HAEMOGLOBIN

INTRODUCTION STRUCTURE TYPES SYNTHESIS FATE DERIVATIVES CLINICAL- ANAEMIAS - HAEMOGLOBINOPATHIES - PORPHYRIAS

 Pigment

present inside RBCs

Carries

O2 and CO2

Responsible

for red color of blood

Deficiency

is called anaemia

Disadvantages of Free Hb
increase in blood viscosity, causing a rise in blood pressure. osmotic pressure of plasma to @ 100mm Hg which interferes with fluid exchange between capillaries & tissue spaces. Loss in urine (Haemoglobinuria) kidney damage by forming acid haematin. Taken up & rapidly destroyed by the tissue Macrophase system.

Normal values
In

foetus- just before birth the Hb concentration of blood from umbilical cord is =16.5 to 18.5 gm/dl. After birth=23gm% this occurs due to transfusion of cells from placenta to infant & haemoconcentration by reduction of plasma volume At the end of 3 months = 10.5 gm/dl At the end of 1 year = 12.5gm/dl Adult male =14-18 gm/dl Adult female =12-15.5 gm/dl Clinically 14.8gm% Hb is considered 100% Hb

Quantity of Haemoglobin in the Cells & amount of O2 carried.

Men: Hb: 15 grams per 100 ml of blood; and Women: Hb: 14 grams per 100 ml. one gram of haemoglobin carries 1.34 ml of oxygen when fully saturated. Therefore, in man 100 ml of blood can carry @ 20 ml of oxygen and in women@ 19 ml of oxygen.

Haemoglobin

Human hemoglobin A, present in adults, consists of four subunits:

two -subunits and two -subunits.

The - and -subunits are homologous and have similar threedimensional structures.

The capacity of hemoglobin to bind oxygen depends on the presence of a bound prosthetic group (heme).

The heme group is responsible for the distinctive red color of blood.

The heme group consists of a protoporphyrin, and a central iron atom.

Protoporphyrin is made up of four pyrrole rings

linked by methene bridges to form a tetrapyrrole ring.

Four methyl groups, two vinyl groups, and two

propionate side chains are attached.

STRUCTURE OF HEMOGLOBIN
Iron-protoporhyrin-globin 4

forms a subunit

subunits join to form a molecule of Hb is in ferrous ( Fe2+) form

Iron

Fe2+

binds 4 pyrrole rings, polypeptide chain and a molecule of O2

pyrroles join to form a ring called porphyrins 4 polypeptide chains constitute globin ,, and are four important polypeptide chains chain has 141 a.a and chain have 146 a.a

GLOBIN (protein part or apoprotein):

It is composed of four polypeptide chains 2 and 2 chains. The -chain contains 141 amino acids and -chain contains 146 amino acids. Each -polypeptide chain is folded into 8 right handed helices ( starting from NH2-terminal) -subunit is folded into 7-helices. The ratio of haem to globin is 1:1. So each haem moiety is linked to one peptide chain.

Attachment of haem to globin

One Hb molecule contain 4 units of Haem each attached to one of the 4 polypeptide chains, constituting globin. Thus one Hb mol. Can carry 4 molecules (8 atoms) of oxygen. Oxygenation of 1st haem mol. In Hb increases the affinity of second mol. for O2

Attachment of haem to globin

and affinity for O2 in 3rd haem mol. Therefore 4th haem-gr of Hb has highest affinity for O2. This shifting affinity of Hb for O2 result in sigmoid shape of O2-Hb curve. Oxygenation of Hb is very rapid <.01 seconds. Hb at its normal con. Increases the O2 carying capacity of blood 70 folds

Types
4 types of poly peptide chains based on amino acid composition and sequence. alpha, beta, gamma, delta Adult Hb Hb A = 2 alpha (141 AA)+ 2 beta (146 AA) chains (22 ) Hb A2 = 2 alpha (141 AA)+ 2 delta (146 AA) chains (2.5%) 1 (22) (10 AA differ) Fetal Hb Hb F = 2 alpha (141 AA)+ 2 gamma (146 AA) chains ( 22) (37 AA differ) 99% replaced with adult Hb with in a year of birth.

TYPES OF HEMOGLOBIN
HbA-

Adult Type- 22

HbA2 HbF-

2% of adult Hb- 22

Fetal Type- 22

gower1- 22 Hb gower2- 22
Hb

 HbA It

Adult Type- 22

is the main form of normal adult Hb.

