Proteins and ligands

Question 1

What is the function of myoglobin?

Answer 1

Stores (binds and releases) oxygen in the muscles

Question 2

What kind of protein are haemoglobin and myoglobin?

Answer 2

Globular

Question 3

What is the structure of foetal haemoglobin?

Answer 3

• made up of two alpha chains and two gamma chains
• Each chain binds a haeme group

Question 4

What is the general structure of myoglobin?

Answer 4

Formed from one chain and one haeme group

Question 5

What sort of coordinate complexes can Fe²⁺ form?

Answer 5

Penta and hexa

Question 6

What type of ligand is haeme?

Answer 6

Polydentate

Question 7

What are the 6 ligands haeme binds to?

Answer 7

• 4 haeme nitrogens
• 1 His side chain
• 1 O₂

Question 8

What technique can be used to measure oxygen binding to myoglobin and haemoglobin?

Answer 8

A spectrophotometer

Question 9

How does spectrophotometry allow oxygen binding to myoglobin and haemoglobin to be measured?

Answer 9

• 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

Question 10

What shape is the oxygen binding curve for haemoglobin?

Answer 10

Sigmoidal

Question 11

What shape is the oxygen binding curve for myoglobin?

Answer 11

Rectangular hypobola

Question 12

What does a small Kd mean in terms of binding and affinity?

Answer 12

A small Kd means tighter binding to a protein and a stronger affinity

Question 13

What is Kd of a protein?

Answer 13

The concentration of ligand at which half the protein is bound to ligand

Question 14

What represents the fraction of a protein bound to ligand?

Answer 14

Y

Question 15

How can you calculate Y?

Answer 15

[Ligand]/K_d x [Ligand]

Question 16

What is the effect of the binding curves of myoglobin and haemoglobin on O₂ release?

Answer 16

The sigmodial shape of the Hb binding curve allows Hb to deliver around 10x as much O₂ to exercising tissues as Mb

Question 17

How many structural forms of Hb are there?

Answer 17

2

Question 18

What are the two structural forms of Hb?

Answer 18

Deoxyhaemoglobin (Tense) and oxyhaemoglobin (Relaxed)

Question 19

Which form (tense or relaxed) of Hb has a low affinity for O2 ?

Answer 19

Deoxyhaemoglobin (Tense)

Question 20

How is cooperativity diagnosed?

Answer 20

Using a Hill plot

Question 21

What feature does binding of O₂ to haemoglobin show?

Answer 21

Positive cooperativity

Question 22

What is positive cooperativity?

Answer 22

Binding of O₂ to one subunit increases the bidning affinity of the other sites

Question 23

What value of n from a Hill plot indicates no cooperativity?

Answer 23

1

Question 24

What is P₅₀?

Answer 24

The concentration of O₂ required to occupy half the binding sites

Question 25

Give a compound which can bind to Hb allosterically

Answer 25

2,3-bisphosphoglycerate

Question 26

Compare the binding of 2,3-bisphosphoglycerate in deoxy and oxy hb

Answer 26

Binds more tightly to deoxy-Hb than oxy-Hb

Question 27

When is 2,3-BPG over produced?

Answer 27

In response to: * high altitude * airway obstruction * congestive heart failure

Question 28

What is the effect of 2,3-BPG on T-state (deoxy) Hb? What is the effect of this?

Answer 28

Stabilises T-state Hb. This increases the P₅₀

Question 29

What are the structural similarities between haemoglobin and myoglobin?

Answer 29

* Both can bind, transport, and detoxify nitric oxide (NO) * Both are globular proteins with tightly packed cores * Both contain haem

Question 30

What roles does nitric oxide have in the body?

Answer 30

It is a vasodilator and inhibitor of platelet aggregation

Question 31

What is the effect of binding of O₂ on the atomic radius of Fe²⁺ in haeme?

Answer 31

When O₂ is bound the Fe²⁺ has a smaller atomic radius

Question 32

Why does O₂ in oxyhaemoglobin form a H bond with another His side chain?

