8. Hemoglobin Flashcards
which protein binds O2 to a heme group for transport in the blood
hemoglobin
what is hemoglobin
a protein in red blood cells used for oxygen transport
what is oxygen bound to in hemoglobin
heme group
how many times bigger is hemoglobin than myoglobin
4x bigger
what’s the molecular weight of Hb
Mr=64,500
how many polypeptides make up Hb
4 (it’s a tretramer)
T or F: each of the 4 polypeptides in Hb have a heme prosthetic group
true
describe the types of polypeptides that make up Hb
two chains are alpha type, two are beta
T or F: the two subunit types of Hb (alpha and beta) look very different in their tertiary structure
false; they look very similar
what naming system do we use for the Hb helices
the same naming system as Mb
why do we use the same naming system for Hb and Mb
the Hb monomers are very similar to Mb in tertiary structure
T or F: the primary structures of Hb and Mb are very similar
false; only 27 of 150 positions are the same
does Hb have distal and proximal His like Mb does
yes
in the quaternary structure of Hb, which subunits have strong interactions
the unlike subunits (alpha with beta)
give an example how the unlike subunits of Hb have strong interactions between them
alpha1beta1 interface has 30 interacting residues
T or F: it’s easy to separate the a1 monomer and the b1 monomer in Hb
false! they are the unlike subunits and have strong interactions
T or F: it’s easy to separate a1b1 dimer from the a2b2 dimer of Hb
true; mild reagents can be used
what are the interactions between the unlike subunits of Hb
salt bridges, hydrogen bonds, etc
how many conformations does Hb have
2
what are the conformations of Hb
T (tense) and R (relaxed)
which conformation of Hb has a hollow center
T
which conformation of Hb has a filled in center
R
T or F: oxygen will bind to heme when Hb is in either conformation
true
which conformation of Hb has a significantly higher affinity for O2
R
describe what occurs when O2 binds to Hb in the T state
O2 binding in the T state triggers a conformational change of that subunit to the R state
which state is most predominant form for oxyhemoglobin
R
which state is most predominant form for deoxyhemoglobin
T
in which conformation is the heme porphyrin ring slightly puckered
T
what is the outcome of the heme ring being puckered in the T state
the heme iron protrudes a bit towards the proximal his (His F8)
what happens to the heme ring when O2 binds to the puckered ring in the T state
binding of O2 causes the heme to be more planar, and this shifts the proximal his, the entire F helix, and the relative position of the entire subunit (domino effect of movement= conformational change of T to R)
how does binding of the O2 to the puckered T state change the subunit interactions
the a1b1 and a2b2 dimers rotate 15 degrees with respect to one another (dimers themselves are relatively unchanged), and we’re now in the R conformation
T or F: Mb and Hb have very similar ligand binding curves
false; they’re significantly different
why would the ligand binding curves of Mb and Hb be different
because one is more suited for O2 storage, and the other for O2 transport
describe ligand affinity and Kd for Mb
high ligand affinity with a low Kd (very steep curve right at the beginning)
describe the implications of Mb having a high ligand affinity and low Kd
it’s very good at keeping O2 bound to the heme and only releases O2 under very low pO2 conditions
why would Mb make a poor transport molecule
low Kd=high affinity for O2, so it wouldn’t want to let it go when it’s time to release it
what is the shape of the Hb ligand curve
sigmoidal (S shape kinda)
what does it mean when a curve is sigmoidal (in regards of ligand affinity)
the binding of one ligand increases the likelihood of a ligand binding to another occupied site
what does the sigmoidal curve represent
represents a hybrid curve as the protein transitions from an initial low affinity state to a high affinity state as more O2 is bound
for Hb, what conformation is the low affinity state and which is the high affinity state
low affinity state: T
high affinity state: R
which part of the body would have low pO2
tissues
which part of the body would have high pO2
lungs
why can’t Mb produce a sigmoidal curve (2 reasons)
it’s a monomer with only one binding site, and each molecule of O2 binds independently
define positive cooperative binding of a ligand
the O2 affinity of Hb increases as each O2 binds to each subsequent subunit
describe the process of cooperativity
O2 weakly binds with deoxyhemoglobin subunit 1 in the T state. Change of subunit 1 from T to R. A change in the subunit interface between subunit 1 and other subunits. O2 has a higher affinity to bind to subunit 2
is cooperativity direct or indirect
indirect
why is cooperativity indirect and not direct
it all happens through conformational changes in quaternary structure and interactions between subunits, because heme groups are too far away from each other for direct interactions
where in the body are Mb and Hb both fully saturated
in the lungs
in the tissues, which of Mb and Hb have a lower O2 affinity
Hb has a much lower O2 affinity than Mb in the tissues
because Hb has a lower affinity for O2 in tissues, what does Hb do in tissues?
