Red Blood Cells:
 Primary function – Transport O2 and CO2
Major Topics
 Production of erythrocytes
 Specialized pathways for energy metabolism
 Maintenance of heme iron in ferrous state (Fe2+)
 Pathways that protect from oxidative injury
Why do we need erythrocytes vs. Hb alone in solution?
 Separate cellular compartment – metabolism regulates transport of O2 and CO2
 Control redox state of compartment (reducing) separate from serum (oxidizing)
 Regulate binding of O2 to Hb with intracellular effectors
 In solution alone, more dilute Hb tetramers dissociate into inactive Hb dimers
Properties of Erythrocytes:
 Renewed every 120 days, cleared by spleen
 Lack nuclei, mitochondria, intracellular organelles
o Lack synthesis of macromolecules (no cell division), lipids, etc.
 Energy from glycolysis
 Reductive power from PPP (NADPH)
 Pathways to prevent oxidative damage
Shape:
 Biconcave
o Increase surface area – gas/solute exchange
o Increases deformability – can pass through tight spots
 Dependent on: cytoskeleton, metabolic pathways, structure of membrane
Erythropoiesis: Generating new RBC
 Pluripotent stem cells for RBC are also precursors for:
o Platelets, neutrophils, monocytes (macrophages), eosinophils, basophils, lymphocytes (B,
T)
 Reticulocyte – has lost nucleus
 Erythrocyte – once in circulation; mature RBC
 Regulated by Oxygen
o HIF-1 – Hypoxia-inducible transcription factor
 Hypoxia inhibits degradation of HIF-1
 HIF-1 increases in kidney, stimulates transcription of EPO gene
 EPO released in blood, stimulates erythropoiesis in bone marrow
Erythropoietin
 Glycoprotein – synthesized in peritubular kidney cells
 Suppresses apoptosis, stimulates growth, & globin synthesis in RBC precursors
 Synthesis regulated by oxygen (HIF-1)
 DRUG:
o Recombinant Erythropoietin (Procrit, Epogen, Eprex) – treat decreased erythrocyte
production
 Dec. due to end-stage kidney disease, reverse transcriptase inhibitors (HIV
patients), chemotherapy (cancer patients), blood loss (surgery)

*Only Erythropoietin (& Recombinant EPO drugs) can stimulate RBC production –
HIF-1 cannot (it’s an intracellular protein, not in circulation)
Energy Metabolism
 Glycolysis
o Production of 2 ATP
o Produces NADH
 To keep iron in ferrous (Fe2+) state
o Produces 2,3 BPG
 Promotes release of O2 from Hb (T state)
 Regulated by pH
 GAPD binding to BPGM or PGK (whether
or not to make 2,3 BPG)
 Regulated by Feedback inhibition of BPG mutase
(1,3 BPG  2,3 BPG)
 Synthesis increased by anoxia (lack of O2)

Pentose Phosphate Pathway
o Produces 2 NADPH
 Reduces metHb (ferric Fe
3+) to Hb (ferrous Fe2+)
 Reduces oxidized
Glutathione (via
Glutathione Reductase)
 Prevents oxidative
damage
 Flux increased when
oxidation of
glutathione
increases (feedback inhibition of G6PD by NADPH)
o Produces 2 ATP
 When products (Ribose-5-P & fructose-6-P) re-enter glycolysis
 Ribose-6-P  Gly-3-P  Pyruvate + 2 ATP
Oxidative Damage:
Glutathione
 Function – protect against oxidative damage
o Detoxifies H2O2 – Glutathione Peroxidase
o Reduces oxidized protein thiols
o Reduces metHb to Hb (Fe2+)
 CLINICAL:
o Cyanosis
 Sx: Blue color at fingertips
 Patho: Hb oxidized to metHb (Fe 3+) – does not bind/transport O2
 Oxidative Stress (peroxides, spontaneous, drugs [nitrates, anti-malarials,
topical anesthetics), toxic oxidants (tobacco smoke), inherited deficiency
Reduction of MetHb
 MetHb Reductase I (cytochrome b5 reductase)
o Majority of metHb reduction – uses NADH
 Transfers e- from NADH to cytochrome b5, cytochrome b5 reduces metHb
 MetHb Reductase II
o Minor pathway – uses NADPH
o Lacking in “blue people” with methemoglobinemia
 CLINICAL:
o Inherited Methemoglobinemia
 Patho:
 MetHb reductase II defect – more susceptible to oxidant stress
 MetHb reductase I (cytochrome b5) defect/deficiency
 Hb M – mutations in α/β subunits, Fe more prone to oxidation
 Glutathione reductase deficiency
 Dealing with Oxidative Damage
o Superoxide Dismutase
 Superoxide anion – highly reactive oxygen species
 One e- reduction to H2O2; one e- reduction to O2
o Antioxidants
 Vitamin C (Ascorbate)
 Vitamin E
 Methylene blue (given to “blue people” to turn them pink)
 CLINICAL:
o G-6-P Dehydrogenase Deficiency
 It’s the rate limiting enzyme in PPP – regenerates NADPH
 NADPH important to regenerate Glutathione to reduce H2O2 and other
oxidative stress!
