erythrocytes catalyzes the formation of carbonic acid which is rapidly dissociated into bicarbonate and a proton.

A buffering system absorbs these excess protons to avoid the increasing acidity of blood. Hemoglobin binds two protons for every four oxygen molecules. 3. In the lungs, the process is reversed i.e. when oxygen binds to deoxygenated hemoglobin, protons are released and combines with bicarbonate forming carbonic acid which is exhaled.

Thus, the binding of oxygen forces the exhalation of CO₂. This reversible phenomenon is called the Bohr effect. Myoglobin does not exhibit Bohr effect.

Essay # 6. 2, 3-Biphosphoglycerate (BPG) Stabilizes the T Structure of Hemoglobin:

i. The increased accumulation of 2, 3- biphosphoglycerate is caused by an oxygen shortage in peripheral tissues. BPG is formed from 1, 3-biphosphoglycerate in the glycolytic pathway. One molecule of BPG is bound to central cavity formed by all four subunits of hemoglobin.

This cavity is of sufficient size for BPG only when hemoglobin is in the T form. BPG is bound by salt bridges between its oxygen atoms and both chains as well as by Lys EF6 and His H21. Thus, BPG stabilizes the T or deoxygenated form of hemoglobin.

ii. Fetal hemoglobin is more weakly bound to BPG because the H21 residue of the Ƴ chain of HbF is Her rather than His and cannot form a salt bridge with BPG. Hence, BPG has a less profound effect on he stabilization of the T form of HbF and is responsible for HbF to have a higher affinity for oxygen than does HbA.

iii. The trigger for the R to T transition of hemoglobin is movement of the iron in and out of the plane of the porphyrin ring.

Essay # 7. De-oxy-hemoglobin S can Form Fibres that Distort Erythrocytes:

i. After the de-oxygenation of hemoglobin S the sticky patch can bind to the complementary patch on another deoxygenated HbS molecule. This binding causes polymerization of de-oxy-hemoglobin S forming long fibrous precipitates. These extend throughout the erythrocyte and mechanically distort it causing lysis and a good number of secondary clinical effects.

ii. De-oxy-hemoglobin A although contains the receptor sites for the sticky patch present on deoxygenated HbS, the binding of sticky hemoglobin S to de-oxy-hemoglobin A cannot extend the polymer. Because de-oxy-hemoglobin A does not have a sticky patch to enhance binding to another hemoglobin molecule.

Therefore, the binding of de-oxy-hemoglobin A to the R or the T form of hemoglobin S will reject polymerization.

iii. The polymer forms a twisted helical fiber whose cross section contains 14 HbS molecules. These tubular fibres distort the erythrocyte.

Essay # 8. Varieties of Human Hemoglobin:

Normal adult hemoglobin or hemoglobin A has a molecular weight of 64,456 and contains two pairs of peptide chains (α & β) of which α chain contains 141 and β chain contains 146 amino acids.

Fetal hemoglobin (F) is present in very small amounts.

All the normal human hemoglobin’s possess a common half-molecule, i.e. a pair of peptide chains (a chains); the other half consists of a pair of different types of peptide chains, one type for each hemoglobin. Hemoglobin A₂ has two δ chains and hemoglobin F has two γ chains; both types of chains contain 136 amino acids and thus are of the same length as the β chain.

Hemoglobin A is represented as α₂Aβ₂A hemoglobin A₂ as α₂A δ₂A and hemoglobin F as α₂Aγ₂A for describing abnormal hemoglobin. In early embryonic life, a fourth hemoglobin a₂Ae₂ exists.

Fetal Hemoglobin:

i. Fetal hemoglobin (F) comprises 50 to 90 per cent of the total hemoglobin in the newborn.

ii. It takes up oxygen more readily at low oxygen tensions and releases carbon dioxide more readily than adult hemoglobin (A).

iii. It is more resistant to denaturation by alkali and is more susceptible to conversion to methemoglobin by nitrites (contaminated water).

iv. Hemoglobin F is gradually replaced by hemoglobin A during the first 6 months of extra uterine life.

v. High concentration of hemoglobin F after two years of age occur in various types of anemia, e.g., sickle cell anemia and thalassemia.

Abnormal Hemoglobin’s:

Over one hundred different types of abnormal hemoglobin’s have been described. Some of these are easily differentiated by their electrophoretic mobilities and have given rise to the concept of “molecular disease” which explains that a defective gene (mutant) may direct the formation of a molecule similar to a normal molecule but differing from it in shape, composition and electrical charge.

One amino acid of the normal hemoglobin is replaced by another amino acid, i.e. acidic amino acid is replaced by a basic or a neutral amino acid for the formation of abnormal hemoglobin. The abnormal hemoglobin’s are named in alphabetic order as C, D, E, F, G, H, K, L, M, N, O, P, Q, S etc.

A. Hemoglobin C:

This occurs in the blood of some Negroes in West Africa. The abnormality is found in the β chain at position 6, the amino acid glutamic acid is replaced by Lysine. It is characterized by the mild anemia with a tendency to infarction.

B. Hemoglobin S:

This appears among the Negroes of Africa. The abnormality occurs in β chain, glutamic acid at position 6 is replaced by valine. Sickle cell anaemia develops and the RBC becomes long and boat-shaped. The blood becomes more viscous which results in reduced blood flow.

C. Hemoglobin F:

HbF is present in fetus and is replaced by adult hemoglobin as the child grows. It is present only in traces in normal adults, it gets hemolysis rapidly producing a severe anemia called “Thalassemia major”.

D. Hemoglobin M:

There are two types of HbM-HbM (Boston) and HbM I Wate which are of clinical interest. The abnormality is found in the α chain, the histidine residues in 58 and 87 position are replaced by tyrosine. Abnormal amounts of methemoglobin are found in the blood of persons affected by this condition. This methemoglobin is not reduced to hemoglobin by reducing agents.

E. Hemoglobin D:

This occurs rarely. It exists in two forms – Dα and Dβ. The persons having HbD do not show any clinical signs and symptoms.

Essay # 9. Technique for Identification of Hemoglobins:

Finger print technique Ingram developed a technique by which the peptide chains in hemoglobin could be broken down into several smaller peptide fragments by digestion with trypsin. Trypsin splits the peptides only at points where only lysine and arginine occur.

A mixture of smaller peptides were obtained. He then separated this mixture using paper electrophoresis technique and paper chromatography. The peptides appeared as spots when ninhydrin was sprayed. Thus peptide maps had been prepared for different hemoglobin’s.