L11 Hemoglobin Structure-Function

L11
Hemoglobin & Myoglobin: Structure and

Functions
Dr. Nelofar Khan
www.gmu.ac.ae COLLEGE OF MEDICINE

Learning Objectives:
At the end of the topic, the student should be able to:
• Describe the structure of normal adult hemoglobin.
• Name the hemoglobin present in the fetal life.
• List the functions of hemoglobin.
• Describe the structure and function of myoglobin.
• Explain the cooperative binding kinetics of hemoglobin.
• Define T and R state of hemoglobin.
• Describe the effect of 2,3 BPG on O2 affinity of hemoglobin.
• Explain why HBF shows decreased sensitivity to 2,3- BPG.
Reading:
Satyanarayana U and Chakrapani U. Biochemistry; Elsevier; 5th Edition; 2020. ISBN- 978-
8131262535. Chapter 10, pages 196 - 200.
Transport and storage of O2 is mediated by two proteins in our body:
1) Hemoglobin (Hb)
2) Myoglobin (Mb)
Hemoglobin is the red blood pigment exclusively present in

erythrocytes (RBC).
Normal concentration of Hb:

In males -- 14 – 16 g/dL
In females -- 13 – 15 g/dL
In the course of the lifetime, 4 different Hb’s
are synthesized in humans
 Hbε: Present in embryonic life ( Hb Gower-1).

Composed of 2 - ζ and 2 - ε chains.
 HbF: Fetal stage (Second trimester till birth). Composed of

2 – α and 2 – γ chains.
 Hb A1: Major normal adult Hb (Starts with birth and

reaches completion by around 6 months). Composed of 2
- α and 2 - β chains.
 Hb A2: Minor normal adult Hb Composed of 2 - α and

2 - δ chains.
Normal adult blood contains (after about 1 year of life):
Hb A1 - 97%
Hb A2 - 2%
Hb F - 1%
• Normal blood contains oxy Hb and deoxy Hb
• Oxidation of the Fe2+ of myoglobin or hemoglobin to Fe3+ destroys

their biologic activity (unable to bind oxygen)
STRUCTURE OF HEMOGLOBIN A1
M.W = 67,000
Conjugated protein: (globin and heme)
Globin (96% of Hb)
 4 polypeptide chains of two different

primary structures:
 2 - α and 2 - β chains
 Each chain has one heme group attached
to it.
Heme (4% of Hb) Hydrophobic & planar structure
 Complex of iron (Fe++) and protoporphyrin IX

 Porphyrin - 4 pyrrole rings
 Fe++: Forms 6 co-ordinated bonds:

4 with the N of porphyrin
On one side of the plane with a.a histidine
(proximal) of the globin
The other side with O2
GLOBIN PROTEINS
• Myoglobin (Mb) - monomeric

• Hemoglobin (Hb) - tetrameric
• Each globin polypeptide is a single chain of ~ 150a.a.
• Each globin molecule contains 1 non-covalently bound heme group in the
hydrophobic pocket.
• Secondary structure – 75% alpha helix arranged in 8 helical segments (Mb & β
chains); α chains – 7 helical segments.
Myoglobin Hemoglobin
• Polar a.a: Located on the exterior surface. Contribute to the high solubility of these
proteins.
• Hydrophobic a.a: Buried within the interior. Stabilize the folding of polypeptide.
Form a hydrophobic pocket for heme.
• Proximal His & distal His play indispensable role in the heme pocket.
• Proximal His - binds directly to the Fe++ of heme.

• The alignment of heme and distal His permits O2 to bind favorably to the Fe++
atom.
• One hemoglobin can bind with 4 molecules of oxygen.

QUARTERNARY STRUCTURE OF HEMOGLOBIN
• Tetramer: α2 β2 subunits.
Organized in a tetrahedral array.
• Multiple noncovalent interactions at the α-β interface.

• Fewer interactions at the α-α or β-β interfaces.
• The actual number and nature of contacts differ in the presence or absence
of O2 and allosteric effectors.
STRUCTURE AND FUNCTION OF MYOGLOBIN
 It is a conjugated protein present in skeletal and cardiac muscles.
 Myoglobin consist of a single polypeptide chain (152 amino acids).
 The polypeptide chain has 1 molecule of heme.
 One molecule of myoglobin can combine with 1 molecule of O2.

