David Biochem Flashcards
Describe Myoglobin.
- monomer
- muscle cells
- takes O2 from the bloodstream and distributes it to muscles
Describe Hemoglobin.
- tetramer
- red blood cells
- takes O2 from our lungs and delivers it to the periphery and back
What is the primary function of Mb and Hb?
Oxygen binding
What was the first protein to be crystallized?
Sperm whale myoglobin
What is the secondary structure of myoglobin?
- it is made up of 8 alpha helices labelled helix A->H
What is the key functional group of myoglobin?
the heme group
What is heme?
- a porphyrin derivative
- made of 4 pyrrole rings
- heme is a planar molecule
Describe the structure of a pyrrole ring?
- 5 membered ring with 1 nitrogen molecule
What is the purpose of pyrrole nitrogen?
Pyrrole N is critical to the function of heme because that is where iron binds
What are the 6 things Fe binds to in a heme in myoglobin?
- 4 pyrrole Ns
- 1 histidine
- O2
Describe the heme complex in myoglobin?
Valine and phenylalanine are two hydrophobic groups that pack against porphyrin and keep your heme groups in place in the protein
Show the reaction that Fe participates in in an isolated heme.
Fe(II) + O2 —> Fe(III) - O - O(-)
- Fe is reduced in the beginning, it donates electrons and gets oxidized in this reaction
- Oxygen accepts electrons and gets reduced in this reaction
What does an isolated heme mean?
A heme not found in myoglobin
What does the protein portion of myoglobin cause?
The protein portion of myoglobin makes O2 binding reversible
In myoglobin what is oxygen binding dependent on?
Binding is dependent on [O2]
- O2 concentration
What else can a heme bind to?
- heme can bind to CO, NO, H2S
- heme binds to these molecules with greater affinity than O2, and this binding is not/not as reversible
- CO poisoning
What does Mb primarily facilitate?
- O2 diffusion into muscle
Describe seal/whale Mb.
- In seals and whales Mb stores O2
- these animals (marine mammals) have 50x higher Mb than humans
What is fractional saturation?
- YO2
- amount of oxygen bound myoglobin over total myoglobin
- (Percent of total Mb bound to oxygen)
What do you graph in an oxygen binding curve?
- YO2 (fractional saturation/percent oxygen bound) on Y axis
- pO2 - partial pressure of oxygen on X axis
What is the dissociation constant for myoglobin?
k = [Mb][O2] / [MbO2]
What is pO2?
partial pressure of O2 / oxygen tension
- measured in torr
What do low and high pO2 indicate in general about YO2?
Low pO2 = low Yo2
High pO2 = high Yo2
What is P50?
- the value of pO2 where 50% of the total Mb is bound to oxygen
What does a lower P50 value indicate?
higher affinity to O2
- less oxygen pressure is required for 50% of Mb to be bound to O2
What is the equation for fractional saturation for Mb?
YO2 = (pO2) / (P50 + pO2)
How is YO2 affected in Mb when there are lower pO2 levels?
When pO2 levels are low, and they change there is a more dramatic change in Yo2
When is O2 released?
As pressure changes from arterial to venous blood
How much O2 is released when Mb travels from arterial to venous blood?
- 6% of bound oxygen is released
- not very efficient at releasing oxygen in our venous system
What is hemoglobin primarily responsible for?
- responsible for O2 transport in blood
- found in red blood cells
Describe the makeup of RBCs.
- 34% of their weight is hemoglobin
- they do not have a nucleus or organelles
What is the general structure of hemoglobin?
alpha2 beta2 tetramer
Describe the similarity of the alpha and beta subunits.
- between alpha and beta there is little 1 degree sequence homology, but high structural similarity (tertiary structures are similar)
How does O2 binding impact hemoglobin?
O2 binding causes a dramatic structural change
What occurs when crystallized deoxy Hb is exposed to O2?
The change in structure will cause it to shatter
What structural changes occur when deoxyhemoglobin turns into oxyhemoglobin (oxygen binds)?
- beta subunits move closer together
- contacts between alpha1-beta2 and alpha2-beta1 change
- there is 15 degree rotation of the alpha beta dimer
Describe the O2 - Mb binding (general).
- hyperbolic O2 binding curve
- non-cooperative (each binding event is independent of the other)
Describe the O2 - Hb binding (general).
- sigmoidal O2 binding curve
- cooperative binding (binding at one site affects other sites)
Describe the sigmoidal binding curve.
- high oxygen bound at high PO2
- low oxygen bound at low PO2
- releases O2 when at low arterial pressure
What is the hill equation?
- Equation that describes YO2 for hemoglobin
What type of binding curve does Hb follow?
Sigmoidal
What is the Hill coefficient and what does an increase Hill coefficient indicate?
- the value of n increases with the degree of cooperativity
What is n = 1, n > 1, and n < 1?
- N = 1 indicates non-cooperative binding and a hyperbolic binding curve
- N > 1 indicates a positively cooperative binding pattern and O2 affinity increases as O2 sites are occupie
- N < 1 indicates a negatively cooperative binding pattern in which binding to one site reduces affinity for other sites
What is the linear hill equation?
For a linear hill equation, what is the slope and what are the x and y intercepts?
