haemoglobin Flashcards
what are examples of allosteric modulators of haemoglobin
oxygen, H+, CO2, organic phosphates
they are also allosteric effectors of myoglobin
where does oxygen bind to in Hb/Mb
oxygen binds reversibly to Fe(II), not Fe(III),
in haem group there is a four membered ring, each member has an N atom pointing towards the iron at the top there at 2 carboxyl groups which let it exist in relatively hydrophillic environment, bottom of the molecule is very hydrophobic
how many subunits do Hb/Mb have
Hb: 4
Mb:1
where are haem groups found
both Mb and Hb
what is the P50, how does it compare for Mb and Hb
P50 is the pressure where oxygen saturation = 50%
Mb: 1 torr
Hb: 26 torr
(Mb has much higher affinity to oxygen than Hb)
describe oxygen saturation curves for Hb and Mb
Hb: sigmoidal (does not bind O2 at low partial pressures)
Mb: hyperbolic
how many molecules of oxygen can Hb bind to
4 since it has 4 harm groups, 1 in each subunit
how do foetal and adult Hb compare
foetal has higher affinity than adult for oxygen
foetal contains 2 alpha subunits and 2 gamma subunits
adult contains 2 alpha subunits and 2 beta subunits
how does to O2/CO2 blood equilibrium compare in the lungs to in other tissues
in lungs equilibrium is on side of CO2 removal and O2 uptake (higher conc of oxygen in air and lower CO2 conc in air)
in tissues equilibrium is on side of O2 release and solubilisation of CO2
describe the reaction equation that carbonic anhydrase catalyses
describe the equation describeing reactions between protons, oxygen and Hb
combine these to equations to get an overall equation
how do these equilibria change in lungs and in tissues
= is a reversible reaction sign
HCO3- + H+ = H2CO3 = CO2 + H2O
(carbonic anhydrase catalyses reaction converting CO2 and H2O into H2CO3)
O2 + HHb+ = H+ + HbO2
overall reaction:
HHb+ + O2 + HCO3- = HbO2 + CO2 + H2O
in the lungs the equilibia for all the reactions favour right hand side, in the tissues they favour left hand side
how does binding of oxygen to Hb affect its visual absorption spectra, how does this compare to Mb
oxygenated Hb is red, deoxyHb is purple
visual absorption spectra changes a lot upon binding of O2
Hb and Mb have very similar visual absorption spectra
what is the physiological benefit of Hb’s O2 binding curve, what is the change in binding affinities called
benefit of sigmoidal binding affinity of Hb is that it has low affinity in respiring tissues so it can offload O2 efficiently, but high affinity in lungs so it can transport oxygen efficiently
change between weak binding and strong binding is known as allostericism
how does binding of O2 effect affinity of Hb to O2
binding of first O2 molecule increases affinity of Hb to bind more O2, increases with more O2 binding
what molecules decrease Hb’s affinity for O2
CO2, BPG, protons, chloride
when CO2 and BPG are added the affinity is reduced to that found in blood
affinity drops at lower pH due to actions of protons
chloride reduces Hb affinity but to lesser extent
how does BPG affect Hbs affinity for O2, what is the process and how does this relate to foeatal Hb
decrease in affinity;
BPG has 5 negative charges, binds to Hb and causes oxygen to release
BPG consists of a glycerol 3 carbon chain with 2 phosphate groups on either end and one carboxyl group in the middle
in T state there is a hole in the middle of Hb, BPG binds to this central hole
BPG binds to his143, lys82 and his2 on each of the beta subunits (6total binding sites)
foetal Hb loses 2 positive charges by replacing its his143 with a ser143, decreasing its affinity for BPG and increasing the oxygen affinity
BPG is all negative charges which bind to positive charges
what is the theory of cooperativity
allosteric enzymes show cooperative substrate binding, the degree of co-operativity is modulated by allosteric effectors
An allosteric inhibitor increases co-operativity to slow down the rate at a specific subtrate concentration
an allosteric activator decreases co-operativity, at high activator concentrations the Vo or rate of the enzyme against substrate concentration becomes hyperbolic
applies to Hb and cooperative enzymes
what are the models concerning Hb binding to O2
concerted model, sequential model
describe the concerted model
Hb can either exist in taut or relaxed state
taut (T) state is deoxyHb and relaxed (R) state is oxy-Hb
Hb is either all relaxed or not at all in concerted model, hybrid states do not exist, independent of how much oxygen is bound, the amount of oxygen bound simply shifts equilibria between T and R states
(applies to aspartate transcarboxylase as well)
describe the sequential model
sigmoidal affinity behaviour of Hb is produced by substrate binding to a subunit, inducing the T to R transition in that subunit
the change in one subunit influences its neighbours
in this model hybrid states must occur, allosteric activators stabilise R state and inhibitors stabilise T state
there is equilibrium between T and R states
in sequential model the states from T to R are:
T4=RT3=R2T2=R3T=R4
each equilibria has a different K value
what ligands promote R/T state in haemoglobin/myoglobin
haemoglobin T state: organic phosphates such as BPG, CO2, chloride and H+
R state: oxygen
Mb: nothing, Mb is not a cooperative protein and so does not have an R/T state
what are symptoms of altitude sickness
dizziness, shortness of breath, headaches and nausea
what is method for protein crystallography
a source of protein is taken from a natural source or expression system
it is purified before it is then crystallised, crystals are usually 0.1-1mm
x ray crystallography is then used
crystal is placed in capillary tube and sealed off at each end with buffer so crystal does not dry out
phases are then estminated using data which can be used to calculate an election density map, using this data you refine the preliminary atomic model to create a final atomic model
what is the structure of myoglobin, how does it compare to Hb
one haem group surrounded by 8 alpha helices, the alpha helices create a hydrophobic pocket which the haem is contained in
function of alpha helices is to provide this pocket for hydrophobic haem
myoglobin and haemoglobin have 8 structurally similar alpha helices labelled A to H making 75% of the secondary structure. Helices A B and E are on top, F passes from top to bottom, G and H are on the bottom. The haem is sandwiched in the angle between helices E and F. E is longer than F.