TRANSPORT OF OXYGEN BY HEMOGLOBIN
pO2 in the alveolar air = 100 mmHg

pO2 in the lung capillary = 90 mmHg
pO2 of tissue capillaries = 40 mmHg
pO2 in capillary bed of active muscle = 20 mmHg
pO2 of muscle under severe physical exercise = 5mmHg
 In the lungs (pO2 is high): Hb gets fully loaded with O 2.
 At the tissue level (pO2 is low): Oxyhemoglobin releases its O 2 for cellular
respiration.
FUNCTIONS OF HEMOGLOBIN
(i) Hb is responsible for transport of O2 from lungs to tissues.
(ii) Also helps to transport CO2 from tissues to lungs for exhalation.
(iii) It acts as a buffer (38 Histidines).

Oxygen Dissociation / Saturation Curve
 The binding ability of Hb with O2

at different partial pressures of O2
(pO2) can be measured by this
graphic representation.
 The ODC for myoglobin is

hyperbolic in shape.
 The ODC for Hemoglobin is

sigmoid in shape.
P50 : Value of PO2 at which half the ligand
sites are occupied (saturated by O2).
Mb has High affinity for O2.

P50 = 4mmHg
Hb A P50 = 26mmHg
Association of chains into a tetrameric

structure (hemoglobin) results in much
greater oxygen delivery than would be
possible with single chains.
Why is myoglobin unsuitable as an O2 transport protein but well
suited for O2 storage?
• The oxygen-binding curve for myoglobin is hyperbolic.
• Myoglobin loads O2 readily at the PO2 of the lung capillary bed (100 mmHg).
• However, since myoglobin releases only a small fraction of its bound O 2 at

the PO2 values typically encountered in active muscle (20 mmHg) or other
tissues (40 mmHg), it represents an ineffective vehicle for delivery of O 2.
• When strenuous exercise lowers the PO 2 of muscle tissue to about 5 mmHg,

the dissociation of O2 from myoglobin permits mitochondrial synthesis of ATP,
and hence muscular activity, to continue.
Hb carries O2 from lungs to tissue capillaries. From there O2 diffuses into tissues.
 In the muscles:
 O2 is taken up and stored by Mb.
 Mb has high affinity for O2 than Hb.
 It cannot deliver bound O2 even at 20 mmHg.
 Under severe physical exercise:

 pO2 in muscle lowers to as little as 5 mmHg.
 So, Mb Releases the bound O2 for use by muscle mitochondria to generate
energy.
 Mb acts as an O2 storage protein in muscles.

The allosteric properties of hemoglobins result from their
quaternary structures
• The properties of individual hemoglobin’s are consequences of their quaternary

as well as of their secondary and tertiary structures.
• The quaternary structure of hemoglobin confers striking additional properties,

absent from monomeric myoglobin, which adapts it to its unique biologic roles.
CO-OPERATIVE BINDING
 The ODC for Hb is sigmoidal in shape. This indicates that binding of O2 to

Hb is cooperative.
 Binding of O2 to one heme increases binding affinity of O2 to other hemes of
the same Hb molecule. It occurs because there is a communication
among heme groups in the hemoglobin function.
 Cooperative interactions is an exclusive property of multimeric proteins.
 On oxygenation, the iron moves into the plane of
heme.
 It’s accompanied by pulling of proximal histidine and

breaking of salt bonds.
OVERALL EFFECT
 Binding of O2 to one heme distorts the globin chain

to which it is attached.
 This causes a distortion in the neighboring globin

chain and its heme.
 The result is that O2 can bind more easily to it.

Deoxyhemoglobin exists in a T (Tense) form. The hydrogen and ionic
bonds limit the movement of monomers; therefore, it has low affinity for
O2 .
The binding of O2 destabilizes these bonds resulting in a R (relaxed)

form of Hemoglobin. R form has high affinity for O 2.
EFFECT OF 2,3 – BISPHOSPHOGLYCERATE ON
AFFINITY OF Hb FOR O2
• A low PO2 in peripheral tissues promotes the synthesis of 2,3-
bisphosphoglycerate (BPG) in erythrocytes.
• 2,3 – Bisphosphoglycerate (BPG) is produced in erythrocytes

from an intermediate of glycolysis.
• It specifically binds to deoxy Hb at the center and decreases

the affinity of Hb for O2. This facilitates the release of O2 at
the partial pressure found in the tissues.
• One molecule of 2,3 – BPG binds with one molecule of

deoxy Hb. It stabilizes it by cross linking the β chains.
• On oxygenation of Hb, 2,3 – BPG is expelled from the pocket

and oxy Hb attains the R-form.
Difference between HbF & HbA
• HbF shows decreased sensitivity to 2,3 BPG.
Reason:
• Replacement of His-143 of β chain by Serine in γ.
• Two of the cationic groups that participate in the binding of 2,3-BPG are no longer present.
• Interaction of 2,3-BPG with HbF is weaker resulting in increased affinity for O2.
• It results in more efficient transfer of O2 from maternal HbA to fetal HbF.

