RBC2 Flashcards
Transport of CO2 as Carbamate
(where does CO2 bind on hemoglobin to be transported in RBC, what state of Hb does this stabilize, and what percent of CO2 is transported this way)
Transport of CO2 as CARBAMATE
-The “N-terminal” amino group of Hb gets carbomylated
-This stabilizes the LOW Affinity “T-state”
(so Hb is has lower binding affinity for oxygen and releases the oxygen into the cells in peripheral tissues due to the CO2 from those tissues binding to Hb as well as due to other effects)
-About 15% of CO2 transport occurs this way (as carbamate)
Bohr Effect (due to increase CO2)
CO2 increases the H+ concentration (it decreases the pH) and this decrease in pH (increase in H+ conc) results in hemoglobin having lower affinity for oxygen. Therefore the Bohr Effect is decrease in pH, lower affinity of oxygen to Hb, and SHIFTS CURVE TO RIGHT (since lower affinity of Hb to oxygen = right shift)
NOTE: This pH change is INSIDE THE RBC (we are NOT talking about changes in pH in the blood or outsdie of the cells, but just inside the red blood cells!)
Carbamoylation (due to increase CO2)
N-terminus of Hb gets carbamylated (CO2 gets added onto the amino end), and this stabilizes the “T state = reduced affinity of Hb for oxygen” which leads to SHIFT TO RIGHT of Hb/Oxygen curve on graph, causing oxygen to be released due to lower affinity.
This method of carbamoylation will further shift curve to the right (even if pH is constant)…the pH lowering due to CO2 that shifts curve to right is Bohr effect, and this carbamoylation is another way CO2 reduces Hb affinity for oxygen/shifts curve to the right. Both will occur on RBCs when higher concentration of CO2 is present (in peripheral tissues)
O2 and CO2 Transport - Summary
Erythrocytes in Lungs:
-Higher pH (lower H+ conc), High O2, Low CO2 …all these three lead to O2 binding to Hb, CO2 released from Hb carbamate, and HCO3- (bicarbonate) converted into CO2 (which will be exhaled as air leaving the lungs)
Erythrocytes in Pheripheral Tissue:
-Lower pH (higher H+ conc), Low O2, High CO2….all these three lead to O2 released from Hb, CO2 carbam0ylates Hb, CO2 converted to HCO3- Ibicarbonate)
Most important function of Erythrocytes (most important function carried out by Hb)
TRANSPORT OF O2 AND CO2!
Structure and Function of the Erythrocyte/RBC Membrane
**Lipids in the Plasma Membrane (Slide)
Lipids in the Plasma Membrane:
- Sides of membrane (inner and outer) have DIFFERENT lipid compositions; affects properties
- LATERAL diffusion (molecules/lipids moving side-to-side across membrane) is RAPID
- TRANSVERSE movement is VERY SLOW (going from top to bottom/facing inner side to facing outerside will be slow and require special enzymes/flippases or floppases that require Energy)
- “Flippases” exchange Phospholipids (PLs) between leaflets (inner and outer leaflets of membrane); they require ATP
- Cholesterol is in the NONPOLAR INTERIOR (it is partly amphipathic due to it’s head like we know…so similar to miscelles the unesterified cholesterol we are talking about here will have the top part on the edge of the membranes/both sides and the long hydrophobic tail in the middle of the membrane bilayer….so cholesterol is considered to be in Hydrophobic/nonpolar INTERIOR/middle of the cell membrane bilayer)
- **Phosphotidyl-SERINE, -INOSITOL, -ETHANOLAMINE will be facing inside of cell/inner leaflet of membrane!
- **Phosphotidyl-CHOLINE will be on the outer leaflet/facing outside of cell (facing extracellular space), along with Sphingomylin, and Glycolipid
- Cholesterol (which is on both sides of membrane with tail in the middle) is in NONPOLAR INTERIOR; cholesterol INCREASES RIGIDITY of the cell membrane
MEMBRANE PROTEINS
INTEGRAL (membrane proteins):
-Significant part of the protein is INSIDE the membrane
-Contacts hydrophobic layer via NONPOLAR AMINO ACID SIDE CHAINS
-Resists removal from membrane by increased salts
(So integral proteins are firmly embedded/anchored to hydrophobic core and won’t be remove if you change salt concentration)
EXAMPLE OF INTEGRAL membrane protein is “Band 3”=multitasking protein in erythrocytes that has multiple functions
vs.
