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:

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

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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.

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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.

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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.

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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) They contain only 1 gram atom of iron per mole.

C. Catalases:

(a) They are iron poiphyrin enzymes.

(b) They have been obtained in crystal­line form.

(c) Their molecular weight is about 225,000.

(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.

(c) They are iron porphyrins and act as electron transfer agents in oxidation-reduction reactions.

(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.

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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.

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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.

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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.

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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.

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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.