1. Hemoglobin Structure & Function

2. Objectives of the Lecture
1- Understanding the main structural & functional details of hemoglobin as one of the hemoproteins. 2- Identify types & relative concentrations of normal adult hemoglobin with reference to HBA1c with its clinical application. 3- Recognize some of the main genetic & biochemical aspects of methemoglobinopathies with some implications on clinical features (with focusing on thalassemias).

3. Hemoglobin is a globular hemoprotein
Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group.
Heme is a complex of protoporphyrin IX & ferrous iron (Fe2+) .
The iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring.
The heme Fe2+ can form two additional bonds, one on each side of the planar porphyrin ring.
One of these positions is coordinated to the side chain of a histidine amino acid of the globin molecule, whereas the other position is available to bind oxygen

4. Globin of hemoglobin is a globular protein with a quaternary structure

5. Structure of heme
Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+).
The iron is held in the center of the heme molecule by bonds of the four nitrogens of the protoporphrin ring.
Heme F2+ can form two additional bonds, one on each side of
the porphyrin ring.
One of these positions is coordinated to the side chain of histidine amino acid of the globin molecule, whereas the other position is available to bind oxygen.

6. Structure & function of hemoglobin
Hemoglobin is found exclusively in RBCs.
Its main function is to transport oxygen from lungs to the tissues & carbon dioxide & hydrogen protons from tissues to lungs.
Hemoglobin A is the major hemoglobin in adults, is composed of four polypeptide chains, 2 alpha (a) & 2 beta (b) chains, held together by noncovalent interactions
Each day, 6-7 grams of hemoglobin is synthesized to replace lost through normal turn over of RBCs.
Each subunit has stretches of a-helical structure & a heme binding pocket.

7. Structure & function of hemoglobin (cont.)

8. Structure & function of hemoglobin (cont.)
Quaternary structure of hemoglobin
The hemoglobin tetramer can be envisioned as being composed of two identical dimers, (αβ)1 and (αβ)2, in which the numbers refer to dimers one and two.
The two polypeptide chains within each dimer are tightly held together, primarily by hydrophobic interactions
In contrast, the two dimers are able to move with respect to each other, being held together primarily by polar bonds.
The weaker interactions between these mobile dimers result in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin

9. oxygenation & deoxygenation of hemoglobin (oxyhemoglobin & deoxyhemoglobin)
Taut structure
Oxyhemoglobin
Relaxed structure

10. Types of adult hemoglobin
3–6 %
HBA: the major hemoglobin in humans
HBF: normally synthesized only during fetal development
HBA2: first appears 12 weeks after birth- a minor component of normal adult HB
HBA1C : has glucose residues attached to b-globin chains – increased amounts in DM

11. Hemoglobin A1c (HBA1c) Some of hemoglobin A is glycosylated
Extent of glycosylation depends on the plasma concentration of a particular hexose (as glucose).
The most abundant form of glycosylated hemoglobin is HBA1c which has a glucose residues attached to b-globin chains in hemoglobin RBCs.
Increased amounts of HBA1c are found in RBCs of patients with diabetes mellitus (DM).
HbA1c could be used as a monitor for the control of the blood glucose level during the last 2 months for diabetic patients

12. Hemoglobinopathies
Hemoglobinopathies are members of a family of genetic disorders caused by:
1- Production of a structurally abnormal hemoglobin molecule
(Qualitative hemoglobinopathies)
Or: 2- Synthesis of insufficient quantities of normal hemoglobin
(Quantitative hemoglobinopathies)
Or: 3- both (rare).

13. Thalassemias  
Thalassemias are hereditary hemolytic diseases in which an imbalance occurs in the synthesis of globin chains.
They are most common single gene disorders in humans.
Normally, synthesis of a- and b- globin chains are coordinated, so that each a-globin chain has a b-globin chain partner.
This leads to the formation of a2b2 (HbA).
 In thalassemias, the synthesis of either the a- or b-globin chain is defective.

14. Thalassemias (cont.)  
Thalassemia can be caused by a variety of mutations, including:
1- Entire gene deletions (whole gene is absent)
Or: 2- Substitutions or deletions of one or more nucleotides in the DNA.
Each thalassemia can be classified as either:
1- A disorder in which no globin chains are produced
(ao- or bo -thalassemia)
Or: 2- Some b-chains are synthesized, but at a reduced rate.
(a+- or b+- thalassemia).

17. Thalassemias (cont.) 1- b-thalassemias:
Synthesis of b-globin chains are decreased or absent, whereas a-globin synthesis is normal.
a-globin chains cannot form stable tetramers & therefore precipitate causing premature death of RBCs ending in chronic hemolytic anemia
Also, a2g2 (HbF) & a2d2 (HbA2 ) are accumulated.

