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Blood chemistryHb Structure & FunctionsHb Structure & Functions

Dr. Vishnu Kumar AwasthiDr. Vishnu Kumar Awasthi

Assistant Professor – In – Charge HLS, Assistant Professor – In – Charge HLS, Department of BiochemistryDepartment of Biochemistry

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ObjectivesObjectives Structure of hemoglobinStructure of hemoglobin Transport of oxygen by HbTransport of oxygen by Hb Oxygen dissociation curve (ODC)Oxygen dissociation curve (ODC) Factors affecting ODCFactors affecting ODC Heme-heme interaction and co-operativityHeme-heme interaction and co-operativity Effect of pH and pCO2Effect of pH and pCO2 The Bohr EffectThe Bohr Effect The chloride shiftThe chloride shift Effect of temperature Effect of temperature Effect of 2,3-BPGEffect of 2,3-BPG Transport of COTransport of CO22

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HaemoglobinHaemoglobin structurestructure

Haemoglobin (Hb) is the most abundant Haemoglobin (Hb) is the most abundant porphyrin – containing compound.porphyrin – containing compound.

It is a tetramer made up of four subunits.It is a tetramer made up of four subunits.

Each subunit contains a heme group Each subunit contains a heme group and a polypeptide chain.and a polypeptide chain.

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Haemoglobin structureHaemoglobin structure

Normal level of Hb in blood of males is 14 – 16 Normal level of Hb in blood of males is 14 – 16 g/dl and in females, 13 – 15 g/dl. g/dl and in females, 13 – 15 g/dl.

Normal adult blood contains 97% HbA, about 2% Normal adult blood contains 97% HbA, about 2% HbAHbA22 and about 1% HbF. and about 1% HbF.

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CHHC

NH

CN

CHHCNH

C

HC

CCN

HC

HC CH

HN

CC CH

CHC

CH

HC

HC

HC C

CH

CH

PyrrolePorphyrin

What is Porphyrin ?

N

HemeHeme

Heme = Porphyrin + ironHeme = Porphyrin + iron

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Hemoglobin is a OHemoglobin is a O22 and CO and CO22 transport transport protein found in the RBCsprotein found in the RBCs

Hemoglobin is an oligomeric protein made Hemoglobin is an oligomeric protein made up of 2 α β dimers, a total of 4 polypeptide up of 2 α β dimers, a total of 4 polypeptide chains: αchains: α11 β β11 α α22 β β22..

Total Total Mr Mr of hemoglobin is 64,500.of hemoglobin is 64,500.

The α (141 aa) and β (146 aa) subunits The α (141 aa) and β (146 aa) subunits have < 50 % identity.have < 50 % identity.

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• • The 3D- structures of α (141 aa) and β The 3D- structures of α (141 aa) and β (146 aa) subunits of hemoglobin and the (146 aa) subunits of hemoglobin and the single polypeptide of myoglobin are verysingle polypeptide of myoglobin are very

similar; all three are members of the similar; all three are members of the globin family.globin family.

• • Each subunit has a haem-binding pocketEach subunit has a haem-binding pocket

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The polypeptide chains are of five types The polypeptide chains are of five types viz. α, β, γ, δ and εviz. α, β, γ, δ and ε

The α chain is made up of 141 amino The α chain is made up of 141 amino acids.acids.

The β , γ, δ and ε chains are made up The β , γ, δ and ε chains are made up of 146 amino acid residues each.of 146 amino acid residues each.

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Normal adult haemoglobin Normal adult haemoglobin (HbA)(HbA) is is made up of four haem groups, two α made up of four haem groups, two α chains and two β chains, and is chains and two β chains, and is represented as α2 β2.represented as α2 β2.

A small amount of HbA2 is also found in A small amount of HbA2 is also found in adults which is α2 δ2.adults which is α2 δ2.

Foetal haemoglobin (HbF) is Foetal haemoglobin (HbF) is α2 γ2α2 γ2

Embryonic haemoglobin isEmbryonic haemoglobin is α2 ε2α2 ε2

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The histidine residues linked to iron are The histidine residues linked to iron are present at positions 58 and 87 in α chains present at positions 58 and 87 in α chains and at positions 63 and 92 in other chains.and at positions 63 and 92 in other chains.

The bond between iron and the distal The bond between iron and the distal histidine residue (His87 or His92) is histidine residue (His87 or His92) is unstable.unstable.

