B7/12 RBCs and Hemoglobin Flashcards
General strcture of RBC
-biconcave shape (thinner in the middle for a large diffusion SA
-flexible to squeeze through blood vessels
Describe the sequence of erythropoiesis + changes that occur throughout it
- proerythroblast
- early and then late erythroblast
- normoblast
- reticulocyte
- FINAL ERYTHROCYTE
CHANGES:
-accumulation of Hb
-ejection of nucleus (between 2/3)
-loss of ribosomes (between 4/5)
Describe the positive ad negative regulator of erythropoiesis
POSITIVE:
-EPO
-testosterone (increases kidney EPO production kidney)
-low blood O2
NEGATIVE:
-increasing oxygen carrying capacity (for negative feedback regulation)
What 3 factors are needed for the correct progression of erythropoiesis (+ role)
- EPO (and other cytokines) - have most receptors expressed on progenitor cells
- Folic acid
- B12
(a lack of which cause abnormal / diminished DNA and hence failure of nucleur maturation and cell division)
Describe the synthesis and regulation of release of EPO
SYNTHESIS:
-glycoprotein, 199 aa
-released from kidney in response to low O2 (stimulated by tissue hypoxia)
ROLE:
-binds to dimerised EPO receptors on progenitor cells and leads to signal EPOR phosphorylation: prevention of erythroid apoptosis + establishes normal biological function
REGULATION:
NORMAL O2: HIFa (hypoxia inducible factor) is hydroxylated, ubiquitinated and then proteosomally degraded (no activation)
HYPOXIA: HIF is no longer degraded and hence activates the EPO genes to increase EPO production (for increased RBC production)
physiological hematorit levels
HCT: 43-53% male and 37-43% female
(ratio of blood cell to non cellular content)
describe the composition of phospholipids on RBC membrane
-mainly cholesterol and glycolipids (containing RBC antigens)
ASSYMETRICALLY ARRANGED:
-PC/SM = outer leaflet
-PE/PS = inner leaflet
(which explains why a triggering of PS from inner to outer leaflet can cause apoptosis)
Describe the structure of RBC membrane
- INTEGRAL PROTEINS:
-2 families: GLYCOPHORINS (negative charge to avoid clumpng) and BAND 3 proteins (transport)
- extracellular domains are glycosylated (determines blood type)
- PERIPHERAL PROTEINS:
-found intracellularly
-SPECTRIN alpha and beta run parallel to cell membrane –> can be remodelled to alter cell shape and allow flexibity. Connect membrane and cytoskeletal elements.
2 protein complexes that anchor spectrin to bilayer:
ADDUCIN/4.1 PROTEIN complex: bind to
ANKYRIN/4.2 PROTEIN complex: bind to spectrin beta
RBC lifespan
120 days
What are the functions of RBC enzymes
-provide energy for metabolic reactions
-membrane integrity
-maintenance of Hb (in ferrous and not ferric state)
-ion gradients
-biconcave shape maintenance
!! all of these are crucial for RBC to survive its 120 day lifespan under physiological conditions
General summary of RBC metabolism:
!! only use glucose not FA bcos no mitochondira (and also insulin independent bcos they have GLUT1/3 receptors)
INITIALLY: all glucose is phosphorylated into G6P
- 90% G6P: anaerobic glycolysis to produce ATP and lactate (transport to liver to keep glycolytic flux)
- Deviation of glycolysis for 2,3BPG formation, affects Hb affinity for O2
- 5-10& G6P: enters PPP for NADH production (which helps antioxidant mechanisms using glutathione but DOESNT make FAs bcos RBCs dont have mitochondria)
Describe the antioxidant function of RBCs
REDUNDANT( for max protection);
-NADPH made from PPP allows conversion of GSSG (ox) to GSH (red)
-the large amounts of reduced glutathione produced allow antioxidant activity (eg. against superoxide ions, H2O2 which could result in defective Hb)
Describe the process of 2,3 BPG formation
DEVIATION OF GLYCOLYSIS:
-triose is diverted through a shunt
-formation of 2,3 BiPhosphoGlycerol via BPG mutase
!! VERY NEGATIVE MOLECULE bcos it has one negative charge and 2 phosphate groups –> slots into the allosteric pocket of HB and is a negative modulator for the Hb/O affinity
How are aged RBCs destroyed
general characteristics:
-more rigid
-slower glycolysis and falling ATP levels
-membrane lipid reduction
Apoptosis and phagocytosis: PS flipped to outer leaflet, certain phagocytic markers brought to the extraceullar space, causes ingestion via macrophages (spleen and liver)
What is redundancy?
the same response being elicited by multiple different mechanisms - allows regulation, and the continuation of the function is one of them is stopped / defective / mutated
Describe the structure of Hb
-globular protein
TETRAMER OF 4 GLOBIN SUBUNITS: each one containing 153 aa, an N and C terminal, and a heme prosthetic group
DIMER OF a/b units
-2 idential alpha globins
-2 identical beta globins
!! dimerisation isnt via covalent bonds, so energy input isnt required to break them/ change the structure
-contains a central allosteric site (pocket) with positive lateral side chains to bind to 2,3BPG
FAVISM (clinical drop)
- genetic deficiency in G6PDH (protects RBC from oxidative damage)
-upon Fava bean consumption, oxidative stress is triggered leading to hemolysis (can be mild or severe manifestations)
What are the isoforms of Hb
Hb A -95% adult
Hb B - 2-5% adult
Hb F - fetal Hb
compare and contrast Hb and myoglobin
- both are globular proteins of the globulin family and have an almost identical structure
- only Hb has allosteric site for 2,3BPG binding
- myoglobin is found in skeletal muscles are O2 reserve and is stained dark red/black, Hb found in bloostream
- Mb has a higher affinity for O2 than Hb (curve for Mb shifted left and up)
Why do we need Hb?
