Session 4
What are the specific methods for separating proteins depending on their size?
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What needs to happen for iron to reversibly bind O2?
it has to be maintained in its ferrous (Fe2+) form, the ferric (Fe3+) form readily bind a water molecule (H2O) and does not bind O2).
Where is myoglobin found?
mainly in muscles
At high altitudes, body makes (less/more) BPG so it can bind to the beta-chain of hemoglobin though a positively charged histidine residue. (compensates for less O2 received from atmosphere).
more
Heme is a prosthetic group for hemoglobin, ___________ and __________
myoglobin and cytochrome b, c, and c1
Stored blood has less BPG and therefore ......
needs to be reconstituted with 5mm BPG so blood will deliver O2, not just bind it.
What charge is BPG?
negative
The iron component of heme is held in position by binding to 4________ of the Protophorphyrin IX ring.
nitrogens
Does a reduced o2 availability have a great affect on the fetus?
no because metabolic requirements for oxygen are low
What happens if Glutamate is replaced with lysine in hemoglobin C?
no significant effect on Hemoglobin function
How is Heme bonded in a hydrophobic crevice in myoglobin and hemoglobin?
non-covalently
Why can proteins be separated by ionic charge?
polypeptides will move in an electric field depending on their net charge and size. At isoelectric point a peptide has no net charge and will remain stationary
In protein denaturation which structure is retained?
primary
What is the backbone of heme?
protophorphyrin IX
What is hemoglobin responsible for?
responsible for the transportation of O2 between lungs and other tissues.
in the gamma chain of fetal Hemoglobin, histidine is replaced by _______ residue and as such BPG doesn't strongly bind to fetal hemoglobin?
serine
Myoglobin is mainly responsible for what?
storage of O2 in muscle because of its very high affinity to O2
It takes 3-5 days to adjust to new altitude or produce __________ __________ to fully compensate for the shortfall in O2.
sufficient BPG
When O2 is not binding to Hemoglobin, which is the more stable state?
the T-state is more stable and is therefore the predominant confirmation
What is the clinical application of denatured proteins?
the amount of protein in urine, serum , cerebrospinal fluid etc can be used to assess for different pathological conditions
What decreases the affinity of hemoglobin for O2?
the binding of BPG (2,3 biphosphoglycerate)
What other binding of hemoglobin can cause the release of O2 and why?
the covalent binding of CO2 to hemoglobin in the tissues causes the release of O2 because carbamates are formed and the T state is stabilized, facilitating O2 release.
What does denaturation of a protein involve?
the destruction and loss of higher level structural organization (secondary, tertiary and quaternary) without causing hydrolysis of peptide bonds.
What is allosteric regulation?
the effect of a molecule binding to one subunit to a binding site on another subunit
What is the Bohr Effect?
the effect of pH on hemoglobin binding of O2
What is the the relationship between protein structure and function ?
the role of myoglobin and hemoglobin as oxygen (O2) carriers
What is an example of a heat stable protein that can have evolved to function at very high temperatures?
thermophilic bacteria
How do organic solvents, urea and detergents denature proteins?
they act primarily by disrupting the hydrophobic interactions of proteins
What is adult hemoglobin made up of?
2 alpha and 2 beta chains
What does the tertiary structure of myoglobin include?
8 alpha helices named A-H
How does extreme of pH denature proteins?
: Extremes of pH alter the net charge on the proteins (remember proteins contain ionizable amino acids that have different pKa values). Changes in net charge result in disruption of electrostatic and hydrogen-bonding interactions.
Hemoglobin can bind to _____ and _______.
CO2 and H+
Why can proteins be separated by binding specificity?
Due to their different tertiary and quaternary structures, different proteins will differentially bind to specific antibodies and complementary proteins
____________ is incorporated in heme.
Fe2+
What is more released in tissues when BPG is bound to hemoglobin?
O2
Heme is the ________________________ common to Mb and Hb
O2 - binding molecule
What is Thalassemias?
One or more genes coding for Hemoglobin chains are deleted, called alpha or beta depending on which chain is affected.
What is the consequence of Valine replacing glutamate (which results in sickle cell hemoglobin)?
Place A non - polar residue on the outside of HbS which markedly reduce solubility of deoxy HbS. But has little effect on oxy - HbS (causes Hb to clump when deoxygenated) Creates sticky patches on the outside surface of each β -chains that are not normally present in adult Hb (not present HbA) The sticky patches interact with complementary sites of another HbS (foxy) forming large aggregates that distort the whole RBC structure Thus, at low oxygen concentrations, deoxy HbS polymerizes forming fibers that distorts erythrocytes into sickle shapes. Because of their rigidity and fragility these red blood cells are destroyed in the spleen resulting in anaemia.
