Proteins and ligands Flashcards
What is the function of myoglobin?
Stores (binds and releases) oxygen in the muscles
What kind of protein are haemoglobin and myoglobin?
Globular
What is the structure of foetal haemoglobin?
- made up of two alpha chains and two gamma chains
- Each chain binds a haeme group
What is the general structure of myoglobin?
Formed from one chain and one haeme group
What sort of coordinate complexes can Fe2+ form?
Penta and hexa
What type of ligand is haeme?
Polydentate
What are the 6 ligands haeme binds to?
- 4 haeme nitrogens
- 1 His side chain
- 1 O2
What technique can be used to measure oxygen binding to myoglobin and haemoglobin?
A spectrophotometer
How does spectrophotometry allow oxygen binding to myoglobin and haemoglobin to be measured?
- when bound to haeme the Fe2+ has some Fe3+ character
- The change in electronic configuration of Fe2+ on binding O2 results in a colour change
What shape is the oxygen binding curve for haemoglobin?
Sigmoidal
What shape is the oxygen binding curve for myoglobin?
Rectangular hypobola
What does a small Kd mean in terms of binding and affinity?
A small Kd means tighter binding to a protein and a stronger affinity
What is Kd of a protein?
The concentration of ligand at which half the protein is bound to ligand
What represents the fraction of a protein bound to ligand?
Y
How can you calculate Y?
[Ligand]/Kd x [Ligand]
What is the effect of the binding curves of myoglobin and haemoglobin on O2 release?
The sigmodial shape of the Hb binding curve allows Hb to deliver around 10x as much O2 to exercising tissues as Mb
How many structural forms of Hb are there?
2
What are the two structural forms of Hb?
Deoxyhaemoglobin (Tense) and oxyhaemoglobin (Relaxed)
Which form (tense or relaxed) of Hb has a low affinity for O2 ?
Deoxyhaemoglobin (Tense)
How is cooperativity diagnosed?
Using a Hill plot
What feature does binding of O2 to haemoglobin show?
Positive cooperativity
What is positive cooperativity?
Binding of O2 to one subunit increases the bidning affinity of the other sites
What value of n from a Hill plot indicates no cooperativity?
1
What is P50?
The concentration of O2 required to occupy half the binding sites
Give a compound which can bind to Hb allosterically
2,3-bisphosphoglycerate
Compare the binding of 2,3-bisphosphoglycerate in deoxy and oxy hb
Binds more tightly to deoxy-Hb than oxy-Hb
When is 2,3-BPG over produced?
In response to:
- high altitude
- airway obstruction
- congestive heart failure
What is the effect of 2,3-BPG on T-state (deoxy) Hb?
What is the effect of this?
Stabilises T-state Hb. This increases the P50
What are the structural similarities between haemoglobin and myoglobin?
- Both can bind, transport, and detoxify nitric oxide (NO)
- Both are globular proteins with tightly packed cores
- Both contain haem
What roles does nitric oxide have in the body?
It is a vasodilator and inhibitor of platelet aggregation
What is the effect of binding of O2 on the atomic radius of Fe2+ in haeme?
When O2 is bound the Fe2+ has a smaller atomic radius
Why does O2 in oxyhaemoglobin form a H bond with another His side chain?
It stabilises the O2-Fe2+ interaction
What is the formula for Kd?
[protein] x [ligand]/ [protein bound to ligand]
Compare the P50 of myoglobin and haemoglobin binding to O2
Myoglobin has a P50 of 2 tor whilst haemoglobin has a P50 of 26 tor
How does binding of O2 to one subunit of haemoglobin affect the Kd of the other subunits of haemoglobin?
Reduces Kd
What is on the y axis of a Hill plot?
Log (Y/1-Y)
What is on the x axis of a Hill plot?
log(pO2)
Is deoxy-Hb referred to as tense or relaxed?
Tense
Does relaxed Hb have a high or low affinity for oxygen?
High
Which form of haemoglobin is favoured when the [O2] is low?
T state
Which form of Hb is favoured when [O2] is high?
R state
Which form of Hb binds O2 more tightly?
R state
What happens to the equilibrium between T state and R state Hb when O2 binds?
The equilibirum shifts from favouring the T state to favouring the R state
Which state of Hb does 2,3-BPG bind more tightly to?
T state
What happens to oxygen release if [2,3-BPG] is increased?
More O2 is released
Does foetal Hb bind to 2,3-BPG as tightly as Hb?
No
Where does 2,3-BPG bind on Hb?
At the interface of β1 and β2 subunits
Which form of Hb do H+ preferentially bind to?
deoxy/T state
What is the effect of increased [H+] on O2 release?
If [H+] increases more O2 is released
Where does the formation of HCO3- occur?
In red blood cells in the capillaries of tissues
Where does formation of CO2 from HCO3- occur?
In red blood cells in the capillaries of the lungs
Outline how Bohr shift occurs
- CO2 diffuses into RBCs where it combines with H2O to form HCO3- and H+
- H+ binds to oxy-Hb causing O2 to be released
- HCO3- diffuses into plasma and circulates until reaches the lungs
- in lungs more oxy-Hb is formed so H+ are released
- H+ combines with HCO3- to form H2O and CO2
- CO2 diffuses out of red blood cells into lungs
Which amino acids do salt bridges which stabilise the T state of Hb form between?
- Lys40 forms a salt bridge to His146 which in turn also forms a salt bridge with Asp94
- These salt bridges occur between subunits
Why are the salt bridges only formed between the amino acid residues in the T state?
Only in the T state are the 3 residues correctly orientated to enable the formation of 2 salt bridges
At a lower pH what is the effect of the fractional saturation curve?
It is shifted to the right so Hb binds O2 less tightly
What are the 2 ways red blood cells carry CO2?
- as HCO3-
- Formation of carbamate
How can CO2 form carbamate?
It can bind directly to the amino terminal groups of Hb
How does formation of carbamate reduce the P50 of Hb for O2?
Carbamate participates in salt bridges which help to further stabilise the T state
What conditions reduces the affinity for O2 the most?
Low pH and prescence of CO2
Which mutation causes sickle cell anaemia?
β E6V
How does sickle cell anaemia confer some resistance to malaria?
It reduces the half life of red blood cells so helps to break the life cycle of the plasmodium
How does sickle cell anaemia cause problems?
- mutation produces a hydrophobic patch on the surface of Hb
- this causes β subunits to stick together
- Hb aggregates into fibres
