12
Haemoglobin 2
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
Mr(tetramer) = 64 000 Average values of pO2:
Concentration in blood: 2.15­2.65 mmol/L (tetramer) Alveoli of the lung: 13­15 kPa
Binding of oxygen: at totals saturation 4 mol O2/mol Hb Arterial blood: 9­13 kPa
Saturation of Hb with oxygen: arterial blood ~ 0.97 Mixed venous blood: > 5 kPa
venous blood ~ 0.73 (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 mL)
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 Structure of Haem
of Globin Chain
4. Characterize the secondary and tertiary structure of globin chain.
5. Characterize the structure of haem and its bindings to the globin chain.
13
Binding of Oxygen to Haem
Electronic configuration of Fe2+
(complete)
26Fe2+
1s2
...................................................
High spin state (the number of coordination 5) Low spin state (the number of coordination 6)
6. What 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 R-Conformation
3d 3d
N
N
F-helix His F8
N
N
F-helix His F8
FeII
FeII
O
O
His E7
Porphyrin
plane
2 H2O + 2 CO2
1
2
2 1
+
+
+
+
+
+
-
+-
+
-
-+
+
1
2
12
4 O2
4 O2 2 H+
+ 2,3-BPG
2 H+
+ 2,3-BPG
2 H2CO3
2 H+
+ 2 HCO3
2
H2CO32 H2O + 2 CO2
Tissues
Lungs
2 H+
+ 2 HCO3
-
O2
O2
O2
O2
Carbonic anhydrase
Carbonic anhydrase
14
SaturationofHb(%)
8. Describe the main types of non-covalent interactions between haemoglobin subunits in
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
HHbO2 HbO2 + H+
pKA ~ 6.2
12. Which of the two forms of haemoglobin (Hb or HbO2) 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
14. Mark areas corresponding to the pO2 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 character
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 pCO2 d) increase of temperature
15
Types of Human Haemoglobin
Type Structure Proportion of the total Hb in adults
HbAo 2 2 (partly HbA-Glc)
HbA ~ 97 %
HbA1 2 2 (glycation on terminal ­NH2 group of -globin)
HbA2 2 2 ~ 2.5 %
HbF 2 2 ~ 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 O2; CO2 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
22. Which factors will affect the amount of glycated haemoglobin?
O
CH2 HN CH2 Protein
Glycated protein
+
H2O
neenzymová reakce
irreverzibilní
aldimin
(labilní)
Amadoriho
p esmyk
C
H O
C OHH
R
Glc
C
H
C OHH
N CH2
Protein
R
glykovaný protein
ketoamin
aminoderivát Fru
C
C O
N CH2H2
Protein
R
H
H2N CH2 Protein
Non-enzymic reaction
(irreversible)
Amadori
rearrangement
Ketamine
Aminoderivative
of fructose
Aldimine
(labile)
16
Glycated Haemoglobin
HbA1 glycation on terminal ­NH2 (Val) group of -chains
4­6 % of the total HbA
HbA-Glc glycation in other sites of Hb: e.g. terminal -NH2 group of -globin or
at -NH2 (Lys) of -globin
Inherited Abnormalities of Haemoglobin Synthesis
Causes
Point mutation Absent or defective synthesis of one
of the Hb chains
Abnormal haemoglobins -Thalassaemia -Thalassaemia
(haemoglobinopathies, more than 200 forms)
Examples: HbS 2 2
6 Glu Val
HbC 2 2
6 Glu Lys
HbM 2 2
.. His Tyr
2 2
67 Val Glu
23. What is the molecular principle of sickle cell anaemia?
24. What is the cause of sickle shape of erythrocytes?