The binding of O2 to hemoglobin in the T state triggers a conformational change to the ______ state?
R
What are the two conformational states that hemoglobin exists in?
T (tense) and R (relaxed) states
When hemoglobin binds to H+, it stimulates the release of O2. How?
a decreased pH decreases the affinity of hemoglobin to O2 and increases its release
What is hemoglobin made up of?
a protein (globin) component and a prosthetic group called heme
What causes sickle cell hemoglobin?
a single amino acid mutation the mutation is replaced of glutamate reside on beta chain of hemoglobin with a valine residue (a charged amino acid is replaced with a non-polar hydrophobic residue) HbA= Val- His - Leu - Thr - Pro - Glu - Glu - Lys HbS= Val- His - Leu - Thr - Pro - Val - Glu - Lys
What is myoglobin made up of?
a single polypeptide of 153 animo acids and one molecule of heme which is very compact (has a globular structure)
What is fetal hemoglobin?
a tetramer of 2 alpha and 2 gamma chains
Hemoglobin needs to bind O2 with high _______ in the lung and offload the O2 in the____________.
affinity and offload in the tissues
How can proteins be separated?
based on molecular size, ionic charge, binding specificity, solubility
How is the binding of O2 to hemoglobin both homotropic and heterotropic regulation?
binding of O2 to O2 is homotropic and binding of O2 to 2,3 BPG, H+ and CO2 is heterotropic.
Hemes are dome shaped, and when O2 binds, the iron pulls down into the protophorphyrin ring and pulls down the alpha-helical F, which does what?
breaks the salt bridge
How is the T-state stabilized?
by multiple hydrogen and salt bridges along the alpha-beta interface
What does it mean when we say that hemoglobin is a tetrameric structure?
contains 4 subunits with a similar tertiary structure to myoglobin
Since myoglobin is a monomer it does not show ___________ like hemoglobin
cooperativity
Since fetal hemoglobin has a much higher affinity than adult hemoglobin, it is easier for fetal Hemoglobin to extract O2 from the placenta, and it means offloading of O2 in the fetal tissues is __________.
decreased
The coordinate nitrogen atoms prevent formation of ferric (Fe3+) state- Why?
due to their electron donating characteristic
When does Heme become an integral part of the global proteins?
during polypeptide synthesis
How does heat denature proteins?
heat breaks hydrogen-bonds and other weal interaction forces that stabilize the 3D structure of protein
What are the agents of denaturation?
heat, extreme of pH, organic solvents, urea and detergents
Each time an O2 molecule binds to hemoglobin, what happens in terms of hemoglobin affinity?
hemoglobin affinity increases each time an O2 molecule binds because there is a transition from low affinity state (T) to a high affinity state (R)
What happens if hemoglobin is placed in urea?
hemoglobin breaks into alpha-beta dimers suggesting that alpha-beta interactions are much stronger than alpha-alpha and beta-beta interactions
Hemoglobin has a (high/low) affinity to carbon monoxide?
high- 200 times higher than O2 which explains why carbon monoxide is extremely dangerous
O2 has a significantly (higher/lower) affinity for hemoglobin in the R (relaxed) state as compared to the T (tense state).
higher
Do smokers have higher or lower BPG levels as compared to non smokers?
higher because in smokers, carbon monoxide ties up some hemoglobin and decreases O2 availability
What is the process of renaturation?
if a denatured protein returns to its native state after the denaturation is removed
Where is hemoglobin found ?
in erythrocytes (red blood cells)
If O2 binds to two free heme molecules, this would result in a permanent conversion of ____________________. This is prevented by sequestering the heme deep within the protein structure thereby restricting access to the two remaining bonding sites for iron.
iron to the ferric state
Because fetal hemoglobin does not bind to BPG, what does that mean in terms of affinity for O2?
it has a much higher affinity for O2 as compared to adult hemoglobin
What are the properties of a denatured protein?
they have reduced solubility and pronounced propensity for precipitation- this occurs due to loss of the hydration shell and the unfolding of protein molecules with concomitant exposure of hydrophobic radicals and neutralization of charged polar group. configurational alteration of the protein molecule Loss of biological activity evoked by the disarrangement of the native structural molecular organization
What is heterotropic regulation?
when a ligand influences the binding of molecules for a different molecule.
What is homotropic regulation?
when a ligand influences the binding of molecules similar to itself
When Salt bridges that stabilizes the T-state are broken and new ones are formed does the state change?.
yes to the R state
