Haemoglobin

O2 O2 O2

98% travels in oxyhaemoglobin (in red blood cells)

2% is dissolved in plasma (compared to carbon dioxide, oxygen is relatively insoluble in plasma)

O2 is not very soluble – thus needs a carrier !

O2 O2 O2

Myoglobin, Haemoglobin, Cytochromes bind O2

Oxygen is transported from lungs to various tissuesvia blood in association with haemoglobin

In muscle, haemoglobin gives up O2 to myoglobinwhich has a higher affinity for O2 than heamoglobin

Cytochromes participate in redox reactions and arecomponents of the electron transport chain

Haemoglobin (Hb or Hgb) is the primaryconstituent of RBCs

This molecule gives the characteristic red colourto erythrocytes and to the blood

The primary function of haemoglobin is totransport oxygen (O2) from the lungs to thetissue cells of the body and to carry carbondioxide (CO2)

Hemoglobin (tetramer) iscomposed of the protein globin,made up of two alpha chains (141a.a) and two beta chains (146 a.a),each bound to a heme group

Alpha and beta are similar but notidentical in a.a. sequence

Each heme group bears an atomof iron, which can bind to oneoxygen molecule

Each hemoglobin molecule cantransport 4 molecules of oxygen

Abundant in skeletalmuscles

Consists of one hemeand globin consists ofsingle polypeptidechain (monomeric:153 aa; 17,200 MW)

1. Hb A:o Makes up about 95%-98% of Hb found in adults;

o contains two alpha (α) protein chains and two

beta (β) protein chains

2. Hb A2:o Makes up about 2%-3% of Hb;

o Has two alpha (α) and two delta (δ) protein chains

3. Hb F:

o Makes up to 2% of Hb found in adults;

o Has two alpha (α) and two gamma (γ) protein

chains;

o The primary haemoglobin produced by the

fetus during pregnancy, its production usually

falls to a low level shortly after birth

o Foetal Hb has a higher affinity for oxygen than

adult haemoglobin

o This means that the fetus can receive oxygen

from the mother across the placenta.

Responsible for the O2-

binding capacity of Hb

Consists of an iron (Fe) ion held in a heterocyclic ring, known as a porphyrin

The protoporphyrin made up of four pyrrole rings linked by methane bridges

A Fe atom in its ferrous state (Fe+2) is at the center of protoporphyrin

Fe+2 has 6 coordination bondso 4 bonded to the 4 pyrrole N

atoms (The nucleophilic N prevent oxidation of Fe+2)

o The 2 additional binding sites are one on either side of the hemeplane:

✓ One of these is occupied by the imidazole group of His

✓ The second site can be reversibly occupied by O2

When Hb is bound to O2, it is called oxyHb. This is the relaxed (R ) state

The form with a vacant O2 binding site is called deoxyHb and corresponds to the tense (T) state

If iron is in the oxidized state as Fe+3, it is unable to bind O2

R state has a higher affinity for O2

T state is more stable in the absence of O2

conformational change

The subunits slide and rotate making the central cavity smaller

O2-binding curves show Hb saturation as a function of the partial pressure for O2

4 subunits, so 4O2-binding sites: If one heme group has a bound O2, it increases the ability of the other heme groups to bind O2 (last O2

affinity is 300 times greater than its affinity for 1st O2)

Cooperative binding

Segmoidal curve

Myoglobin has a higher O2

affinity than Hb

Myoglobin O2 dissociation curve is hyperbolic

A number of factors reduce the affinity of Hb for O2 so that more O2 is released to tissues

As the curve shifts from A to B (to right) the affinity for O2 decreases

H+, PCO2, and BPG modify the structure of Hb and alter its affinity for oxygen:

Increases of these factors:

◦ Decrease hemoglobin’s affinity for oxygen

◦ Enhance oxygen unloading from the blood

Decreases act in the opposite manner

A number of factors reduce the affinity of Hb for O2 so that more O2 is released to tissues

Increasing temperature also shift the curve to the right

CO2 in blood present in 3 forms:

1. 7% dissolved in plasma

2. 70% travels as HCO3- ions

(hydrogencarbonate ions)

3. 23% travels as carboamino compounds

In red blood cells

CO2 = waste product of cellular metabolism (the end-product)