- slope = n
- y-intercept = -nlogP50
- x-intercept = logP50
What is the y-axis and the x-axis in an hill graph?
y-axis = log(Yo2 / (1 - Yo2)) x-axis = logpO2
What are the 3 zones of Hb linear hill graph?
1. YO2 < .1 y-axis < -.095 non-cooperative slope = 1 p50 = 30 torr 2. .1 < YO2 < .9, -.095 < y-axis < .095 cooperative slope = 2.8 p50 = 26 torr 3. .9 < YO2 .095 < y-axis slope = 1 p50 = 0.3 torr non-cooperative
How is binding at one site affected by binding at another?
- binding information is transferred from one site to another through protein movement
- conformational changes
What is T and R Hb?
T - no O2, deoxyHb
R - O2 bound, oxyHb
What structural changes occur in the T-R transition?
- Fe(II) moves into the plane
- Iron drags Histidine F8 (bound to iron) -> causing the F-helix to tilt
- Shifts alpha 1, beta 2 and alpha 2, beta 1 interfaces
- c-termini ion-pair interactions: the electrostatic interactions holding the molecule together are disrupted
What structural changes occur in the T-R transition?
- Fe(II) moves into the plane
- Iron drags Histidine F8 (bound to iron) -> causing the F-helix to tilt
- Shifts alpha 1, beta 2 and alpha 2, beta 1 interfaces
- c-termini ion-pair interactions: the electrostatic interactions holding the molecule together are disrupted
What 3 things affect T ->R transition?
- pH (Bohr effect)
- CO2 levels
- BPG
Explain the Bohr Effect.
- O2 affinity increases as pH increases
- electrostatic interactions involved the N-term amino group and the Histidine residue in C-term (too high of a pH de-protonates these molecules)
- as pH increases salt bridges are broken, which favors the R-state (higher O2 affinity)
What are the two ways that CO2 levels can affect T - R?
- the Bohr Effect (pH)
- covalent modification
What is the acid/base reaction that occurs in our blood and what is it catalyzed by?
[CO2] [HCO3-] + [H+]
This conversion is catalyzed by carbonic anhydrase
Describe CO2 levels in the muscle and how this impacts T - R.
- In the muscle there are HIGH CO2 levels, this increases bicarbonate and H+
- Increase in H+ lowers pH
- Lower pH lowers O2 affinity, more T state Hb
Describe CO2 levels in the lungs and how this impacts T - R.
- In the muscle there are LOW CO2 levels, this decreases bicarbonate and H+
- Decrease in H+ increases pH
- Higher pH increases O2 affinity, more R state Hb
Explain covalent bonding of CO2 in Hb.
- covalent modification of N-term (of T-state Hb) by CO2
- this occurs when CO2 is high
- shifts towards more T-state, lower O2 affinity
Which state of Hb is more likely to be modified by Hb binding of CO2?
T-state
Which state of Hb does BPG bind to and why?
- binds to T state
- T - R transition narrows binding pocket so BPG can no longer fit
How does BPG bind to Hb?
non-covalently
How do high levels of BPG impact T - R equilibrium?
- Increase in BPG increases the T-state
- BPG binds to the T state and causes the equilibrium to shift towards the T - state
What happens to our blood at high altitude?
- BPG concentration in our bloodstream increases when we go from low to high altitude
- This is because arterial pressure drops at high altitude, so BPG concentration must increase to increase the P50 value
Describe the structural differences in fetal hemoglobin.
- has alpha2, gamma 2 subunit instead of alpha 2 beta 2
- Has a serine instead of a histidine
- Histidine is important for BPG binding so fetal hemoglobin doesnt bind BPG
Describe the different function of fetal hemoglobin.
- Fetal hemoglobin doesn’t bind BPG
- During pregnancy the mothers BPG is increased so mothers blood has a lower affinity for O2, but fetal Hb is not affected by this increase bc it doesn’t bind BPG
- ensures transfer of O2 from mother to fetus
What is the mutation in sickle cell anemia?
- A glutamate to valine
- causes 2 mutant beta globin chains
- the chains interact and cause the cells to sickle
- valine binds a hydrophobic pocket in another Hb unit which leads to aggregation of Hb molecules and linear polymer structures
What is sickle cell Hb called?
Hbs
What must heterozygous sickle cell patients have? Homozygous?
Heterozygous - trait
Homozygous - disease
When does aggregation of Hb molecules occur in sickle cell disease?
When O2 levels are low
How are sickle cell patients treated and what does this do?
- treated with hydroxyurea which increase expression of fetal Hb
- can replace deficient betaS form
What do sickle cell heterozygotes have?
protection from malaria
Explain how heterozygotes have protection from malaria.
- malaria is caused by plasmodium which infects RBCs and lowers their pH
- lower pH causes hemoglobin to shift to T-state
- carriers express some Hbs, as T state increases Hbs aggregates and parasite infected RBCs sickle (lower oxygen affinity state)
- spleen can selectively remove sickled cells that are infected
What is the allosteric effect?
- binding of a ligand at one site affects binding of another ligand at a different site
What are the two models for the allosteric effect?
symmetry model and sequential model
What is the symmetry model?
- the 4 subunits can only exist as 4 in the T-state or 4 in the R-state
What is the sequential model?
- binding in one section causes small conformational changes in adjacent subunits