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The distal iron-histidine bond is broken The distal iron-histidine bond is broken when haemoglobin is exposed to high when haemoglobin is exposed to high oxygen tensionoxygen tension

This results in the formation of an iron-This results in the formation of an iron-oxygen bondoxygen bond

The binding of oxygen to haemoglobin changes The binding of oxygen to haemoglobin changes the conformation of haemoglobinthe conformation of haemoglobin

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Two conformations have been described, T Two conformations have been described, T (taut) and R (relaxed).(taut) and R (relaxed).

Deoxygenated Hb exists in T form which is Deoxygenated Hb exists in T form which is stabilised by 2,3-bisphosphoglycerate (2,3-BPG) stabilised by 2,3-bisphosphoglycerate (2,3-BPG) which is formed from 1, 3-BPG (an intermediate in which is formed from 1, 3-BPG (an intermediate in glycolytic pathway) when there is a deficiency of glycolytic pathway) when there is a deficiency of oxygen in the tissues.oxygen in the tissues.

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T and R states of HemoglobinT and R states of Hemoglobin • • HemoglobinHemoglobin exists in two major exists in two major

conformationalconformational states: Relaxed (R ) and Taut or Tense (T)states: Relaxed (R ) and Taut or Tense (T) • • R state has a higher affinity for O2.R state has a higher affinity for O2. • • In the absence of O2, T state is more stable; In the absence of O2, T state is more stable;

when O2when O2 binds, R state is more stable, so hemoglobinbinds, R state is more stable, so hemoglobin undergoes a conformational change to the R undergoes a conformational change to the R

state.state. • • The structural change involves readjustment The structural change involves readjustment

of interactions between subunitsof interactions between subunits

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There is a central cavity in the haemoglobin There is a central cavity in the haemoglobin molecule surrounded by the four polypeptide chains.molecule surrounded by the four polypeptide chains.

2,3-BPG enters this cavity and cross links the two β 2,3-BPG enters this cavity and cross links the two β chains.chains.

When oxygen tension increases, 2,3-BPG is When oxygen tension increases, 2,3-BPG is displaced and the T form changes into R form.displaced and the T form changes into R form.

During this transition, one pair of α and β subunitsDuring this transition, one pair of α and β subunits rotates by 15° relative to the other pair.rotates by 15° relative to the other pair.

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Each subunit of haemoglobin can bind one Each subunit of haemoglobin can bind one oxygen molecule.oxygen molecule.

Since there are four subunits in a molecule Since there are four subunits in a molecule of haemoglobin, one molecule can bind of haemoglobin, one molecule can bind four oxygen molecules.four oxygen molecules.

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Transport of oxygen by HbTransport of oxygen by Hb

Hb has all the requirements of an idealHb has all the requirements of an ideal

respiratory pigment:respiratory pigment: It can transport large quantities of oxygenIt can transport large quantities of oxygen It has great solubilityIt has great solubility It can take up an release oxygen at It can take up an release oxygen at

appropriate partial pressureappropriate partial pressure It is powerful buffer.It is powerful buffer.

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Oxygen dissociation curveOxygen dissociation curve

i. The ability of Hb to load and unload i. The ability of Hb to load and unload oxygen at physiological pOoxygen at physiological pO22 is shown by is shown by

oxygen dissociation curve (ODC)oxygen dissociation curve (ODC)

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AA

Per

cent

age

Per

cent

age

satu

rati

onsa

tura

tion

pO2 in mm of Hg

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BB

Per

cent

age

Per

cent

age

satu

rati

onsa

tura

tion

pO2 in mm of Hg

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CC

Per

cent

age

Per

cent

age

satu

rati

onsa

tura

tion

pO2 in mm of Hg

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DD

Per

cent

age

Per

cent

age

satu

rati

onsa

tura

tion

pO2 in mm of Hg

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A. A. Theoretical curve as per mass action.Theoretical curve as per mass action.

B. Sigmoid curve, due to heme-heme interaction (hill B. Sigmoid curve, due to heme-heme interaction (hill effect).effect).

C. C. Further shift to right due to carbon dioxide (Bohr Further shift to right due to carbon dioxide (Bohr effect) and BPG. This curve represents the pattern effect) and BPG. This curve represents the pattern under normal conditions.under normal conditions.