Adaptation to high altitude
Prolonged exposure to high altitude include:

increase in RBC number, their conc. of hemoglobin and BPG.
Elevated BPG lowers the affinity of HbA for oxygen (decreases P50),
which enhances release of O2 at the tissues.
1. MYOGLOBIN : Storage
Conjugated protein
Single polypeptide chain + 1 heme.
One Mb can bind 1 O2.
O2 storage protein in muscles.
3. Cooperative Binding of O2.
ODC for myoglobin is hyperbolic in shape.
4. Deoxyhemoglobin exists in a T form. It has
2. HEMOGLOBIN : Transport
low O2 affinity.
Conjugated protein : globin and heme.
5. The binding of O2 destabilizes these
Globin : 4 polypeptides ( 2 α and 2 β). bonds resulting in a R form of Hb. R
Each chain has one heme group. form has high O2 affinity.
Heme: (Complex of iron and porphyrin) 6. 2,3 – BPG: Binds to deoxy Hb and
decreases the O2 affinity to Hb.
Iron (Fe++) forms 6 co-ordinated bonds.
• The levels of 2,3 – BPG are related to
The ODC for Hb is sigmoidal in shape. tissue demands of O2 supply eg
Hypoxia, Anemia.
Thank You

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L11

Hemoglobin & Myoglobin: Structure and

Dr. Nelofar Khan

www.gmu.ac.ae COLLEGE OF MEDICINE

Hemoglobin is the red blood pigment exclusively present in

Normal concentration of Hb:

 Hbε: Present in embryonic life ( Hb Gower-1).

 HbF: Fetal stage (Second trimester till birth). Composed of

 Hb A1: Major normal adult Hb (Starts with birth and

 Hb A2: Minor normal adult Hb Composed of 2 - α and

• Normal blood contains oxy Hb and deoxy Hb

• Oxidation of the Fe2+ of myoglobin or hemoglobin to Fe3+ destroys

Globin (96% of Hb)

 4 polypeptide chains of two different

 Complex of iron (Fe++) and protoporphyrin IX

 Fe++: Forms 6 co-ordinated bonds:

• Myoglobin (Mb) - monomeric

• Proximal His - binds directly to the Fe++ of heme.

• One hemoglobin can bind with 4 molecules of oxygen.

• Multiple noncovalent interactions at the α-β interface.

 It is a conjugated protein present in skeletal and cardiac muscles.

 Myoglobin consist of a single polypeptide chain (152 amino acids).

 The polypeptide chain has 1 molecule of heme.

 One molecule of myoglobin can combine with 1 molecule of O2.

pO2 in the alveolar air = 100 mmHg

 In the lungs (pO2 is high): Hb gets fully loaded with O 2.

(i) Hb is responsible for transport of O2 from lungs to tissues.

(iii) It acts as a buffer (38 Histidines).

 The binding ability of Hb with O2

 The ODC for myoglobin is

 The ODC for Hemoglobin is

Mb has High affinity for O2.

Association of chains into a tetrameric

• However, since myoglobin releases only a small fraction of its bound O 2 at

• When strenuous exercise lowers the PO 2 of muscle tissue to about 5 mmHg,

 Under severe physical exercise:

 Mb acts as an O2 storage protein in muscles.

• The properties of individual hemoglobin’s are consequences of their quaternary

• The quaternary structure of hemoglobin confers striking additional properties,

 The ODC for Hb is sigmoidal in shape. This indicates that binding of O2 to

 It’s accompanied by pulling of proximal histidine and

 Binding of O2 to one heme distorts the globin chain

 This causes a distortion in the neighboring globin

 The result is that O2 can bind more easily to it.

The binding of O2 destabilizes these bonds resulting in a R (relaxed)

• 2,3 – Bisphosphoglycerate (BPG) is produced in erythrocytes

• It specifically binds to deoxy Hb at the center and decreases

• One molecule of 2,3 – BPG binds with one molecule of

• On oxygenation of Hb, 2,3 – BPG is expelled from the pocket

• HbF shows decreased sensitivity to 2,3 BPG.

• It results in more efficient transfer of O2 from maternal HbA to fetal HbF.

Prolonged exposure to high altitude include:

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