18. Thalassemias (cont.)  
There are only two copies of the b -globin gene in each cell (one on
each chromosome 11).
So, individuals with b -globin gene defects have either:
1- b-thalassemia minor (b -thalassemia trait):
if they have only one defective b-globin gene.
2- b- thalassemia major (Colley anemia):
if both genes are defective.

19. Thalassemias (cont.) b-thalassemia Mutation in both Mutation in one of
b-globin genes
b-thalassemia
major
Mutation in one of
b-globin genes
b-thalassemia
minor
b-thalassemia

20. Thalassemias (cont.) Some clinical aspects of b-thamassemias:
1- As b-globin gene is not expressed until late fetal gestation, the physical
manifestations of b -thalassemias appear only after birth.
2- Individuals with b -thalassemias minor, make some b-chains, and
usually require no specific treatment.
3- Infants born with b - thalassemias major seem healthy at birth, but
become severely anemic during the first or second years of life.
They require regular transfusions of blood.

21. Thalassemias (cont.) 2- a-thalassemia:
Synthesis of a-globin chains is decreased or absent.
Each individual's genome contains four copies of the a-globin (two
on each chromosome 16), there are several levels of a-globin chain
deficiencies

22. Thalassemias (cont.) Types: If one of the four genes is defective
If one of the four genes is defective
The individual is termed a silent carrier of a- thalassemia as no physical manifestations of the
disease occur.
If two a-globin genes are defective,
The individual is designated as having a-thalassemia trait.
If three a-globin genes are defective;
Synthesis of unaffected g- and then b- globin chains continues, resulting in the accumulation of
g tetramer in the newborn (g4, Hb Bart's) or b-tetramers (b4, HbH).
The subunits do not show heme-heme interactions. So, they have very high oxygen affinities. Thus,
they are essentially useless as oxygen carriers to tissues (clinically severe).
If four a-globin genes are defective,
hydrops fetalis & fetal death occurs as a-globin chains are required for the synthesis of HbF

23. Thalassemias (cont.)  
Types of a-thalassemias

24. Sickle cell anemia Definition: Inheritance of sickle cell anemia:
Sickle cell anemia is a genetic disorder of the blood caused by
a single nucleotide alteration (a point mutation) in the b-globin gene.
Inheritance of sickle cell anemia:
Sickle cell disease is a homozygous recessive disorder:
i.e. It occurs in individuals who have inherited two mutant genes (one from each
parent) that code for synthesis of the b chains of the globin molecule.
RBCs of homozygous is totally HB S (a2bs2 )
Heterozygotes individuals:
Have one normal and one sickle cell gene.
RBCs of heterozygotes contain both HB S (a2bs2 ) & HB A (a2b2 )
These individuals have sickle cell trait

25. Sickle Cell Disease
Results from a single genetic mutation in which a nucleotide in the coding sequence of a beta-globin gene is mutated from adenosine to thymidine
This mutation occurs in the middle of the triplet that codes for normally glutamic acid as the 6th AA of the beta-chain of hemoglobin. The single base change substitutes Valine for glutamic acid.
Dr. Aly Samy , MLT,PSMCHS
DR. ALY SAMY,MLT,PSMCHS
GENETIC SYNDROME CLSDR. ALY SAMY,MLT,PSMCHS 25

26. Sickle Cell Disease
The resulting mutated hemoglobin has decreased solubility and abnormal polymerization properties
If only 1 beta-globin gene is mutated= heterozygous state which is referred to as sickle cell trait
If both genes are mutated resulting in homozygous state and called sickle cell anemia or sickle cell disease.
Dr. Aly Samy , MLT,PSMCHS
DR. ALY SAMY,MLT,PSMCHS
GENETIC SYNDROME CLSDR. ALY SAMY,MLT,PSMCHS 26

27. Sickle Cell Mutation From Robbins (2005)
This is the prototype mutation in human genetics. Whenever a new analytical technique is discovered, it is first applied to this mutation. RFLP analysis, PCR analysis, etc, have all been validated using this mutation. In fact, the term molecular disease has its origins in the analysis of this mutation.
From Robbins (2005)

28. Sickle Cell Mutation
Substitution of valine (a hydrophobic amino acid) for glutamic acid (a charged amino acid) at amino acid position six can have dramatic effects on protein structure and function. This cartoon illustrates the ball-and-stick-structures for these two amino acids.

30. What is Sickle Cell Anemia (SCA)?
First described in Chicago in 1910 by James Herrick as an inherited condition that results in a decrease in the ability of red blood cells to carry oxygen throughout the body
Sickle red blood cells become hard and irregularly shaped (resembling a sickle)
Become clogged in the small blood vessels and therefore do not deliver oxygen to the tissues.
Lack of tissue oxygenation can cause excruciating pain, damage to body organs and even death.