It

is spheroidal molecule with a molecular weight of 68000 2% of adult Hb- 22

Hb A2 10 individual Amino Acids differ from those in beta chain of HbA

HbA2 In

III. Fetal haemoglobin = HbF (2 2):

It is present normally in newborn and early fetal life and at age of 5 months 90 % of fetal haemoglobin is replaced by adult haemoglobin ( HbA1)

- It consists of 2 alpha chains and 2 gamma chains. - In gamma chain there is 37 amino acid different from those in HB A.

- HbF has a great affinity for O2 because chains do not bind 2,3 DPG well. DPG is responsible for lowering the O2 affinity of Hb and allowing Hb to release O2 at the low PO2 of tissues.

Fetal Hb-(HB-F)
Due to this movement of o2 from maternal to fetal circulation is facilitated. At PO2 20 mmHg ,Hb-F is 70% saturated while Hb-A is only 30-35% saturated. Life span of Hb-F is much less (80 days) as compared to that of Hb-A(120 days)

Hemoglobin Genes and Gene Products

Hemoglobin (Hb) Red, oxygen carrying pigment present in RBCs. Heme (4%) Globin (96%) Quantity 700-900g in body 29-32 pico gram/RBC(MCH) RBCs (MCHC) Male= 36g/100ml Female = 34g/100ml Molecular Weight 68,000

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Myoglobin and Hemoglobin Structure

oxyMb (MbO2) deoxyMb deoxyHb

O2

DERIVATIVES OF Hb
1) Oxyhaemoglobin (HbO2) oxygenation reaction combination with Fe 2+ 2) Carbaminohaemoglobin ( CO2Hb) carbon dioxide combines with globin part 3) Carboxyhaemoglobin (COHb) carbon monoxide binds with Fe 2+ { 250 times more affinity than oxygen}

4) Methhaemoglobin ( HHb) oxidized haemoglobin

5) Sulfhaemoglobin 6) Glycated haemoglobin (HbA1c) Glucose attached to terminal Valine in chain. integrated index of Diabetic control over 4 to 6 weeks

Methemoglobin

This is oxidized Hb,

The Fe2+ normally present in heme being replaced by Fe3+,the ability to react as an O2 carrier is lost.
The normal erythrocyte contains small amount of met Hb, formed by spontaneous oxidation of Hb. Met Hb is normally reconverted to Hb by reducing systems in the RBC, the most important of which is NADH-methemoglobin reductase.

Congenital methemoglobinemias A. Hemoglobin M (Hb-M): It is a congenital condition due to mutation in globin biosynthesis in which distal or proximal histidine is replaced by tyrosine. B. Deficiency of NADH cytochrome b5 methemoglobin reductase system Acquired (toxic) methemoglobinemea Usually arises following the ingestion of large amounts of drugs e.g. phenacetin or the sulphonamides, excess of nitrites or certain oxidizing agents present in the diet.

II. Glycosylated

haemoglobin (Hb A1c):

It is modified form of haemoglobin . it contains glucose linked to amino group present on the NH2 terminal ends. The reaction is non enzymatic and its rate depends on the concentration of glucose .It is present in normal value -5% of the total haemoglobin. This glycohaemoglobin gives an idea about the blood glucose level during the last three months and is useful in the assessment of diabetic control This percentage is increased in diabetic patients up to 8-14%.

FUNCTIONS OF HEMOGLOBIN
1. Transports O2 from lungs to tissues in the form of oxy-hemoglobin 2. Transports CO2 from tissues to lungs in the form of carbamino-hemoglobin:- 30% of total CO2 transport 3. Acts as a buffer- important in acid-base balance- 6 times more than plasma proteins

Hemoglobin (Hb)

250 million Hb molecules / RBC So carry 1 billion oxygen molecules / RBC Synthesis of Hb Starts at proerythroblastic stage Synthesis steps: Heme is made from acetic acid and glycine in mitochondria Acetic Acid -ketoglutaric Acid Succinyl Co A (Krebs Cycle) Globin (polypeptide chain) is synthesized by Ribosomes

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SYNTHESIS
SUCCINYL Co-A + GLYCINE
ALA synthase

-AMINOLEVUNIC ACID PORPHOBILINOGEN PROTOPORPHYRIN IX

Fe2+
GLOBIN

haem synthase

HAEM

HAEMOGLOBIN

FATE OF HAEMOGLOBIN

RBCs are destroyed in RES ( mainly spleen and bone marrow) after 120 days of life.
Macrophages phagocytose hemolyse degrade haemoglobin form Bilirubin transport to liver with albumin conjugated and detoxified secreted in bile.