PERIPHERAL (membrane proteins)
- Bind to the exterior OR the interior of membrane
- No significant contact with the hydrophobic layer
- Removed (easily removed) by INCREASED SALT that does not disrupt the lipid bilayer
EXAMPLEs OF PERIPHERAL membrane proteins are “Spectrin”(=part of cytoskeleton) and “Ankyrin”(=peripheral protein that binds to Band 3)…both easily removed by increasing salt.
Membrane Proteins
- Membrane proteins are ASYMETRIC
- Integral membrane proteins are frequently glycosylated on exterior surface
- Transmembrane proteins span the membrane
- Solute transporters are examples
- Hydrophobic side chains exposed to the hydrocarbon layer of the membrane
- Significant intra- or extracellular domains exposed to cytosol or extracellular fluid.
How do we know/study the PROTEINS (that are on red blood cell membranes)…name and describe the lab technique
SDS-Polyacrylamide Gel Electrophoresis
- SDS=sodium dodecyl sulfate (strong anionic detergent)
- Boil sample in SDS containing a STRONG REDUCING AGENT
- Denatures protein; reduces disulfide bonds; solubolizes lipids
- Protein binds 1 SDS molecule per 2 peptide bonds; all proteins have essentially same charge-to-mass ratio
- SEPARATED in polymerized, cross-linked acrylamide gel BASED ON MOLECULAR WEIGHT
- Stain with “Coomassie Blue”
Describe what we do when using SDS to study RBC/erythrocyte CELL MEMBRANE proteins?
- Since majority percent that makes up RBC is mainly all hemoglobin, and we want to study the membrane proteins (not all the hemoglobin on the inside), we first rupture the cell.
- We go from isotonic solution to “hypotonic solution” to rupture the cell membrane…the erythrocyte swells/bursts/releases soluble protein components
- It leaves behind the “Erythrocyte Ghosts”=erythrocyte plasma membrane with attached cytoskeletal elements (all these attached membrane proteins will still be on the membrane since we keep it at low salt)….and now we can take this, boil in SDS, etc to study the membrane proteins.
**Spectrin (alpha form is band 2, beta form is band 2), Ankyrin (band 2.1-2.6 due to different forms), and Actin (band 5) are all cytoskeletal proteins (all are PERIPHERAL PROTEINS) that are part of the erythrocyte membrane/Ghost…so we can now take high salt concentration to extract these peripheral proteins out from the ghost membrane to study these peripheral cytoskeleton components….Spectrin is largest (so will be at top of cystoskeletal gel/blot/image….Ankrin is second largest of the three (also close to the top but below spectrin)…and then the rest of proteins are smaller… a lot further toward the bottom is Actin (since it is very small peripheral/cytoskeletal protein)….so larger band number=closer to bottom=smaller protein molecule
Two key INTEGRAL proteins we should know:
***BAND 3 (= “anion exchange protein”) is an INTEGRAL PROTEIN
***“Glycoporins” are SMALLEST and are INTEGRAL membrane proteins. (no band listed but toward bottom)
Cytoskeleton maintains erythrocyte shape
Cytoskeleton maintains erythrocyte shape
- Erythrocytes deform to fit through narrow capillaries
- In venules, they return to original shape (disk shape)
- Resistance to shape change depends on the cytoskeleton
Spectrin
- Long, 200nm fiber
- made of two subunits that form a tetramer (alpha2, beta2)
- part of cytoskeleton on INNER face of the membrane (so Spectrin is an inner face, peripheral/cytoskeletal membrane protein)
- Spectrin links to Band 3 (via ankryin) and it links to glycoporin C (via actin and band 4.1)
- Spectrin maintains shape of erythrocyte
Hereditary Spherocytosis
40min.42sec finish
list diseases
after 40.42 a few diseases (horizontal vs vertical changes and stuff) were mentions
HERE IS WHAT YOU SHOULD KNOW
Here is what you should know:
- Glycophorin C linked to spectrin via adducin. Actin and other proteins also involved
- Spectrin linked to Band 3 and Glycophorin A via ankyrin/protein 4.2 complex
- Disrupting above complexes leads to loss of vertical interactions (= spherocytosis)
- Disrupting spectrin/spectrin interactions leads to loss of horizontal interactions (=eliptocytosis or =ovulocytosis). Band 3 and 4.2 also involved.