31. Red blood cells Going through Vessels

32. Result: Balanced polymorphism
E.g., Sickle Cell Anemia: Mutation = single amino acid subst. in beta chain of hemoglobin --> single a.a. difference.
Sickle blood cells
Normal blood cells

33. Sickle Cell Anemia
Homozygotes for sickle mutation (HsHs): lethal

34. Heterozygotes (HsHn): resistant to malaria,
Sickle Cell Anemia
Heterozygotes (HsHn): resistant to malaria,
selected for in malaria-infested regions,
selected against where malaria not present.

35.                                                     
Harvard Med Sch

36. Normal and Sickle Cell Hemoglobin
website

37. ORGAN/TISSUE INVOLVED PROBLEMS CAUSED
KIDNEY
Hematuria Urinary frequency
SPLEEN
Serious infections Abdominal pain
LUNGS
Pneumonia Chest problems
BONES
Infection Necrosis
BRAIN
Stroke
Headache
LIVER
Hepatomegaly Jaundice

38. Complications of Sickle Cell Disease
NCBI
bookshelf

39. Clinical manifestations of sickle cell anemia
Homozygous individuals
An infant (first 2 years of life) begins show manifestations when sufficient HbF
is replaced by HbS
Clinical manifestations:
- Chronic hemolytic anemia
- Lifelong episodes of pain
- Increased susceptibility to infection.
- Acute chest syndrome
- Stroke
- Splenic & renal dysfunction
- Bone changes due to bone marrow hyperplasia
Heterozygote individuals
Usually do not show clinical symptoms

40. Amino acid substitution in HB S b chains
HB S contains two mutant b-globin chains (bs ).
In mutant chains, glutamate (polar) at position 6 is replaced with valine (nonpolar) resulting in:
Formation of a protrusion on the b-globin that fit into a complementary site on the a chain of another
hemoglobin molecule in the cell.
In low oxygen tension, deoxy HB S polymerize inside the red blood cell leading to stiffening & distorting of the cell ending in production of rigid misshapen RBCs.
Sickle cells block the flow of blood in narrow capillaries resulting in interruption of oxygen supply (localized anoxia) in tissues causing pains.
Finally, cell death occurs due to anoxia (infarction)
Also, RBCs of HB S have shorter life span than normal RBCs (less than 20 days, compared to 120 of normal)
Hence, anemia is a consequence of HB S.

41. sickle cell anemia

42. Factors that increase sickling
The extent of sickling is increased by any factor that increases the proportion of HB S in the deoxy state as in cases of
1- Decreased oxygen tension:
in high altitudes
flying in a nonpressurized plane
2- Increased pCO2
3- Decrease pH
4- Increased 2,3- BPG in RBCs

43. Diagnosis of HB S Hemoglobin Electrophoresis HB S migrates more slowly
towards anode (+ve electrode) than normal hemoglobin
due to absence of negatively charged
glutamate resulting in decrease of
negativity of hemoglobin

44. 1-2-3  Hb A (normal) no other hemoglobin present.
4-5Hb A (normal) present Hb S and C (abnormal) they have sickle cell disease.
6control, HbA and HbF is present (normal in low density 2%), HbS and C (abnormal).
7 Hb A (normal), HbS and C (abnormal).
8 HbA, HbS, HbA2/C (abnormal) sickle cell disease.
11HbA(normal),HbC, HbS(abnormal)
12 HbA (normal) HbF, HbC, HbS(abnormal)

45. Selective advantage of the heterozygote state
Heterozygotes individuals for sickle cell anemia are less suscibtiple to malaria caused by the parasite Plasmodium falciparum as their RBCs have shorter lifespan than normal , the parasite cannot complete its natural stage of development in RBCs. HB S gene is highly frequent in Africa in which malaria is also highly frequent.

46. Methemoglobinemia Causes of oxidation of ferrous ions:
Methemoglobin results from oxidation of the ferrous ion (Fe2+) of heme of hemoglobin to ferric (Fe3+) ion
Methemoglobinemia is characterized by “chocolate cyanosis” i.e. brown-blue coloration of skin & membranes & chocolate colored blood
Causes of oxidation of ferrous ions:
1- Drugs as nitrates
2- Endogenous products (as reactive oxygen species )
3- Inherited defects (as in certain mutations of a or b chains)
4- Deficiency of NADH-Met HB reductase :enzyme for reduction of Fe3+ of Met HB
RBCs of newborns have ½ capacity of adults to reduce Met HB & therefore they are more susceptible to Met HB formation by previous factors.
Clinically, symptoms are due to tissue hypoxia
Treatment: Methylene blue (to reduce the ferric ions)