8 - Haemoglobin and Cooperativity

Question 1

What is the structure of haemoglobin?

Answer 1

Four helical subunits - two alpha and two beta. Each containing a haem group.

Question 2

What is the structure of myoglobin?

Answer 2

A single subunit with a haem group, comparative to one of the haemoglobin subunits.

Question 3

What is AHSP?

Answer 3

Alpha-haemoglobin Stabilising Protein - a chaperone which stabilises the alpha haemoglobin subunits prior to formation into the haemoglobin complex.

Question 4

What is the structure of the haem group?

Answer 4

It is a prophyrin ring, comprising four pyrrole rings whose endocyclic nitrogens coordinate the iron atom.

Question 5

What state is the haem iron in when not bound to oxygen?

Answer 5

Ferrous, +2

Question 6

How many coordination bonds can the anoxygenic ferrous iron form?

Answer 6

Six.

Question 7

How many of the coordinate bonds of the ferrous iron are fulfilled by the porphyrin ring?

Answer 7

Four

Question 8

What part of the subunit binds to the fifth coordination site of the ferrous iron, and what effect does this have?

Answer 8

The proximal histidine on the F-helix, whose coordination pulls the iron downwards, puckering the ring.

Question 9

How does oxygen reach its binding site?

Answer 9

The haem group is deeply buried, so the O2 only reaches it due to local breathing of the protein.

Question 10

What occurs when the oxygen binds to the ferrous iron?

Answer 10

It pulls the iron back into plane with the porphyrin ring, undoing the puckering. It also partially donates an electron producing ferric (+3) iron and becoming a superoxide radical.

Question 11

What binding event stabilises the superoxide radical?

Answer 11

Hydrogen bonding of the second oxygen atom with the distal histidine on the E-helix.

Question 12

What are the forms of the oxygen affinity curves for myoglobin and haemoglobin?

Answer 12

Myoglobin - rectangular hyperbola

Haemoglobin - sigmoidal

Question 13

What is the percentage change in oxygen affinity for myoglobin and haemoglobin between oxygen partial pressures at the tissues and lungs?

Answer 13

7% for myoglobin

66% for haemoglobin

Question 14

What would the oxygen affinity curve look like for haemoglobin if it did not show cooperative binding?

Answer 14

Much higher Km than Hb, to the point where it does not plateau until well after the maximum physiclogical concentration. It has a 38% change in affinity over the difference between lung and tissue O2 pp.

Question 15

What is the mechanism for haemoglobin cooperativity?

Answer 15

When oxygen binds to a subunit it causes a conformational change which effects a 15° rotation relative to the other subunits. It is this that increases the oxygen affinity of the adjacent chains.

Question 16

What are the two models of three models of cooperativity?

Answer 16

Concerted
Sequential
Mnemonic

Question 17

What is the concerted model of cooperativity in the context of haemoglobin?

Answer 17

The concerted model postulates that the haemoglobin subunits can either all be tense or all relaxed, and that oxygen binding to more subunits shifts the equilibrium towards the relaxed state.

Question 18

What is the sequential model of cooperativity in the context of haemoglobin?

Answer 18

The sequential model assumes that the subunits can exist in different states within the same Hb protein, with oxygen binding to one of them converting it to the relaxed state which affects the likelihood of the adjacent subunits relaxing.

Question 19

What is used to measure the cooperativity of the binding of a protein?

Answer 19

Its Hill Coefficient, derived from a Hill plot.

Question 20

What coefficients are Hill plots derived from?

Answer 20

Hill equation requires logarithmic transformation of an equation involving number of binding sites, ligand concentration and Kd.

Question 21

How is the Hill coefficient derived from a Hill plot?

Answer 21

It is the gradient of the line.

Question 22

What does a Hill coefficient of 1 entail?

Answer 22

Lack of cooperativity.

Question 23

What do high and low Hill coefficients describe?

Answer 23

> 1 = positive cooperativity

Question 24

What are the Hill coefficients of myoglobin and haemoglobin?

Answer 24

Haemoglobin = 2.8

Myoglobin = 1

Question 25

What generally happens with the oxygen affinity curve as the Hill coefficient increases?

Answer 25

It becomes more sigmoidal, with a steeper central region.

Question 26

What binds to deoxygenated haemoglobin?

Answer 26

2,3-Bisphosphoglycerate

Question 27

Where does 2,3-Bisphosphoglycerate bind?

Answer 27

A single one binds in the centre between all subunits by its terminal phosphate groups and central carbonyl.

Question 28

What effect does 2,3-Bisphosphoglycerate have on the haemoglobin?

Answer 28

It binds it only when all the subunits are deoxygenated, and shifts the equilibrium towards the tense state.

Question 29

What benefit does 2,3-Bisphosphoglycerate grant?

Answer 29

It shifts the affinity curve of haemoglobin so that the steepest region occurs more over the physiological range. Without it the Hb affinity curve is almost our of the steep middle region of its curve at tissue O2 pp.

Question 30

What is the Bohr Effect?

Answer 30

The decrease in Hb O2 affinity with increased acidity or [CO2], the former being a product of the latter.

Question 31

Why does an increased concentration of CO2 lead to a decrease in pH?

Answer 31

Because erythrocytic carbonic anhydrase converts the carbon dioxide to carbonic acid.

Question 32

Why is the Bohr effect useful?

Answer 32

Rather than returning empty, haemoglobin is also used to carry CO2 from the cells to the lungs for excretion, from a high concentration to a low concentration respectively. The Bohr effect ensures that oxygen is released and carbon dioxide bound at the tissues, thus aiding in both roles.

Question 33

How is CO2 disposed of?

Answer 33

Gaseous exchange in the lungs, and conversion to carbonic anhydrase and subsequent excretion via the renal system.

Question 34

Why is most carbon dioxide converted to carbonic acid?

Answer 34

CO2 is not very soluble, so would bubble out if the concentrations were as high as they would be if this were not done.

Question 35

How does CO2 bind to haemoglobin?

Answer 35

It reacts with the terminal amine group of each of the chains to form a carbamate group, displacing a proton and hence contributing the pH decrease regardless.

Question 36

How does increased acidity decrease oxygen’s binding affinity?

Answer 36

Protonates various residues leading to a conformation change that stabilises the tense state.

Question 37

Where are the chains protonated and what are the consequences of these?

Answer 37

The His-146 on the B1 chain, forms an ionic bond with Asp-94 on the same chain.
Lys-40 on the a1 chain, forms an ion pair with the B1 C-terminus acid.

Question 38

What mutation causes sickle-cell anaemia?

Answer 38

ß-haemoglobin gene, Glu-6 to Val-6.

Question 39

What does the sickle cell anaemia mutation cause?

Answer 39

Introduction of the hydrophobic residue allows for crystallisation of the haemoglobin which ‘sickles’ the red blood cell limiting functionality.