it will pass some of its O2 to Mb for effective diffusion to tissues and storage
T or F: Hb fraction saturation (Y axis) stays relatively the same when Hb reaches the tissues from the lungs
false; it is really sensitive over that range
why is Hb sensitive when it reaches the tissues
tissues have different pO2 levels depending on their activity, so when Hb reaches a really active tissue it will release much more O2 for us
what is the equation for ligand association when there are multiple binding sites
P + nL = PLn
what is the Hill equation
log(y/1-y)=nlog[L]-logKd
what would a hill plot have on the x axis
log[L]
what would a hill plot have on the y axis
log(y/1-y)
what would the slope be on a hill plot
nH
what values will nH have
between 1 and 4
what would it mean if nH was 1
no cooperativity. The four subunits do not communicate or influence each other
what would it mean if nH was 4
full cooperativity. the four subunits fully cooperate (all protein binding sites bind ligand simultaneously)
what would it mean if nH was between 1 and 4
partial cooperativity. The subunits influence each other and promote more O2 binding, but Hb can exist as partially saturated
what would it mean if nH was less than 1
negative cooperativity
since we’re dealing with pO2 and not [O2], what is the new hill equation
log(y/1-y)=nlog[pO2]-log(P50)
what is the fraction saturation for a hill plot
0 on the y axis (you get this when you plug 0.5 into the left of the hill equation)
T or F: on a hill plot, when you use 0 on the y axis to find fraction saturation, the x axis represents Kd
false, it represents log of Kd
what are the two models that describe the transition from T to R
concerted model and sequential model
what happens in the concerted model of T –> R
all subunits transition from T to R simultaneously. When one subunit goes, they all go
describe the process of the concerted model
O2 binding can happen in either conformation, but the successive binding makes the transition from T to R more likely. At each level of O2 loading, an equilibrium exists between T and R states. The equilibrium shifts favoring T when no O2 is bound to favoring R when O2 is bound to all subunits
what happens in the sequential model of T –> R
individual subunits can change one at a time from T to R
describe the process of the sequential model
the conformational change of T to R in one subunit as O2 binds makes both a similar conformational change in an adjacent subunit and O2 binding in an adjacent subunit more likely
between the two models of T to R, which one does Hb more closely follow
both! neither model in its pure form fully accounts for the behavior of Hb
describe how Hb follows both models of T to R
kind of concerted: the tetramer with 3 O2 bound is almost always fully in the R state. The binding affinity of the final empty subunit has increased over 20x from plain deoxyhemoglobin
its kind of sequential because the tetramer with 1 O2 bound is almost always still fully in the T state, but still has a higher affinity for O2 compared to plain deoxyhemoglobin
what makes Hb an allosteric protein
the binding affinity of O2 to a Hb subunit can be affected by the binding of a ligand to another subunit
when discussing allostery, the ligand binding elsewhere is referred to as a ___
modulator
what is a modulator
a term for a ligand binding elsewhere to affect the affinity of O2 to a subunit
T or F: a modulator can be an activator or an inhibitor
true
define homotropic allostery
when the normal ligand and the modulator are the same chemical
define heterotopic allostery
when the normal ligand and the modulator are different chemicals
in regards to modulators, what is the term for O2 in relation to Hb
a homotropic activator
other than O2, what can Hb carry
H+ and CO2 (products of cell respiration)
where does Hb carry H+ and CO2
from the tissues to the lungs and kidneys
T or F: CO2 is not very soluble in blood
true
since CO2 is not soluble in blood, how does it travel in the blood
travels as bicarbonate
what is the reaction to make bicarbonate
CO2 + H2O –> H+ + HCO3-
what enzyme catalyzes bicarbonate formation
carbonic anhydrase
where is the enzyme carbonic anhydrase located
red blood cells
why is carbonic anhydrase located in the blood
it catalyzes bicarbonate formation so CO2 has a way to travel through the blood
