1. Erythrocyte metabolism Alice Skoumalová

2. Erythrocytes  deliver oxygen to body tissues and remove carbon dioxide and protons  biconcave 7.7μm  lack cell organelles  120 days  women 4,2-5,4 million/μl, men 4,6-6,2 million/μl

3. The erythrocyte membrane 50% lipid bilayer (phospholipids, cholesterol) 50% proteins SDS-PAGE:separation of proteins (band 1-7) isolation and analysis (10 main proteins) Integral: Anion exchanger protein, Glycophorin A, B, C Peripheral: Spectrin, Ankyrin, Actin

4. Spectrin: the most prominent component (two isoforms α,β; a tetramer; a meshwork ) fixed to the membrane- ankyrin binding sites for several other proteins (glycophorin C, actin, band 4.1, adducin) This organization keeps the erythrocyte shape.

5. 4 protein chains + 4 haem groups Haemoglobin

6. Haem

7. Movements of the heme and the F helix during the T – R transition in hemoglobin:

9. Hemoglobin saturation curves:

10. Hemoglobin autooxidation O 2 binds Fe 2+ - an intermediate structure - an electron is delocalized between the iron ion and the O 2 the side effect - every so often a molecule of oxyhaemoglobin undergoes decomposition and release superoxide Hem - Fe 2+ - O 2 Hem - Fe 3+ - O 2 - 3% of the haemoglobin undergoes oxidation every day Methemoglobin (Fe 3+ ) is unable to bind O 2 (methaemoglobin reductase)

11. Erythrocyte exceptions They lack organelles no ATP production in oxidative phosphorylation no ability to replace damaged lipids and proteins (low metabolic activities, with no ability to synthesize new proteins or lipids) Free radicals exposure haemoglobin autoxidation (O 2 - release) a cell membrane rich in polyunsaturated fatty acids (susceptible to lipid peroxidation) deformation in tiny capillaries; catalytic ions leakage (cause of lipid peroxidation)

12. Erythrocyte metabolism Glucose as a source of energy Glycolysis generates ATP and 2,3-bisphosphoglycerate The pentose phosphate pathway produces NADPH Glutathione synthesis- the antioxidant defence system

14. Glucose- source of energy Glucose transporter: integral membrane protein (12 membrane-spanning helices) a channel for the glucose transport insulin-independent transporter Glycolysis in erythrocytes 1. Source of ATP Lactate- the end product Cover energy requirement 2. Generate 2,3-bisphosphoglycerate (2,3-BPG) a major reaction pathway for the consumption of glucose in erythrocytes the specific binding of 2,3-BPG to deoxyhemoglobin decreases the oxygen affinity of hemoglobin and facilites oxygen release in tissues

15. 2,3-bisphosphoglycerate Allosteric effector of haemoglobin: –binds to deoxyhaemoglobin (a central cavity capable of binding 2,3- BPG) –decreases haemoglobin‘s O 2 affinity Clinical aspects: –In people with high-altitude adaptation or smokers the concentration of 2,3-BPG in the blood is increased (low oxygen supply) –Fetal haemoglobin has low BPG affinity - the higher O 2 affinity - facilitates the transfer of O 2 to the fetus via the placenta

16. Glutathione synthesis in erythrocytes

17. Glutathione Elimination of H 2 O 2 and organic hydroperoxides 1. Cofactor for the glutathione peroxidase (removes H 2 O 2 formed in erythrocytes) 2. Involved in ascorbic acid metabolism 3. Prevents protein –SH groups from oxidizing and cross-linking Gly Cys Glu Gly Cys Glu Gly Cys SH Glu S Oxidized form of glutathione (dimer, disulphide) Reduced form of glutathione (monomer) Glutathione peroxidase Glutathione reductase + R-O-O-H + H 2 O + NADPH

18. The pentose phosphate pathway in erythrocytes Generates NADPH - reduction of glutathione (eliminates H 2 O 2 formed in erythrocytes) Clinical apect: Glucose-6-phosphate dehydrogenase deficiency –Causes hemolytic anemia (decreased production of NADPH - reduced protection against oxidative stress - haemoglobin oxidation and Heinz bodies formation, membrane lipid peroxidation and hemolysis) –Hemolytic crises are evocated by drugs (primaquine, sulphonamide antibiotics) and foods (broad beans) –The most common enzyme deficiency disease in the world (100 million people)

19. Haemoglobin Oxyhaemoglobin Methaemoglobin Methaemoglobin reductase O2O2 Superoxide Superoxide dismutase H2O2H2O2 Catalase ½ O 2 +H 2 O Glutathione peroxidase H2OH2O GSH GSSG Glutathione reductase NADPH NADP + Pentose phosphate pathway GSH-reduced form; GSSG-oxidized form of glutathione

20. Haemoglobinopathy abnormal structure of the haemoglobin (mutation) large number of haemoglobin mutations, a fraction has deleterious effects sickling, change in O 2 affinity, heme loss or dissociation of tetramer haemoglobin M and S, and thalassemias Haemoglobin M replacement of the histidine (E8 or F7) in α or β-chain by the tyrosine the iron in the heme group is in the Fe 3+ state (methaemoglobin) stabilized by the tyrosine methaemoglobin can not bind oxygen Thalassemias genetic defects- α or β-chains are not produced (α or β-thalassemia)

21. Haemoglobin S (sickle-cell) Causes a sickle-cell anemia Erythrocytes adopt an elongated sickle shape due to the aggregation of the haemoglobin S

22. Replacing Glu with the less polar amino acid Val - forming „an adhesive region“ of the β chain Hg proteins aggregate into a long rodlike helical fiber Cross section

23. Red blood cells adopt a sickle shape in a consequence of the forming haemoglobin S fibers The high incidence of sickle-cell disease coincides with a high incidence of malaria Individuals heterozygous in haemoglobin S have a higher resistance to malaria; the malarial parasite spends a portion of its life cycle in red cells, and the increased fragility of the sickled cells tends to interrupt this cycle Scanning electron micrograph of a sickled erythrocyte. The haemoglobin S fibers can be seen within the distorted cell. The cell has ruptured and haemoglobin fibers are spilling out.

25. Hemoglobin switching:

26. Glycosylated haemoglobin (HbA 1 ) formed by hemoglobin's exposure to high plasma levels of glucose non-enzymatic glycolysation (glycation)- sugar bonding to a protein normal level HbA 1 - 5%; a buildup of HbA 1 - increased glucose concentration the HbA 1 level is proportional to average blood glucose concentration over previous weeks; in individuals with poorly controlled diabetes, increases in the quantities of these glycated hemoglobins are noted (patients monitoring) SugarCHO + NH 2 CH 2 Protein SugarCHNCH 2 Protein Sugar CH 2 NH CH 2 Protein Schiff base Glycosylated protein Amadori reaction

27. Summary 1)The erythrocyte membrane 2)Haemoglobin –O 2 binding 3)Haemoglobin – allosteric efectors 4)Free radicals in erythrocytes 5)Metabolic pathways in erythrocytes 6)Haemoglobinopathy 7)Glycosylated haemoglobin

28. Pictures used in the presentation: Marks´ Basic Medical Biochemistry, A Clinical Approach, third edition, 2009 (M. Lieberman, A.D. Marks) Principles of Biochemistry, 2008, (Voet D, Voet J.G., and Pratt C.W) Color Atlas of Biochemistry, second edition, 2005 (J. Koolman and K.H. Roehm)