D. D. further shift to right when temp is increased to further shift to right when temp is increased to 424200C.C.

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ii. At the oxygen tension in the pulmonary ii. At the oxygen tension in the pulmonary alveoli, the Hb is 97% saturated with alveoli, the Hb is 97% saturated with oxygen. Normal blood with 15 gm/dl of Hb oxygen. Normal blood with 15 gm/dl of Hb can carry 20 ml of oxygen /dl of blood.can carry 20 ml of oxygen /dl of blood.

iii.iii. In the tissue capillaries, where the pO2 In the tissue capillaries, where the pO2 is only 40 mmHg, theoretically Hb is only 40 mmHg, theoretically Hb saturation is 75%. Thus under STP saturation is 75%. Thus under STP conditions, blood can release only 22%.conditions, blood can release only 22%.

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Factors affecting ODCFactors affecting ODC

1.1. Heme-heme interaction and co-Heme-heme interaction and co-operativity:-operativity:-

AA. the sigmoid shape of ODC – due to . the sigmoid shape of ODC – due to allosteric effect, or co-operativity.allosteric effect, or co-operativity.

equilibrium of Hb=O2 equilibrium of Hb=O2

Hill equation (A V Hill, nobel prize,1922)Hill equation (A V Hill, nobel prize,1922)

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B. Positive co operativityB. Positive co operativity

HbHb HbOHbO22 HbOHbO44 HbOHbO66 HbOHbO88

Homotropic interactionHomotropic interaction

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c. c. Each successive addition of OEach successive addition of O22, increase , increase

the affinity of Hb to Othe affinity of Hb to O22 synergistically. synergistically.

D. Similarly, binding of 2, 3 – BPG at a site D. Similarly, binding of 2, 3 – BPG at a site other than the oxygen binding site, lowers other than the oxygen binding site, lowers the affinity for oxygen (heterotropic the affinity for oxygen (heterotropic interaction).interaction).

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Alteration of structureAlteration of structureDiagrammatic representation of subunit interaction in Hemoglobin

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During oxygen uptake, the T form to the R During oxygen uptake, the T form to the R form with disruption of the salt bridges . form with disruption of the salt bridges .

The Hb subunits are moved relative to one The Hb subunits are moved relative to one another.another.

During oxygenation, the During oxygenation, the αα11 - - ββ22 interface interface shows movement.shows movement.

The two subunits slip over each other. The two subunits slip over each other. The quaternary structure of oxy Hb is The quaternary structure of oxy Hb is

described as described as R formR form; and that of de-oxy Hb ; and that of de-oxy Hb is is T form. T form.

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When oxygenation occurs the salt When oxygenation occurs the salt

bonds are broken successively. Thus on bonds are broken successively. Thus on

oxygenation, the Hb molecule can form oxygenation, the Hb molecule can form

two similar dimers.two similar dimers.

(2x alpha)+(2x beta)→2x(alpha-beta)(2x alpha)+(2x beta)→2x(alpha-beta)

(Deoxy-Hb) (oxy-Hb(Deoxy-Hb) (oxy-Hb))

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3. The Bohr Effect3. The Bohr Effecti.i. The influence of pH and pCOThe influence of pH and pCO2 2 to facilitate to facilitate

oxygenation of Hb in the lungs and oxygenation of Hb in the lungs and deoxygenation at the tissues is known as the deoxygenation at the tissues is known as the Bohr effect (1904).Bohr effect (1904).

ii.ii. Binding of COBinding of CO22 forces the release of O forces the release of O22

iii.iii. When the pCOWhen the pCO22 high, CO high, CO22 diffuses into the diffuses into the RBCsRBCs

COCO2 2 + H+ H22O O → → HH22COCO33 → → HH++ + HCO + HCO33--

Carbonic Carbonic

AnhydraseAnhydrase

Iv. When carbonic acid is ionizes, the intracellular Iv. When carbonic acid is ionizes, the intracellular pH falls. The affinity of Hb for oxygen is pH falls. The affinity of Hb for oxygen is decreased and oxygen is unloadeddecreased and oxygen is unloaded

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4. The chloride shift4. The chloride shift

CO2H2O + CO2

H2CO3

Carbonic anhydrase

HCO3-

H+

HHb +O2

Cl-

Cl shift (in tissues)

Cl-

HCO3-

O2- To cells

When CO2 is taken up ----When CO2 is taken up ----HCO3- ↑HCO3- ↑

N N

HbO2

Chloride enters into RBC

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When the blood reaches the lungs, reverse When the blood reaches the lungs, reverse reaction takes placereaction takes place

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4. The chloride shift4. The chloride shift