CO2 reacts directly with Hb to form the carboaminoHb;

reversible reaction

Small quantity of CO2 reacts with plasma proteins -

less significant (quantity of proteins 1/4th that of Hb)

R N H

H

+ CO2 R N H

COO-

+ H+

Carboamino compoundProtein

Dissolved CO2 in blood reacts with water to form Carbonic Acid

CO2 + H2O H2CO3 H+ + HCO3-

Carbonic Anhydrase present inside RBCs (but not plasma) catalyzes this reaction

Carbonic acid rapidly dissociates into ion H+ and bicarbonate ion

Bohr ShiftThe relationship between the binding of O2, H

+, CO2 to hemoglobin (allosteric site), is knowing as Bohr effect

H2CO3

(carbonic acid)

H+

Trapped in cytoplasm

acidification in the red blood cell

Bohr Shift

HCO-3

Leaves the red blood cell

CO2 enters the

blood

Tissues

With high level of activity

Hb

affinity with O2

Release of O2

The dissociation curve moves to the right at higherconcentration of carbon dioxide. This shows that carbondioxide lowers the affinity of Hb for oxygen.

Hb tends to give up O2 in area of high CO2 such as therespiring tissues that need it most.

The build up of hydrogen carbonate ions causesthem to diffuse out of the RBC leaving the inside ofthe RBC positively charged

In order to balance this electric charge chloride ionsdiffuse into the RBCs from the plasma this isknown as the chloride shift

When blood gets to the lungs, all the reactions are reversed

The hydrogen carbonate and hydrogen ions recombine releasing CO2

The chloride shift is reversed

Carbamino-haemoglobin breaks down to release CO2

Figure 27–7

Hemoglobin acts as a buffer in blood by picking up CO2 or H+

In tissues:

Hemoglobin becomes more basic when it is deoxygenated, i.e. it binds H+ more tightly

In the lung:

Hemoglobin is oxygenated, becomes more acidic, (i.e. it is a more powerful H+ donor), and releases its H+

SenescentRBCs

LIVER

Bilirubin diglucuronide(water-soluble)

2 UDP-glucuronic acid

via bile duct to intestine

Stercobilinexcreted in feces

Glucuronic acid is removed and bilirubin is converted to urobilinogen which is then oxidized by intestinal bacteria

KIDNEY

Urobilinexcreted in urine

CO

Biliverdin

Heme oxygenase

O2

Bilirubin (water-insoluble)

NADP+

NADPH

Biliverdinreductase

Heme

Globin

Hemoglobin

reabsorbedinto blood (Portion of

urobilinogen)

Bilirubin (water-insoluble)

via blood to the liver (complexed with albumin)

INTESTINE

Catabolism of hemoglobin

Unconjugated bilirubin Toxic to tissues

Not soluble in aqueous solutions

Tightly complexed to albumin

Cannot be excreted in the urine even when blood levels are high

Conjugated bilirubin Water-soluble

Non-toxic

Loosely bound to albumin

Excreted in urine (bilirubinuria)

Jaundice describes the yellowing of sclera, skin and mucosal membranes due to increased circulating bilirubin in the plasma

This becomes clinically evident when serum bilirubin reaches about 80-100 mol/l.

A. Hemolytic anemia

excess hemolysis

unconjugated bilirubin(in blood)

conjugated bilirubin (released to bile duct)

B. Hepatitis C. Biliary duct stone

unconjugated bilirubin(in blood)

conjugated bilirubin (in blood)

Figure : Examples of hyperbilirubinemia

Hemolytic Jaundice Hepatic jaundice Obstructive jaundice

unconjugated bilirubin(in blood)

conjugated bilirubin secreted by liver into bile

Anemia is due to deficiency of Hb in blood due to lack of erythrocytes and/or their Hbcontent

Normal Hb concentration

o Adult male =14g/dl (14-17)

o Adult female not pregnant = 12g/dl (12-14)

o Adult female pregnant = 11g/dl (11-12)

What is anemia?

The most common symptom of anemia is tiredness.

Other signs and symptoms of anemia include:

1. Weakness,

2. pale skin,

3. brittle nails,

4. Dizziness,

5. irritability.