what is the result of changes in H+ (and pH) and CO2 concentrations
changes can greatly influence the ability of Hb to bind O2
where on Hb does O2 bind
to the heme iron
where on Hb does H+ bind
can bind to any amino acid R groups that are positively charged (basic)
where on Hb does CO2 bind
binds to the amino group at the N-terminus of each polypeptide as carbamate adducts
T or F: CO2 binding to Hb produces more H+
true
why does CO2 binding to Hb cause more H+ to form
when CO2 attaches to the amino group at the N-term of each polypeptide, it forms a carbamate adduct, which releases H+ from the amino group
what does the bohr effect describe
the effect of pH and CO2 concentration on the binding and release of O2 by Hb
what is the relationship between H+/CO2 binding and O2 binding
they’re inversely related
describe Hb’s affinity for O2 when we have low pH (high H+) and high CO2
low affinity for O2 (O2 is released into tissues)
describe Hb’s affinity for O2 when we have high pH (low H+) and low CO2
high affinity for O2 (O2 binds to Hb for transport to tissues)
name a major site for H+ binding
His 146 aka His HC3
what is His146 aka His HC3
it’s in the linker of helix H between H and it’s C terminus
what happens when H+ protonates His HC3
His HC3 forms one of the ion pairs (to Asp FG1) across subunits to help stabilize the T state
what is the result of His HC3 being protonated and forming one of the ion pairs across subunits
pKa rises, so it’s very unlikely for it to be deprotonated
since the T state is very stabilized (due to His HC3 protonation), what is the R state like
in the R state, His HC3 ion pair cannot form
in regards to His HC3, what happens as H+ concentration rises
protonation of His HC3 promotes the release of oxygen by favoring a transition to the T state
what is 2,3BPG (not the name but the role)
it’s a heterotrophic modulator of mammalian Hb
where is BPG
erythrocytes
what does BPG do
it raises the P50 of Hb
what is the relationship between O2 binding and BPG binding
inverse relationship
what is the formula for BPG formation
HbBPG + O2 -> HbO2 + BPG
where on Hb does BPG bind
binds the central cavity of the Hb tetramer between the two beta subunits
what type of charges does BPG have
negative
what type of charges does the binding pocket of Hb have (this is where BPG binds)
positive
which state is stabilized when BPG is bound
T
which state is stabilized when BPG is unbound
R
describe Hb saturation when BPG is absent
Hb is nearly saturated
what would happen is BPG was not present in blood
Hb would not release O2 within body tissues
T or F: BPG plays a big role in what happens when you move from sea level to higher altitudes
true
how would high altitude affect your O2 delivery
limited O2 in the lungs to pick up –> won’t have Hb leave lungs 100% saturated with O2
what happens to BPG levels when we move to higher altitudes
red blood cells produce more BPG
how does the increase in BPG affect the curve
more BPG=lower O2 affinity (sounds bad), but it causes fraction saturation to be lower. While overall affinity is lower, your drop off rate has increased (which is good)
what are the isoforms of embryo Hb
ζ2ε2, ζ2γ2, or α2 ε2 (aka no beta)
what is the isoform for fetus Hb
α2γ2 (no beta)
describe the P50 for embryo and fetus Hb
embryo Hb: low P50
fetus Hb: intermediate P50
why is it important that embryo and fetus Hb have low and intermediate P50
fetal Hb will have a higher affinity for O2 than adult Hb
what type of mutation produces sickle cell anemia
single amino acid change
what amino acid change occurs to produce sickle cell anemia
glutamine to valine (E to V)
where does the amino acid change occur to produce sickle cell anemia
at position 6 in the two beta chains
what is the result of the amino acid change to produce sickle cell anemia
negative aa to hydrophobic aa: creates sticky hydrophobic contact point on the outer surface of the molecule, so in the deoxygenated form the molecules stick together and form aggregates
what are some symptoms of sickle cell
weakness, dizziness, shortness of breath, heart murmurs, low erythrocyte count, blocked capillaries
what happens if you’re heterozygous for the sickle cell allele
you have a mild condition called sickle cell trait (only 1% of erythrocytes are sickle shaped)