 Similar distribution to malaria, 11% African Americans
 Protects against malaria
 More sensitive to oxidative stress
 Fava beans
 Antimalarial drugs
 Etc. etc.…p 252 (19)
 Sx: neonatal jaundice, acute hemolytic anemia
 *Which test would establish G6PD deficiency?
 G6PD activity in lysed RBC (bc it is intracellular)
Regulation of O2 and CO2 Transport:
 2,3 BPG
o Stabilizes low affinity T state; promotes release of O2 (right shift)
o Low O2 increases [2,3 BPG]
 High altitude, suffocation, heart failure, exercise, anemia, chronic obstructive
pulmonary disorder (COPD), cystic fibrosis, congenital heart disease,
hyperthyroidism (increases basal metabolic rate, using more O2)
o CLINICAL:
 Hexokinase Deficiency – rare
 Decreased rate of glycolysis  reduce 2,3, BPG
 Left shift, increased O2 binding
 Sx: Chronic hemolytic anemia
 Pyruvate Kinase Deficiency -- common
 At end of glycolysis pathway: Reduced end steps of glycolysis but beginning
steps still moving forward
o Less PEP  pyruvate = less ATP made
o Increased 2,3 BPG
 Right Shift
 Sx: Chronic hemolytic anemia
 O2 Transport to Fetus
o Fetal Hb (α2,γ2)
 Higher affinity for 2,3 BPG; higher affinity for O2
 Transport of CO2
o As Bicarbonate: 85%
o As Carbamate: 15%
 N-terminal of Hb carbamolyated
 Stabilizes T state  bc it produces H+  Bohr Effect
o CO2 favors Low Affinity T State
 Increases H+  Bohr Effect
 Hb carbamoylation produces H+
o Bohr Effect --- CO2, H+ decreasing Hb affinity for O2
o Haldane Effect --- O2 decreasing Hb affinity for CO2
Structure and Function of Erythrocyte Membrane:
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Lipids in Plasma Membrane
o Rapid lateral diffusion, slow transverse diffusion
o Flippases exchange PLs between leaflets, require ATP
o Cholesterol in non-polar interior – increases membrane rigidity
Glycoproteins
o Carbohydrate side chain
Membrane Proteins
o Integral
 Significant part of protein inside hydrophobic membrane (nonpolar)
 Resists removal from membrane from increased salt (band 3)
 Often glycosylated on exterior surface
o Peripheral
 Bind to exterior or interior surface of membrane, no significant contact with
hydrophobic layer (polar)
 Removed by increased salt that doesn’t disrupt lipid bilayer (spectrin or ankyrin)
o Asymmetric – polar and nonpolar regions
o Transmembrane proteins – span the membrane (can be more than once)
 Ex. solute transporters
SDS-Polyacrylamide Gel Electrophoresis
o Separates based on relative size
o Procedure:
 Boil sample in SDS containing a strong reducing agent
 Denatures protein, reduces disulfide bonds
 Protein binds 1 SDS molecule per 2 peptide bonds – all proteins have same chargeto-mass ratio
 Separate in polymerized, cross-linked acrylamide gel based on MW
 Stain with Coomassie Blue
o Prep of RBC Membranes
 Put in hypotonic soln, RBC swells/bursts, releasing soluble components
 Left with RBC ghosts – plasma membrane with attached cytoskeletal proteins
 Extract cytoskeletal elements before boiling in SDS
o Isolate integral proteins only (Spectrin, Ankyrin, Actin) in a salt
solution that removes peripheral proteins
o Stain carbohydrates of integral glycoproteins with PAS stain
Major Proteins Associated with RBC Membrane – p.267
 Spectrin & Ankyrin – larger
 Anion Exchange Protein - large
 Protein 4.1
 Actin
 G3PD
 Tropomyosin
 Glycophorin

o Overall Interactions:
 Band 3 linked to Spectrin via Proteins 4.1, 4.2, Ankyrin, Adducin, Actin
 Actin/4.1/Adducin Complex linked to Glycoprotein C (and band 3?)
