2Objectives 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).
3Hemoglobin 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
4Globin of hemoglobin is a globular protein with a quaternary structure
5Structure of hemeHeme 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 ofthe 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.
6Structure & 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 interactionsEach 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.
8Structure & function of hemoglobin (cont.) Quaternary structure of hemoglobinThe 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 interactionsIn 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
10Types of adult hemoglobin 3–6 %HBA: the major hemoglobin in humansHBF: normally synthesized only during fetal developmentHBA2: first appears 12 weeks after birth- a minor component of normal adult HBHBA1C : has glucose residues attached to b-globin chains – increased amounts in DM
11Hemoglobin 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
12HemoglobinopathiesHemoglobinopathies 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).
13Thalassemias 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.
14Thalassemias (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).
17Thalassemias (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 anemiaAlso, a2g2 (HbF) & a2d2 (HbA2 ) are accumulated.
18Thalassemias (cont.) There are only two copies of the b -globin gene in each cell (one oneach 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.
19Thalassemias (cont.) b-thalassemia Mutation in both Mutation in one of b-globin genesb-thalassemiamajorMutation in one ofb-globin genesb-thalassemiaminorb-thalassemia
20Thalassemias (cont.) Some clinical aspects of b-thamassemias: 1- As b-globin gene is not expressed until late fetal gestation, the physicalmanifestations of b -thalassemias appear only after birth.2- Individuals with b -thalassemias minor, make some b-chains, andusually require no specific treatment.3- Infants born with b - thalassemias major seem healthy at birth, butbecome severely anemic during the first or second years of life.They require regular transfusions of blood.
21Thalassemias (cont.) 2- a-thalassemia: Synthesis of a-globin chains is decreased or absent.Each individual's genome contains four copies of the a-globin (twoon each chromosome 16), there are several levels of a-globin chaindeficiencies
22Thalassemias (cont.) Types: If one of the four genes is defective If one of the four genes is defectiveThe individual is termed a silent carrier of a- thalassemia as no physical manifestations of thedisease 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 ofg 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
24Sickle cell anemia Definition: Inheritance of sickle cell anemia: Sickle cell anemia is a genetic disorder of the blood caused bya 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 eachparent) 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
25Sickle Cell DiseaseResults from a single genetic mutation in which a nucleotide in the coding sequence of a beta-globin gene is mutated from adenosine to thymidineThis 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,PSMCHSDR. ALY SAMY,MLT,PSMCHSGENETIC SYNDROME CLSDR. ALY SAMY,MLT,PSMCHS25
26Sickle Cell DiseaseThe resulting mutated hemoglobin has decreased solubility and abnormal polymerization propertiesIf only 1 beta-globin gene is mutated= heterozygous state which is referred to as sickle cell traitIf both genes are mutated resulting in homozygous state and called sickle cell anemia or sickle cell disease.Dr. Aly Samy , MLT,PSMCHSDR. ALY SAMY,MLT,PSMCHSGENETIC SYNDROME CLSDR. ALY SAMY,MLT,PSMCHS26
27Sickle 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)
28Sickle Cell MutationSubstitution 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.
30What 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 bodySickle 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.
38Complications of Sickle Cell Disease NCBIbookshelf
39Clinical manifestations of sickle cell anemia Homozygous individualsAn infant (first 2 years of life) begins show manifestations when sufficient HbFis replaced by HbSClinical 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 hyperplasiaHeterozygote individualsUsually do not show clinical symptoms
40Amino 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 anotherhemoglobin 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.
42Factors 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 of1- Decreased oxygen tension:in high altitudesflying in a nonpressurized plane2- Increased pCO23- Decrease pH4- Increased 2,3- BPG in RBCs
43Diagnosis of HB S Hemoglobin Electrophoresis HB S migrates more slowly towards anode (+ve electrode) than normal hemoglobindue to absence of negatively chargedglutamate resulting in decrease ofnegativity of hemoglobin
441-2-3 Hb A (normal) no other hemoglobin present. 4-5Hb A (normal) present Hb S and C (abnormal) they have sickle cell disease.6control, 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.11HbA(normal),HbC, HbS(abnormal)12 HbA (normal) HbF, HbC, HbS(abnormal)
45Selective 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.
46Methemoglobinemia Causes of oxidation of ferrous ions: Methemoglobin results from oxidation of the ferrous ion (Fe2+) of heme of hemoglobin to ferric (Fe3+) ionMethemoglobinemia is characterized by “chocolate cyanosis” i.e. brown-blue coloration of skin & membranes & chocolate colored bloodCauses of oxidation of ferrous ions:1- Drugs as nitrates2- 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 HBRBCs 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 hypoxiaTreatment: Methylene blue (to reduce the ferric ions)