1. Myoglobin and hemoglobin
Lecture 3
Dr. Malik ALQUB MD. PHD.

2. Hemoglobin and Myoglobin
First protein to be crystallized
First protein to have its mass accurately measured.
First protein to be studied by ultracentrifugation.
First protein to be associated with function
First protein to show that a point mutation can cause a disease.
First proteins to have his X-ray structure.

3. Structure of myoglobin

4. The heme group
Fe in Mb is Fe2+ - ferrous iron - the form that binds oxygen +
Protoporphyrin IX

5. Overveiw of heme synthesis
Protoporphyrin IX
Succinyl CoA + Glycine
ALA synthase
Protoporphyrinogen IX
-aminolevulinic acid
Coproporphyrinogen III
mitochondrial matrix
cytoplasm
-aminolevulinic acid
Porphobilinogen
Uroporphyrinogen III
Coproporphyrinogen III
Uroporphyrinogen I
Coproporphyrinogen I
Heme synthesis occurs in all cells due to the requirement for heme as a prosthetic group on enzymes and electron transport chain. By weight, the major locations of heme synthesis are the liver and the erythroid progenitor cells of the bone marrow.

6. Binding of oxygen induce conformational changes

7. Binding of oxygen induce conformational changes

8. The Conformation Change
Oxygen binding changes the Mb conformation
Without oxygen bound, Fe is out of heme plane
Oxygen binding pulls the Fe into the heme plane
Fe pulls its His F8 ligand along with it
The F helix moves when oxygen binds
Total movement of Fe is nm A
This change means little to Mb, but lots to Hb!

9. Hemoglobin structure

10. This reaction is reversible.
How is oxygen carried ?
Complex protein containing heme (iron)
1 molecule of hemoglobuline combines with 4 molecules of oxygen to form oxyhaemoglobin
Hb + O2 HbO8
Haemoglobin
oxygen
oxyhaemoglobin
This reaction is reversible.

11. Cooperativity
The haemoglobin molecule is a tetramer composed of 4 protein subunits, each of which can bind a single molecule to O2
These subunits exhibit cooperativity when they bind to O2
i.e. the binding of O2 to any subunits increases the likelihood that the other subunits will bind to O2

12. Hemoglobin structure

13. Oxygen-Hemoglobin Dissociation Curve
Shows the amount of O2 that is bound to haemoglobin (Y-axis) as a function of the partial pressure of O2 in the blood plasma (X-axis).
Because of the cooperativity, this dissociation curve is S-shaped

14. Significance of the S-shape curve
100%
Plateau:
► haemoglobin highly saturated
► favour the loading of O2 in lung
% saturation of haemoglobin
Steep slope:
► small drop of O2 partial pressure leads to a rapid decrease in % saturation of haemoglobin
► favour the release of O2 in tissue cells
partial pressure of O2 (mmHg)
Body tissue
Lung

15. Shift to right= low affinity to oxygen
100%
% saturation of haemoglobin
50%
partial pressure of O2 (mmHg)
26 mmHg

16. Shift to left= high affinity to oxygen
100%
% saturation of haemoglobin
50%
partial pressure of O2 (mmHg)
26 mmHg

17. Hemoglobin structure

18. Binding of CO2 to hemoglobin
Increase the stability of hemoglobin (T form)
Decrease the affinity of hemoglobin to oxygen
Shift the Dissociation Curve to right

19. Binding of CO to hemoglobin
Increase the (R form)
Increase the affinity of hemoglobin to oxygen

20. PH and Oxygen-Hemoglobin Dissociation Curve (bohr effect)
About 70% of the blood
CO2 reacts with H2O to form carbonic acid
CO2 + H2O  H2CO3
Carbonic acid rapidly dissociates into
H2CO3  H+ + HCO-3
ion H+ and bicarbonate ion
Carbonic anhydrase

21. Increased PH and Oxygen-Hemoglobin Dissociation Curve (bohr effect)
Ion H+
Increase the stability of hemoglobin (T form)
Decrease the affinity of hemoglobin to oxygen
Shift the Dissociation Curve to right

22. Bohr effect – the effect of [PH] on haemoglobin
100%
Higher [CO2] e.g. tissue cells
► curve shift to the right
% saturation of haemoglobin
► always at a value of lower % saturation of haemoglobin than the curve at the left
► haemoglobin has a lower affinity to O2
partial pressure of O2 (mmHg)
∴ in actively respiring tissue cells, O2 is more easily released by haemoglobin !

