Haemoglobin
Globin - conformation of the molecule, types of chains, interactions between the chains. Haem - structure, binding of oxygen, arrangement in oxygenated and deoxygenated state, binding to globin chain. Haemoglobin (Hb) - binding 2,3-BPG, Bohr effect, allosteric interactions, saturation curve, physiological and abnormal types of haemoglobin. Glycation of proteins.
Haemoglobin
M-(tetramer) = 64 000
Concentration in blood: 2.15-2.65 mmol/L (tetramer) Binding of oxygen: at totals saturation 4 mol 02/mol Hb Saturation of Hb with oxygen:   arterial blood   ~ 0.97
venous blood   ~ 0.73
Average values ofp02: Alveoli of the lung:      13-15 kPa Arterial blood: 9-13 kPa
Mixed venous blood: > 5 kPa (Critical value for hypoxia 3.5 kPa)
1. Give the mass and molar concentration of Hb related to the Hb monomer.
2. Calculate the maximal volume of oxygen that can bind to 1 g of Hb. (1.4 mLj
3. When isolated haem reacts with oxygen, the oxidation of Fe2+ to Fe3+ occurs. Describe the reaction of oxygen with haemoglobin. Explain the difference.
Secondary and Tertiary Structure of Globin Chain
Structure of Haem
4. Characterize the secondary and tertiary structure of globin chain.
5. Characterize the structure of haem and its bindings to the globin chain.
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Binding of Oxygen to Haem
Electronic configuration of Fe2+ (complete)
->r,Fe2+ Is2.
High spin state (the number of coordination 5) 3d
F-helix His F8
-N
t (Fe11
Low spin state (the number of coordination 6)
3dnmnr
Porphyrin plane
F-helix^
His F8
)r-N
0
10.
........His E7
6. Wliat change in haem structure is triggered by binding of oxygen?
7. What change in deoxyHb subunit conformation results from it?
Quaternary Structure of Haemoglobin
T-Conformation 2 FFO + 2 CO-. «-
Lungs
2 H2C03 R-Conformation
Carbonic anhydia.se
2 H+ + 2 HCO3"
4 02 2 H+ + 2,3-BPG 4 02        2 H+ + 2,3-BPG
Tissues
2 H+ + 2 HCO3"
Carbonic anhyclrase
2 FFO + 2 C02-►   2 H2C03
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8. Describe the main types of non-covalent interactions between haemoglobin oxygenated and deoxygenated state.
9. Give the formula of 2,3-bisfosfoglycerate and mark its binding in the T-form of Hb.
10. What is the principle of the Bohr effect?
11. Explain, why is the affinity of Hb to oxygen decreased in the presence of 2,3-BPG.
Dissociation of Haemoglobin
HHb Hb" + H+ pKA~7.8
HHb02 ,.    Hb02" + H+ pATA -6.2
12. Which of the two forms of haemoglobin (Hb or Hb02) is stronger acid?
13. Which of the amino acids is responsible for acid base properties of haem at physiological pH?
Saturation of Haemoglobin by Oxygen
Saturation curve of haemoglobin
p02 (kPa)
14. Mark areas corresponding to the ;;02 in alveoli of lungs and mixed venous blood in the graph What is the saturation of Hb in % at these pressures?
15. Complete the saturation curve for myoglobin into the graph. Explain the differences in charactei of the both curves. Which of the both proteins binds oxygen more tightly?
16. The binding of oxygen to haemoglobin has cooperative character. Explain it.
17. On the saturation curve for Hb mark changes resulting from:
a) lowering of the pH c) decrease of 2,3-BPG concentration
b) decrease of pC02 d) increase of temperature
subunits in
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Types of Human Haemoglobin
Type	Structure	Proportion of the total Hb in adults
HbA0	a2 p2   (partly HbA-Glc)	
HbA		~ 97 %
HbA,	a2 P:   (glycation on terminal	-NH2 group of P-globin)
HbA2	a2 52	~ 2.5 %
HbF	a2 y2	~ 0.5 %
18. Compare the affinities of Hb and HbF to oxygen. What is the cause of this difference? What is its significance?
Derivatives of Haemoglobin
19. Name the derivatives of haemoglobin formed after: a) binding of 02; C02 and CO; b) oxidation.
20. What are the most common causes of CO poisoning? How can be this poisoning detected? What is the first aid in this case?
21. Explain what methaemoglobinemia is and what may cause this disturbance.
Glycation of Haemoglobin
Principle of non-enzymatic glycation H
I
HC—OH
H2N—CH2— Protein
R
Glc
H,0 <+J
Non-enzymic reaction (irreversible)
\    , N—CH2—[ Protein
HC—OH R
Aldimine (labile)
Amadori rearrangement
Protein
H
H2 C—N—CH2-C=0
p Ketamine
Aminoderivative of fnictose
-O
CH^HN-CH— Protein
Glycated protein
22. Which factors will affect the amount of glycated haemoglobin?
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Glycated Haemoglobin
HbA|  glycation on terminal -NH2 (Val) group of p-chains
4-6% of the total HbA
HbA-GIc glycation in other sites of Hb: e.g. terminal -NH2 group of a-globin or
at g-NH2 (Lys) of a, P-globin
Inherited Abnormalities of Haemoglobin Synthesis
Causes
Point mutation Absent or defective synthesis of one
of the Hb chains
Abnormal haemoglobins ct-Thalassaemia P-Thalassaemia
(haemoglobinopathies, more than 200 forms)
Examples: HbS a2p26Glu^Va' HbC a2p26G,u^Lya HbM a2p2'His">Tyr
23. What is the molecular principle of sickle cell anaemia?
24. What is the cause of sickle shape of erythrocytes?
a2 p2
67 Val -> Olu
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