1. Hemoglobin Structure & Function

2. Objectives of the Lecture structuralfunctional 1- Understanding the main structural & functional details of hemoglobin as one of the hemoproteins. types HBA1c 2- Identify types & relative concentrations of normal adult hemoglobin with reference to HBA1c with its clinical application. methemoglobinopathies thalassemias 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 hemeHemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group. Heme protoporphyrin IX ferrous iron (Fe2+).Heme is a complex of protoporphyrin IX & ferrous iron (Fe2+). ironThe iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring. heme Fe2+The heme Fe2+ can form two additional bonds, one on each side of the planar porphyrin ring. histidine amino acid oxygen 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 Hemeprotoporphyrin IX ferrous iron (Fe2+). Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+). iron 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. histidine amino acid oxygen 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 HemoglobinRBCs Hemoglobin is found exclusively in RBCs. function Its main function is to transport oxygen from lungs to the tissues & carbon dioxide & hydrogen protons from tissues to lungs. Hemoglobin A Hemoglobin A is the major hemoglobin in adults, is composed of four polypeptide chains, 2 alpha (  ) & 2 beta (  ) 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 Each subunit has stretches of  -helical structure & a heme binding pocket.

7. Structure & function of hemoglobin Structure & function of hemoglobin (cont.)

8. 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. tightlyhydrophobic interactionsThe two polypeptide chains within each dimer are tightly held together, primarily by hydrophobic interactions polar bonds.In contrast, the two dimers are able to move with respect to each other, being held together primarily by polar bonds. weaker deoxyhemoglobin oxyhemoglobinThe weaker interactions between these mobile dimers result in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin Structure & function of hemoglobin Structure & function of hemoglobin (cont.)

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

10. Types of adult hemoglobin 3–6 % HBA: HBA: the major hemoglobin in humans HBA 2 HBA 2 : first appears 12 weeks after birth- a minor component of normal adult HB HBF: HBF: normally synthesized only during fetal development HBA 1C : HBA 1C : has glucose residues attached to  -globin chains – increased amounts in DM

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

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

13. Thalassemias Thalassemias Thalassemias Thalassemias are hereditary hemolytic diseases in which an imbalance occurs in the synthesis of globin chains. single gene disorders They are most common single gene disorders in humans. Normally Normally, synthesis of  - and  - globin chains are coordinated, so that each  -globin chain has a  -globin chain partner. This leads to the formation of  2  2 (HbA). In thalassemias In thalassemias, the synthesis of either the  - or  -globin chain is defective.

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

15. 1-  -thalassemias:  -globindecreasedabsent  Synthesis of  -globin chains are decreased or absent, whereas  -globin synthesis is normal. premature death of RBCsending in chronic hemolytic anemia   -globin chains cannot form stable tetramers & therefore precipitate causing premature death of RBCs ending in chronic hemolytic anemia  Also,  2  2 (HbF) &  2  2 (HbA 2 ) are accumulated. Thalassemias Thalassemias (cont.)

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

17. both Mutation in both  -globin genes  -thalassemiamajor one Mutation in one of  -globin genes  -thalassemiaminor Thalassemias Thalassemias (cont.)  -thalassemia

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

19. 2-  -thalassemia:  -globindecreasedabsent Synthesis of  -globin chains is decreased or absent. Each individual's genome contains four copies of the  -globin (two on each chromosome 16), there are several levels of  -globin chain deficiencies Thalassemias Thalassemias (cont.)

20. Types: If one of the four genes is defective silent carrier of  - thalassemia The individual is termed a silent carrier of  - thalassemia as no physical manifestations of the disease occur. If two  -globin genes are defective,  -thalassemia trait The individual is designated as having  -thalassemia trait. If three  -globin genes are defective; Synthesis of unaffected  - and then  - globin chains continues, resulting in the accumulation of  tetramer in the newborn (  4, Hb Bart's) or  -tetramers (  4, 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  -globin genes are defective, hydrops fetalis & fetal death hydrops fetalis & fetal death occurs as  -globin chains are required for the synthesis of HbF Thalassemias Thalassemias (cont.)

21. Types of  -thalassemias

22. Sickle cell anemia Definition: Sickle cell anemia is a genetic disorder of the blood caused by a single nucleotide alteration (a point mutation) in the  -globin gene. Inheritance of sickle cell anemia: Sickle cell disease is a homozygous recessive disorder: two mutant genes i.e. It occurs in individuals who have inherited two mutant genes (one from each parent) that code for synthesis of the  chains of the globin molecule. RBCs of homozygous is totally HB S (  2  s 2 ) Heterozygotes individuals: one normal and one sickle cell gene Have one normal and one sickle cell gene. RBCs of heterozygotes contain both HB S (  2  s 2 ) & HB A (  2  2 ) sickle cell trait These individuals have sickle cell trait

23. 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: anemia - Chronic hemolytic anemia pain - Lifelong episodes of pain infection - Increased susceptibility to infection. chest - Acute chest syndrome Stroke - Stroke Splenic renal - Splenic & renal dysfunction Bone - Bone changes due to bone marrow hyperplasia Heterozygote individuals not Usually do not show clinical symptoms

24. HB S  s HB S contains two mutant  -globin chains (  s ). In mutant chains, glutamate (polar) at position 6 is replaced with valine (nonpolar) resulting in: Formation of a protrusion on the  -globin t Formation of a protrusion on the  -globin that fit into a complementary site on the a chain of another hemoglobin molecule in the cell. In low oxygen tensiondeoxy HB S rigid misshapen RBCs 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 anoxia Sickle cells block the flow of blood in narrow capillaries resulting in interruption of oxygen supply (localized anoxia) in tissues causing pains. anoxia 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. Amino acid substitution in HB S  chains

25. sickle cell anemia

26. 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 in high altitudes flying in a nonpressurized plane flying in a nonpressurized plane 2- Increased pCO2 2- Increased pCO2 3- Decrease pH 3- Decrease pH 4- Increased 2,3- BPG in RBCs 4- Increased 2,3- BPG in RBCs

27. Diagnosis of HB S Hemoglobin Hemoglobin Electrophoresis 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

28. Heterozygotes individuals 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 malaria HB S gene is highly frequent in Africa in which malaria is also highly frequent. Selective advantage of the heterozygote state

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