Haemoglobin

Haem

Globin

Protoporphyrin

Iron

CO

Amino acid pool

Biliverdin

iron pool

Bilirubin

HAEMOGLOBINOPATHIES
Abnormal formation of Hb Due to disorders of Globin Synthesis Two main types Formation of abnormal polypeptide chain e.g.-Sickle cell Anaemia Supression of synthesis of polypeptide chain e.g- Thalassaemia

Sickle cell anaemia:

Valine replaces glutamate in the 6th position of chain. Is common in African blacks Hb crystallizes & takes sickle shape under hypoxic conditions. Increased tendancy towards haemolysis

Heterozygous Half the circulating hemoglobin is abnormal and half is normal.

Have sickle cell trait

Homozygous all of the hemoglobin is abnormal.

Develop the full blown disease

Sickle Cell Disease

Hemoglobin S (HbS) 50% Hb present.
Homozygotic HbSS (sickle cell anemia) - HbS = 100% Hb present, Giving Sickle cell disease HbSA disease - heterozygote for HbS and HbA, with intermediate clinical severity. It is called Sickle cell trait

Molecular and cellular changes of hemoglobin S

Basic abnormality - glutamic acid is replaced by valine at the sixth position of the -globin chain. 2 normal -globin and 2 abnormal -globin chains forms HbS. HbS carries O2 normally but begins to form semisolid aggregate structures once O2 is unloaded to the tissues. These HbS aggregates distort RBCs and cause them to lose their normal elasticity.

Anemia - reduced O2 carrying capacity of the blood

Abnormal hemoglobin in RBCs:

Sickle Cell - one amino acid in the beta chains is wrong.

In low O2 conditions the beta chains form stiff rods which cause RBCs to sickle blocking small vessels.

Hb-S polymerizes at low O2 tensions, and this causes the red cells to become sickle-shaped, hemolyze, and form aggregates that block blood vessels. The result is the severe hemolytic anemia known as sickle cell anemia.

The sickle cell gene is an example of a gene that has persisted and spread in the population.
It originated in the black population in Africa, and it confers resistance to one type of malaria. Africa = 40% of the black population have the sickle cell trait. In United States 10 %
Treatment: Bone marrow Transplatation Hb-F production by hydroxyurea.

Thalassaemias:

The name is derived from the Greek word thalassa,which means sea.Greek identified this disease present around Mediterranean sea. They are hereditary hemolytic diseases in which the synthesis of either - or - globin chain is defective. This decreased rate of synthesis of the globin chains is due to mutation affecting the regulatory gene rather than the structural gene.

A)

- thalassaemias: there are decreased or absent synthesis of -chains of haemoglobin with compensatory increase in the synthesis of other chains. a.Homozygous -chain thalassaemia (thalassaemia major): Incompatible with life, and present as hydrops foetalis usually die in utero , due to complete absence of chains which are required for synthesis of HbF. b.Heterozygous -chain thalassaemia thalassaemia minor,(trait):

Alpha Thalassemia

Alpha thalassemia is caused by mutations in the alpha chain of the hemoglobin molecule Major:all four alpha chain genes are deleted, which is so severe that death can occur in utero (prior to birth). Minor: two alpha chain genes are deleted Silent Carrier State ,Mild .

3 genes defective & 1 normal

2 genes normal 2 genes defective

All 4 genes deleted

B) - thalassaemias: Synthesis of of -chains is decreased or absent whereas synthesis of -chains is normal and will combine with -chains giving excess of HbA2 (22) or it may combine with -chains producing excess of HbF (22). The abnormal haemoglobin do not function as normal haemoglobin - Homozygous (Thalassaemia - major = Cooley's anaemia = Mediterranian sea anaemia): There is complete absence of -globin chain and there is marked increase of HbF. - Heterozygous thalassaemia (Thalassaemia - minor): There is slow rate of synthesis of -globin chain.

THALASSEMIA DIAGNOSIS

complete blood count (CBC) - a measurement of size, number, and maturity of different blood cells in a specific volume of blood.
hemoglobin electrophoresis with A2 and F quantitation - a lab procedure that differentiates the types of hemoglobin present.

FEP (free-erythrocyte protoporphyrin) and ferritin - to exclude iron deficiency anemia

Other molecules containing Haem:i) Myoglobin present in muscles - combination with single polypeptide chain. ii) Neuroglobin present in CNS iii) Cytochrome enzymes present in mitochondria iv) Peroxidases