CO2H2O + CO2

H2CO3

Carbonic anhydrase

HCO3-

H+

HHb +O2

Cl-

Cl shift (in lungs)

Cl-

HCO3-

O2- Air

When O2 is taken up ----When O2 is taken up ----

N N

HbO2

Chloride comes out of RBC

Air

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5. Effect of temperature5. Effect of temperature

p50 = the pOp50 = the pO22 at which Hb is half saturated at which Hb is half saturated

p50 of normal Hb = 26 mmHg (at 37p50 of normal Hb = 26 mmHg (at 37ooC)C) Elevation of temp. causes 88 % increase in Elevation of temp. causes 88 % increase in

p50p50 ODC shifts to ODC shifts to left left at low temp.at low temp. Under febrile conditions , increased needs Under febrile conditions , increased needs

of oxygen met by a shift in ODC to of oxygen met by a shift in ODC to right.right.

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6. Effect of 2,3-BPG6. Effect of 2,3-BPG

Normal 2,3-BPG level=15 ± 1.5 mg/g Hb.Normal 2,3-BPG level=15 ± 1.5 mg/g Hb. 2,3-BPG == high in children 2,3-BPG == high in children 2,3-BPG is produced from 1,3-BPG, an 2,3-BPG is produced from 1,3-BPG, an

intermediate of glycolytic pathway.intermediate of glycolytic pathway. 2,3-BPG, preferentially binds to deoxyHb 2,3-BPG, preferentially binds to deoxyHb

and stabilizes T formand stabilizes T form When T form reverts to R, 2, 3-BPG ejected When T form reverts to R, 2, 3-BPG ejected During oxygenation, BPG releasedDuring oxygenation, BPG released

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Transport of COTransport of CO22

At rest, about 200 ml of COAt rest, about 200 ml of CO2 2 is produced is produced

/minute in tissues./minute in tissues.

The COThe CO2 2 is carried by the following 3 ways:-is carried by the following 3 ways:-

1. Dissolved form:1. Dissolved form:

COCO2 2 + H+ H22O HO H22COCO33 HCO HCO33-- + H + H++

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2. Isohydric transport of CO2. Isohydric transport of CO22

1.1. Haldane effect: The H+ ions are buffered by Haldane effect: The H+ ions are buffered by the deoxyhemoglobin. the deoxyhemoglobin.

2.2. In tissueIn tissue

3.3. Oxy-Hb is more Oxy-Hb is more – – (negatively) charged than (negatively) charged than deoxy-Hbdeoxy-Hb

4. 4. In the In the LungsLungs

H - Hb + 4OH - Hb + 4O2 2 Hb ( Hb (OO22)4 + H)4 + H++

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5. 5. The proton released in the RBC combine The proton released in the RBC combine with HCOwith HCO33

- - forming Hforming H22COCO3 3 which would which would

dissociate to COdissociate to CO22, that is expelled through , that is expelled through

pulmonary capillaries.pulmonary capillaries.

6. 6. As the HCOAs the HCO33- - level inside the erythrocytes level inside the erythrocytes

falls, more and HCOfalls, more and HCO33-- gets into the RBC, gets into the RBC,

and chloride diffuse out.and chloride diffuse out.

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3. Carriage as carbamino- Hb3. Carriage as carbamino- HbR-NH2 + CO2 R-NH-COOHR-NH2 + CO2 R-NH-COOH

4.4. Clinical Applications.Clinical Applications.

1-1- Hypoxic states,O Hypoxic states,O22 affinity decreased. affinity decreased.

- ODC shift right.- ODC shift right.

- increased in 2,3-BPG increased in RBC.- increased in 2,3-BPG increased in RBC.

2. In anemia, increased oxygen unloading will 2. In anemia, increased oxygen unloading will ensure proper oxygenation of tissues.ensure proper oxygenation of tissues.

3. 2,3-BPG level varies as Hb conc. 3. 2,3-BPG level varies as Hb conc.

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4. The Red cell 2,3-BPG level is decreased in 4. The Red cell 2,3-BPG level is decreased in acidosis and increased in alkalosis, ODCacidosis and increased in alkalosis, ODC

shift to right.shift to right.5. Transfusion of large vol. of stored blood 5. Transfusion of large vol. of stored blood

which has a low level of 2,3-BPG can lead which has a low level of 2,3-BPG can lead sudden hypoxia and a left shifted ODC.sudden hypoxia and a left shifted ODC.