 Disrupting complexes = loss of vertical interaction (concave shape)
 Disrupting spectrin interactions (including Actin) = loss of Horizontal Interaction
(disc shape)
o Vertical Interaction – holds cell in concave shape
 Band 3, Band 4.2, Ankyrin
 Cytoskeleton maintains RBC biconcave shape
o Can deform to fit through narrow capillaries
 CLINICAL:
 Hereditary Spherocytosis
o Defective spectrin-membrane crosslink by mutations in:
 β-spectrin or α-spectrin
 Ankyrin or Band 3 (rare)
o Defective vertical interaction = spherical shape
o Abnormal cells cleared faster by spleen, causes splenomegaly
 Splenectomy a “cure”
o Sx: anemia, jaundice
o Horizontal Interaction – holds cell in disc shape *******CHECK IF ALL HERE
 Spectrin, Ankyrin, Adducin, Actin, Tropomyosin, Band 4.1, Glycoprotein C
 Spectrin
 2 subunits form tetramer - cytoskeletal network on inner surface on
membrane
 Links to Band 3 (via Ankyrin) and Glycoprotein C (via Actin and Band 4.1)
 Maintains shape of RBC
 CLINICAL:
o Hereditary Elliptocytosis
 Mutation: β-spectrin, α-spectrin, protein 4.1, or glycoprotein C
 Loss of horizontal interaction
 Sx: Mild hemolytic anemia
 Some resistance to malaria
o Ovalocytosis
 Defects in spectrin, band 4.2, band 3
 Sx: jaundice in newborns, Asymptomatic in adults
 Loss of horizontal interaction
 Southeast Asian Ovalocytosis:
 Point mutation – Band 3
 Heterozygotes benign, embryonic lethal in homozygotes
 Malaria resistance
o Anion Exchange Protein I (Band 3)
 Transmembrane channel glycoprotein, spans membrane 12 times
Exchanges Cl- for HCO3Both terminals cytoplasmic (N – interacts with Ankyrin, linking spectrin to
membrane)
 Associates with G3PD
RBC Integral Membrane Proteins
 Anion Exchange Protein (Band 3)
 Glycophorins
 Solute transporters
 Surface carbohydrates
 Give surface negative charge – so it won’t stick to each other & vessel wall
 Give ABO blood type
Transporters for Small Molecules
 Polar molecules/ions need help passing membrane
 Desolvation – shed shell of hydration (H2O) to cross membrane then rehydrate on
other side
 Requires a lot of energy
 Solute transporters interact noncovalently with transported molecule to
overcome energy barrier (more favorable pathway)
 O2 and CO2: (small, hydrophobic)
 Simple diffusion – pass membrane without transporter
 Polar and charged solutes
 Facilitated diffusion or Active Transport – requires transporters
o Ex. glucose (glut1), H2O (aquaporin), ions
Types of Solute Transporters
 Uniporter
 Not coupled to transport of another solute
 Driven by conc. gradient
 Ex. Glut1, aquaporin
 Symporter
 Co-transport of two solutes in same direction
 Driven by conc. gradient
 Ex. Na+/sugar transporters
 Antiporter
 Co-transport of two solutes in opposite directions
 Driven by conc. gradient
 Ex. Anion Exchange Protein (band 3)
Band 3 – Anion Exchange Protein
 ANTIPORTER
 Cl- exchanged for HCO3o Periphery: Cl- in, HCO3- out (to remove CO2 from cell)
o Lungs: HCO3- in (bring CO2 in to diffuse out), Cl- in
 Binding Sites:
 Ankyrin and band 4.1
 PGK, aldolase, GAPD at cytoplasmic N-terminal
 Carbonic anhydrase IV at cytoplasmic C-terminal
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o
o
o
o
Senescence of RBC
o Reactive O2 species damage & damaged components accumulate with age of RBC
o Damaged Hb (hemichrome) binds to N-terminal of Band 3
o Hemichrome-Band 3 complexes aggregate
o Auto IgG binds to aggregated Band 3
o Tags cells for clearance by macrophages
RBC Glucose Transport
o Glut1 – integral membrane protein
o Facilitated diffusion (passive UNIPORTER) – driven by conc. gradient
o Does not require ATP
o NOT stimulated by insulin (that is Glut4!)
Aquaporin
o Passive UNIPORTER – does not require ATP
o Permits single-file transport of H2O -- Allows cell to swell/shrink based on osmolality
Na+/K+ ATPase
o Pumps out 3 Na+ for every 2 K+ in
o More K+ inside cell
o More Na+ outside cell
o Needs ATP! Active transport bc moving against the gradient!
Other Membrane Proteins:
Glycophorins
o Give RBC surface negative charge (sialic acid)
o Prevents adhesion to other RBS and vessel walls
o Membrane spanning
o Extracellular domain glycosylated
o A – E; A is most abundant
o If lacking A or B, glycosylation of Band 3 compensates
o Receptors for malaria
ABO Blood Group Antigens
o Ag = oligosaccharides on RBC surface linked to:
o Band 3 (80%)
o Glucose transporter (Glut1)
o Other membrane proteins
o Glycosphingolipids
o Glycosyl Transferase – determines Ag
o H = Fucosyl Transferase
 H is precursor/substrate of A and B
 A & B transferases specific for terminally fucosylated H precursor
o A = N-acetyl-galactosyl Transferase
o B = Galactosyl Transferase
o People form antibodies to the oligosaccharide NOT expressed