A-H happens sequentially from the N terminus ending at the C terminus.
myoglobin contains 153 amino acids, 131 of which are in alpha helices
what is the biochemical process that causes oxygen binding to increase Hbs affinity
haem binds to nitrogen atom of histidine residue called hisF8/ histadine F8
binding of oxygen causes slight conformational change in helix F which allows for the allosteric effect in Hb, this is transmitted through F8
when oxygen binds it pulls iron atom into the plane of the hae, the iron atom then pulls hisF8 and helix F moves with this
how is oxygen access to haem controlled
a distal histidine, his E7 controls oxygen access, which is on E helix
how does the subunit interface structure vary in T and R states
the alpha1beta2 interface switches from the T to R form on oxygenation. The “dove tailed” construction of this interface allows the subunits to readily adopt either of the two conformations.
in T state a salt bridge forms between aspartate G1 of beta subunit and tyrosine C7 of the alpha 1
in R state a salt bridge is formed between aspartate G1 of the alpha 1 and asparagine G4 of the beta 2
in R state the alpha1beta2 and alpha2beta1 dimers move relative to eachother by 15 degrees
how do F,G and C helices relate
extensive subunit interactions occur between FG corners and C helices
the beta 2 FG corner interaction with the alpha C helix is the switch region
the flexible joint region is the interaction between the alpha 1 FG corner and beta 2 helix
the switch region is located below the beta2 C terminus and the joint region is located above the alpha1 C helix terminus
what causes sickle cell anaemia
amino acid mutation from beta Glu6 (hydrophillic) to beta Val6 (hydrophobic)
what is the sickling cycle and what is the molecular basis
(Hb-S is sickle cell Hb)
oxy-Hb-S becomes deoxy Hb-S
deoxy Hb-S precipitates as long fibres, causing sickling of erythrocytes
this causes blockage of the blood vessel, this creates a local region of low oxygen tension which leads to more deoxygenation and more sickling which can lead to a myocardial infarction
molecular basis: valine creates a hydrophobic stick patch on ends of Hb-S, deoxy Hb-S hydrophoic sites form, which the stick patches stick to creating a chain and causing aggregation
what is thalassaemia
absence of a globin chain, either alpha or beta, usually occurs in mediterranean populations
alpha-thalasseaemia haemaglobin is made up of 4 identical beta chains where no or very little alpha chains are formed, the molecule shows non cooperativity and high oxygen affinity, behaves like 4 myoglobin molecules that are connected
what is mechanism for increase in oxygen affinity after binding to Hb
there are 8 key salt bridges in haemoglobin, 2 between each subunit. This helps explain allostericism since breaking the first subunit off requires breaking 4 salt bridges, subunits 2 and 3 require breaking 2 salt bridges and the last one requires no breaking of salt bridges.
BPG creates more salt bridges between beta subunits thus making salt bridges harder to break.
how are subunits in Hb structured
haemoglobin has 4 protein subunits, 2 alpha and 2 beta which associate as alpha1beta2 and alpha2beta1.
no contact between haem groups in Hb, signalling between haems for allosteric effect occurs through protein structure.
what body changes occur at high altitude
the way Hb acclimitises to high altitude is to make more BPG which shifts saturation curve to the right which creates a difference in 37 of the oxygen it carries. Before this happens, at an alititude of 4500 metres it is only 30%, normally at sea level the value is 38%.