Answer 32

It stabilises the O₂-Fe²⁺ interaction

Question 33

What is the formula for Kd?

Answer 33

[protein] x [ligand]/ [protein bound to ligand]

Question 34

Compare the P₅₀ of myoglobin and haemoglobin binding to O₂

Answer 34

Myoglobin has a P₅₀ of 2 tor whilst haemoglobin has a P₅₀ of 26 tor

Question 35

How does binding of O2 to one subunit of haemoglobin affect the Kd of the other subunits of haemoglobin?

Answer 35

Reduces Kd

Question 36

What is on the y axis of a Hill plot?

Answer 36

Log (Y/1-Y)

Question 37

What is on the x axis of a Hill plot?

Answer 37

log(pO₂)

Question 38

Is deoxy-Hb referred to as tense or relaxed?

Answer 38

Tense

Question 39

Does relaxed Hb have a high or low affinity for oxygen?

Answer 39

High

Question 40

Which form of haemoglobin is favoured when the [O₂] is low?

Answer 40

T state

Question 41

Which form of Hb is favoured when [O₂] is high?

Answer 41

R state

Question 42

Which form of Hb binds O₂ more tightly?

Answer 42

R state

Question 43

What happens to the equilibrium between T state and R state Hb when O₂ binds?

Answer 43

The equilibirum shifts from favouring the T state to favouring the R state

Question 44

Which state of Hb does 2,3-BPG bind more tightly to?

Answer 44

T state

Question 45

What happens to oxygen release if [2,3-BPG] is increased?

Answer 45

More O₂ is released

Question 46

Does foetal Hb bind to 2,3-BPG as tightly as Hb?

Answer 46

No

Question 47

Where does 2,3-BPG bind on Hb?

Answer 47

At the interface of β1 and β2 subunits

Question 48

Which form of Hb do H⁺ preferentially bind to?

Answer 48

deoxy/T state

Question 49

What is the effect of increased [H⁺] on O₂ release?

Answer 49

If [H+] increases more O₂ is released

Question 50

Where does the formation of HCO₃^- occur?

Answer 50

In red blood cells in the capillaries of tissues

Question 51

Where does formation of CO₂ from HCO₃^- occur?

Answer 51

In red blood cells in the capillaries of the lungs

Question 52

Outline how Bohr shift occurs

Answer 52

* 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

Question 53

Which amino acids do salt bridges which stabilise the T state of Hb form between?

Answer 53

* Lys40 forms a salt bridge to His146 which in turn also forms a salt bridge with Asp94 * These salt bridges occur between subunits

Question 54

Why are the salt bridges only formed between the amino acid residues in the T state?

Answer 54

Only in the T state are the 3 residues correctly orientated to enable the formation of 2 salt bridges

Question 55

At a lower pH what is the effect of the fractional saturation curve?

Answer 55

It is shifted to the right so Hb binds O₂ less tightly

Question 56

What are the 2 ways red blood cells carry CO₂?

Answer 56

* as HCO₃^- * Formation of carbamate

Question 57

How can CO₂ form carbamate?

Answer 57

It can bind directly to the amino terminal groups of Hb

Question 58

How does formation of carbamate reduce the P₅₀ of Hb for O₂?

Answer 58

Carbamate participates in salt bridges which help to further stabilise the T state

Question 59

What conditions reduces the affinity for O₂ the most?

Answer 59

Low pH **and** prescence of CO₂

Question 60

Which mutation causes sickle cell anaemia?

Answer 60

β E6V

Question 61

How does sickle cell anaemia confer some resistance to malaria?

Answer 61

It reduces the half life of red blood cells so helps to break the life cycle of the plasmodium

Question 62

How does sickle cell anaemia cause problems?

Answer 62

* mutation produces a hydrophobic patch on the surface of Hb * this causes β subunits to stick together * Hb aggregates into fibres