relying solely on diffusion would be completely insufficient for the pO2 in arterial blood
HENCE: Hb is needed to increase the saturation of O2 and avoid hypoxia (by almost 67 times)
describe the structure of a heme group
-4 PYRROLE rings each linking to a N atom
-the 4 N atoms linked to a central Fe ion in the ferrous state (2+)
-pyrrole rings held together laterally via metine bridges
-each pyrrole group has certain side chains to induce molecular assymetry (methyl, vinyl, propionate groups)
!!! POLAR GROUPS: presence allows Hb glycosylation which is important for catabolism
describe the binding of a heme group with Hb subunit
-HisF8 and HisE7 residues on the F and E helix allow the binding to Fe2+ and stabilisation to O2 for binding
-interactions are DATIVE COVALENT: 6 dative covalent bonds formed: 4 on the flat plane of the porphyrin ring and 2 on the perpendicular
describe the colour absorbption of oxy and deoxyHb
- oxyHb has two absorption peaks: roughly at 540 and 580
- deoxyHb only has one absorption peak: at 560
reason: peaks occur due to the electronic orbital overlapping due to the presence of O2, and the distance between the two peaks indicates the distance between the two electron clouds
(no peak for deoxyHb bcos there is no O2)
describe the model for O2 binding on Hb
COOPERATIVE BINDING
-conformational change due to angle change of the ab dimers due to binding of O2
!! binding is an equilibrium between the 2 Hb states:
T (Tense) STATE: unliganded which has low affinity for O2
R (relaxed) STATE: liganded and high affinity for O2
-O2 binding shifts the 4ary structure of Hb from T to R state, and since R state is favoured the capability to bind further oxygen increases
!!! 4 equilibriums are set up bcos there are 4 hemes for O2 binding
-this creates the sigmoid curve of O2 association/ dissociation
-the equilibrium can be changed by allosteric modulators either +vely ot -vely
Describe the Hb-O2 dissociation curve
SIGMOID CURVE:
-p(100) in lungs with 100% saturation
-p(50) = 50% saturation
-p(1%) in respiring tissues = <10% saturation
HENCE: shows that Hb is very efficient in O2 carrying (at high torrs) AND very efficient in O2 release at tissues (at low torrs)
what is the Bohr effect?
the effect that a specific condition/concentration of a specific molecule exerts on the affinity of Hb for O2 and hence the changes of the O2 dissociation curve
-negative modulators: decrease affinity of Hb (hence increased dissociation) –> shift of curve right and down
-positive modulations: increases affinity (we dont have many bcos we WANT efficient O2 release at tissues) –> shift of curve left and up
What is the effect of pH on Hb saturation
H+ = nagative mod (acidic pH means right)
-pH drop due to CO2 released by respiring tissues, which has a higher affinity for Hb than O2
-CO2 spontaneously forms carbamate using N terminal of Hb (to make carbaminoHb), and is also incorporated into the buffer system
What is the effect of 2,3 BPG on Hb saturation
allosteric negative mod = more bound, to the right
-binds to the central allosteric site due to the lateral positive chains and the negativity of the BPG molecule
!!! 2,3 BPG doesnt bind to fetal Hb
What is the effect of TEMP on Hb saturation
temp = negative mod (higher temp means right)
-this can increase in the case of fever or decrease with higher altitudes (eg, mountains)
what is the catabolism of 2,3BPG in the RBC?
reenters the glycolytic pathway (made into 3PG in the adjacent step)
-used for the formation of pyruvate and then either lactate, or breakdown by the TCA cycle into water and CO2
describe the reations involved in the CO2 buffer system
- CO2 enters RBC
- CO2 hydrated to H2CO3 (carbonic acid) by carbonic anhydrases - zinc metalloenzymes
- H2CO3 becomes in equilibrium down into HCO3- and H+
4 H+ interacts with deoxyHb and HCO3- can be transported out of cells via Cl- shift for purposes of electrochemical balance
Describe the effect of CO on Hb
REVERSIBLE BUT HUGE SHIFT TO THE LEFT –> dissociation to tissues is tiny
-CO binds to Fe2+ and increases the O2 binding affinity for heme groups
TREATMENT: 100% O2 for a few hours for the O2 to displace CO from heme
SYMPTOMS: headache, dizziness, drowsiness and confusion
what are methemoglobins + what is their effect
-Hb derivative arising from the oxidation of the Fe ion from ferrous to ferric (2 to3+)
-Unable to bind O2, hence in high levels MetHbs cause cyanosis
-oxidation can either be ingerited (and lead to genetic Methemoglobinopathies) or as a result of ox. stress
CURVE: causes left shift
what is S-nitrosylated Hb
-NO creversibly binds to cysteine residues in the globin part of Hb
-improvement of O2 peripheral delivery, and influence on vascular resistance, BP and resp control
types of mutations that can occur and the diseases that they cause
POINT MUTATIONS: can be silent, but can also cause:
-methemoglobinemia
-sickle cell
-hemolytic anemia
Describe the affinity of fetal Hb
-2 alpha and 2 gamma units
-higher affinity for O2 than Hb (curve shift to the left) –> this is bcos beta subunit in Hb has more positive charges than gamma unit, hence binding more 2,3BGP which negatively modulates affinity
-this enables fetus to retrieve O2 from mothers circulation via placenta
Sickle cell anemia (clinical point)
SEVERE HEMOLYTIC ANEMIA: a single aa in HbS is changed (glutamate becomes valine) - creation of a hydrophobic patch which alters the shape of RBC from biconcave to sickle shape
!! seen often in africa, and has actually been linked with malaria resistance (in the heterozygous state)