23. 2,3 bisphosphoglycerate

24. Response of 2,3BPG to chronic anemia and hypoxia

25. Shift to right = low affinity to oxygen
100%
Increased CO2
decreased PH
Increased 2.3 BPG
% saturation of haemoglobin
50%
partial pressure of O2 (mmHg)
26 mmHg

26. Shift to left= high affinity to oxygen
100%
decresed CO2
increased PH
decresed 2.3 BPG
% saturation of haemoglobin
50%
partial pressure of O2 (mmHg)
26 mmHg

27. Hemoglobin and Myoglobin

28. 1 mL blood at 15 g/dL Hb = 0.5 mg Fe
An Erythrocyte (RBC)
Normal Values
RBCs, male x 106/µL
female x 106/µL
Hb, male g/dL
female g/dL
Hct, male 42-53%
female 37-47%
MCH 29±2 pg
MCV fL
MCHC %
Practical Values
65% of Fe in Hb
1 g Hb = 3.46 mg Fe
1 mL blood at 15 g/dL Hb = 0.5 mg Fe
RBC x 3 = Hb
Hb x 3 = Hct
Microcytic < 81 fL
Macrocytic > 94 fL

29. Hemoglobin A1c Diabetes Increased glucose concentration
Nonenzymatic glycosylation
Associated with diabetic complication

30. Hemoglobin Genes and Gene Products

31. Genetics.
In the first 8 weeks of embryonic life the predominant forms of hemoglobin are:
Hb Gower 1 (ζ2ε2).
Hb Gower 2 (α2ε2).
Hb Portland 1 (ζ2γ2).
By the 12th week embryonic hemoglobin is replaced by Hb F (α2γ2) which represents 70 – 100% of hemoglobin in fetal life.

32. Genetics (2).
Adult hemoglobin Hb A (α2β2) detectable from 16/40, replaces Hb F as predominant hemoglobin by 6/12 after birth, up to 30% of Hb in fetal life.
In normal adults 96 – 98% of hemoglobin is HbA, HbF (<1%) constitute a minor component of the total hemoglobin.

33. Fetal hemoglobin
Is the main oxygen transport protein in the fetus during the last seven months of development in the uterus and in the newborn until roughly 6 months old.
Baby takes about 2 years to completely switch over to adult haemoglobin

34. Function
Fetal hemoglobin differs most from adult hemoglobin in that it is able to bind oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother's blood stream.
The transfer of oxygen is from the mother (less tightly bond) to the baby (more tightly bond).

35. Characteristic
Hemoglobin F is made up of 2 alpha chains and 2 gamma chains
Hemoglobin F does not turn into hemoglobin A.
Hb F and Hb A are completely different hemoglobin
Newborn babies with sickle cell disease make hemoglobin F and hemoglobin S

36. Sickle cell anemia
Hemoglobin molecules in each red blood cell carry oxygen from the lungs to body organs and tissues and bring carbon dioxide back to the lungs.
In sickle cell anemia, the hemoglobin is defective. After hemoglobin molecules give up their oxygen, some may cluster together and form long, rod-like structures. These structures cause red blood cells to become stiff and assume a sickle shape.

37. Characters of Sickled Red Cell
Changing the shape of the RBC from a round disc to a characteristic crescent (sickle) shape.
Sickled red cells cannot squeeze through small blood vessels.
They stack up and cause blockages that deprive organs and tissues of oxygen-carrying blood.
This process produces periodic episodes of pain and ultimately can damage tissues and vital organs and lead to other serious medical problems.

38. Characters of Sickled Red Cell
Normal red blood cells live about 120 days in the bloodstream, but sickled red cells die after about 10 to 20 days.
Because they cannot be replaced fast enough, the blood is chronically short of red blood cells, a condition called anemia.

39. Inheritance
Sickle cell anemia is an autosomal recessive genetic disorder caused by a defect in the HBB gene, which codes for hemoglobin.
The presence of two defective genes (SS) is needed for sickle cell anemia.
Hemoglobin S differs from normal adult hemoglobin (hemoglobin A) only by a single amino acid substitution (a valine replacing a glutamine in the 6th position of the beta chain of globing).
When a person has two copies of the S gene (homozygous SS), he has sickle cell anemia.

40. Inheritance
In sickle cell disease, as much as 80% to 100% of the hemoglobin may be HbS.
A person with one altered S gene will have sickle cell trait.
In those who have sickle cell trait, 20% to 40% of the hemoglobin is HbS.
The person does not generally have any symptoms or health problems but can pass the gene on to his children.

41. Inheritance
If each parent carries one sickle hemoglobin gene (S) and one normal gene (A), each child has a 25% chance of inheriting two defective genes and having sickle cell anemia.
25% chance of inheriting two normal genes and not having the disease.
50% chance of being an unaffected carrier like the parents.

43. Pathophysiology
Normal hemoglobin exists as solitary units whether oxygenated or deoxygenated (upper panel).
In contrast, sickle hemoglobin molecules adhere when they are deoxygenated, forming sickle hemoglobin polymers (lower panel).

44. Hemoglobin C
Hemoglobin C
Alpha
Beta S
Beta C
This results when one of the beta subunits is replaced with beta C
The mutation that causes this change in the beta happens because a glutamic acid residue replaces a lysine residue at the sixth position of the beta globin chain.

45. Hemoglobin Electrophoresis
Start position - +
HbA
HbS
HbC - +
cathode
anode
migration

46. Hemoglobin Electrophoresis

47. Beta Thalassemia
β thal major is homozygosity or compound heterozygosity resulting in severe phenotype.
β thal minor or trait is heterozygosity with asymptomatic phenotype.

48. Alpha Thalassemia α
Father α
Mother