Excess blood loss due to bleeding

Undernutrition: deficiencies of several vitamins and minerals like vitamins A, B2, B6, B12, C, iron, calcium and folic acid along with protein all of which can cause anaemia.

Pregnancy

Others causes: include worm infestation and chronic disease like AIDS, cancer or kidney disease, cancer treatment, and hereditary diseases

Hemolytic anemia is a disorder in which the red blood cells are destroyed prematurely

RBCs are destroyed faster than the bone marrow can produce them

Extrinsic:

Red blood cells are produced healthy but are later destroyed by becoming trapped in the spleen, destroyed by infection, or destroyed from drugs that can affect red blood cells

intrinsic:

oThe destruction of the red blood cells due to a defect within the red blood cells themselves

o Intrinsic hemolytic anemia is often inherited, such as sickle cell anemia and Glucose-6-Phosphate Dehydrogenase deficiency cells

Sickle Cell anemia is a hereditary disease which causes the body to make abnormally shapes red blood cells (“ C “ form)

Causes complications because the blood cells are not able to reach certain parts of the body

The α chains in mutant Hb (HbS) are the same as in normal Hb (HbA)

A point mutation in the Hb β gene is responsible for the sickling of RBCs seen in sickle cell anemia

Substitution of non polar

valine for a charged Glu

Normal hemoglobin Sickle Cell hemoglobin

No oxygen

No Oxygen: stick together No Oxygen: Separate

No Oxygen

Substitution of non polar valine for a charged Glu

Causes tissue anoxia (Interruption in O2 supply)

This blocking can produce micro vascular occlusions which can cause necrosis (death) of the tissue and pain

(25%)(25%)

(50%)

Do not express the disease symptoms

During electrophoresis, HbS moves slowly towards

anode than HbA at alkaline pH

Lysine replaces glutamic acid at position 6 of the β globin gene.

Mild chronic haemolytic anaemia

Mixture of Sickle hemoglobin (Hb S) + (Hb C)

Thalassemia is inherited disorders characterized reduced or absent amounts of hemoglobin

Two major types of thalassemia:

1. Alpha (α): Caused by defect in rate of synthesis of alpha chains (usually caused by gene deletion)

2. Beta (β): Caused by defect in rate of synthesis in beta chains (usually caused by mutation)

Absence of 1 α gene (silent carrier): no symptoms, may be slightly anemia, does not require therapy

Absence of 2 α gene (α Thalassemia trait): no serious symptoms, except slight anemia

Absence of 3 α genes (Hb H disease): microcytic anemia (small RBC), splenomegaly

Absence of 4 α genes (Hydrops fetalis): most serious form, death before birth

Usually caused by point mutations and short insertions or deletions limited to a few nucleotides

Two situations have clearly to be distinguished: 1. βo thalassemia: No β-globin chain is made

2. β+ thalassemia: decreased β-globin chain is made

Disease results in an over-production of α-globin chains, which precipitate in the cells

Inadequate absorption

Inadequate dietary intake of foods high in Fe

Excess loss of iron due to bleeding, some parasites, menstrual loss and gastrointestinal bleeding

In pregnancy iron is taken from mother by growing fetus, so iron supplement must be taken by pregnant women

Folic Acid (also known as vitamin B9) Deficiency causes megablastic anemia (RBCs that are large and fewer in number)

Deficiency can be due to:

1. Poor dietary intake2. Malabsorption syndromes3. Drugs that inhibit absorption4. Alcohol abuse5. Hemodialysis6. Increased requirement (pregnancy)

Vitamin B12 is a water soluble vitamin with a key role in the normal functioning of the brain and nervous system, and for the formation of blood

Dangerous anemia

It is a type of megablastic anaemia due to malabsorption of Vit B12 (decreased gastric intrinsic factor IF which is needed for absorption of vit B12)

The primary defect is a reduction in ordepletion of hematopoietic precursor stemcells with decreased production of all cell lines.

Aplastic anemia is a severe, life threatening

syndrome in which production of erythrocytes,

WBCs, and platelets has failed.

Aplastic anemia may occur in all age groups

and both genders.

Incidence (acquired)

◦ 2/1000000

◦ rare < 1 year; plateaus 20-60 yrs; increase > 60 yrs

The disease is characterized by peripheral

pancytopenia and accompanied by a

hypocellular bone marrow.