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Erythrocyte metabolism
Alice Skoumalová
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
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
The erythrocyte membrane
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.
Hereditary spherocytosis
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autosomal dominant
a deficiency in a spectrin amount and its abnormalities
the presence of spherocytes in the blood
the spleen‘s hemolysis
Haemoglobin
4 protein chains + 4 haem groups
•
•
O2 binds to Fe2+ - an intermediate structure- an electron is delocalized
between the iron ion and the O2
the side effect - every so often a molecule of oxyhaemoglobin
undergoes decomposition and release superoxide
Hem - Fe2+- O2
Hem - Fe3+ - O2•-
Methemoglobin (Fe3+) is unable to bind O2
(methaemoglobin reductase)
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 (O2•- release)
• a cell membrane rich in polyunsaturated fatty acids (susceptible to lipid
peroxidation)
• deformation in tiny capillaries; catalytic ions leakage (cause of lipid
peroxidation)
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
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
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•
Lactate- the end product
Cover 90% of 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
2,3-bisphosphoglycerate
• Allosteric effector of haemoglobin:
– binds to deoxyhaemoglobin (a central cavity capable of binding 2,3BPG)
– decreases haemoglobin‘s O2 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 O2 affinity facilitates the transfer of O2 to the fetus via the placenta
Glutathione synthesis in erythrocytes
Glutathione
Elimination of H2O2 and organic hydroperoxides
1. Cofactor for the glutathione peroxidase (removes H2O2 formed in
erythrocytes)
2. Involved in ascorbic acid metabolism
3. Prevents protein –SH groups from oxidizing and cross-linking
Glutathione peroxidase
Gly
Gly
Gly
+ R-O-O-H
Cys
Cys
SH
+ NADPH
Glu
Glu
S
S
Cys
+ H2O
Glu
Glutathione reductase
Reduced form of glutathione
Oxidized form of glutathione
(monomer)
(dimer, disulphide)
The pentose phosphate pathway in
erythrocytes
•
Generates NADPH - reduction of glutathione (eliminates H2O2 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)
Oxyhaemoglobin
O2
Superoxide dismutase
Haemoglobin
Superoxide
H2O2
Catalase
Methaemoglobin reductase
Methaemoglobin
½ O2+H2O
Pentose phosphate
NADP+
pathway
Glutathione reductase
NADPH
GSH
Glutathione peroxidase
GSSG
H2O
GSH-reduced form; GSSG-oxidized form of glutathione
Haemoglobin autoxidation
•
•
3% of the haemoglobin undergoes oxidation every day
a constant flux of O2•Hem - Fe2+- O2
Hem - Fe3+ - O2•-
Methaemoglobin reductase
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•
Converts methaemoglobin back to ferrous haemoglobin to permit
continued O2 transport
System containing FAD, cytochrome b5 and NADH (glycolysis)
Methaemoglobinemia
1. Congenital type
– methaemoglobin reductase deficiency (AR)
– variant haemoglobin M (HbM)- mutation; tend to be oxidized to
methaemoglobin
2. Acquired type- drugs or chemicals (sulphonamides, aniline)
Visual indicator- a blue tint to the skin (10% of metHb)
Treated- reductants (methylene blue, ascorbic acid)
Superoxide dismutase (SOD)
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•
a high concentration in erythrocytes
accelerates the dismutation O2•- to H2O2
H2O2 remove
1. Catalase
2. Glutathione peroxidase
1. Catalase
•
•
a ferric haem group bound to the active site
catalyses decomposition of H2O2 to water and oxygen:
2H2O2
2H2O+O2
2. Glutathione peroxidase
•
removes H2O2 by coupling its reduction to H2O with oxidation of reduced
glutathione (GSH)
H2O2+2GSH
GSSG+2H2O
Glutathione reductase
• reduces oxidized glutathione back to reduced
GSSG+NADPH+H+
•
2GSH+NADP+
NADPH- the pentose phosphate pathway (glucose-6-phosphate
dehydrogenase)
Cooperation of glutathione peroxidase and catalase
•
The concentration of H2O2 is raised- catalase becomes more important
(high Km for H2O2)
Low-molecular mass antioxidants
α-tocopherol (vitamin E)
• Present in the erythrocyte membrane
• Prevents lipid peroxidation (chain-breaking antioxidant)
α-TocH+LO2•
α-Toc•+LO2H
Ascorbic acid (vitamin C)
• Present in the cytoplasm
• Recycles α-tocopherol
• Dehydroascorbate reductase (GSH-dependent) regenerates
ascorbate
Haemoglobinopathy
• abnormal structure of the haemoglobin (mutation)
• large number of haemoglobin mutations, a fraction has deleterious
effects
• sickling, change in O2 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 Fe3+ 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)
Haemoglobin S (sickle-cell)
•
•
Causes a sickle-cell anemia
Erythrocytes adopt an elongated sickle shape due to the
aggregation of the haemoglobin S
• Replacing Glu with the less polar amino acid Val - forming „an
adhesive region“ of the β chain
• The hydrophobic Val fits to the region of the another β chain in
deoxy (not oxy) haemoglobin and thus adjacent haemoglobin
molecules can fit together and aggregate into a long rodlike helical
fiber
Cross section
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Red blood cells adopt a sickle shape in a consequence of the forming haemoglobin S
fibers
The elongated cells tend to block capillaries, causing inflammation and considerable
pain; they are fragile what leads to anemia
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.
Glycosylated haemoglobin (HbA1)
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•
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formed by hemoglobin's exposure to high plasma levels of glucose
non-enzymatic glycolysation (glycation)- sugar bonding to a protein
normal level HbA1- 5%; a buildup of HbA1- increased glucose concentration
the HbA1 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)
Sugar CHO
Sugar CH
+
NH2
N
CH2
CH2
Protein
Protein
Schiff base
Amadori reaction
Sugar
CH2
NH
CH2
Protein
Glycosylated protein
Summary
 Erythrocytes lack cell organelles; their membranes are rich in
polyunsaturated fatty acids and proteins (fluidity and elasticity)
 Glucose as a energy source
 Glycolysis generates ATP and 2,3-BPG; the pentose phosphate pathway
produces NADPH
 Haemoglobin autoxidation forms free radicals
 Free radicals are removed by the antioxidant defence system with
glutathione and NADPH
 There is a large number of haemoglobin mutations; some of them are
pathological (haemoglobinopathy)