Module 3: V5 - V8

Question 1

Why is the equilibrium association constant for cooperative proteins which have multiple ligand-binding sites different to other proteins which only have one binding site? What is the equation?

Answer 1

this is because the number of binding sites needs to be accounted for in the equation
Ka = [PLn]/[P][L]^n

Question 2

What is the equation for theta during cooperative binding?

Answer 2

θ = [L]^n/[L]^n + Kd

Question 3

What gives the Hill Equation?

Answer 3

taking the log of both sides of the equation for theta gives:
log(θ/1 - θ) = nlog[L] - logKd

Question 4

What does the slope of the Hill Equation give?

Answer 4

gives a measure of the interaction/cooperativity of binding sites in a protein (Hill coefficient)

Question 5

What does it mean if nH > 1?

Answer 5

this indicates positive cooperativity and binding at one site increases binding at other sites on other subunits e.g. Hb/O2

Question 6

What does it mean if nH = 1?

Answer 6

this indicates that binding is not cooperative and the sites are independent

Question 7

What does it mean if nH < 1 (rare)?

Answer 7

this indicates negative cooperativity and binding at one site decreases binding at another site on another subunit

Question 8

What is the theoretical upper limit for nH? Give an example. What do we see experimentally?

Answer 8

the theoretical upper limit for nH is n
e.g., for Hb, n = 4 so the theoretical maximum nH is 4
experimentally, nH places a lower limit on the number of interacting sites: nH is always < n

Question 9

What is nH related to (shown by complex analyses)?

Answer 9

the average occupancy of binding sites

Question 10

What are the two models used to explain cooperativity?

Answer 10

the concerted model and the sequential model

Question 11

What are the characteristics of the concerted model?

Answer 11

all or none
in the absence of L, all subunits of a multimer are thought to be in the inactive T or active R form
the inactive state is destabilised by L binding
successive binding of L to the inactive state makes the transition to the active state more likely

Question 12

What are the characteristics of the sequential model?

Answer 12

each subunit of the multimer can be in either the T or R form
L binding produces a change in conformation of the subunit
a change in conformation in one subunit induces a similar change in an adjacent subunit
therefore binding of a second L is more likely

Question 13

Are the two models used to explain cooperativity mutually exclusive?

Answer 13

no

Question 14

How is H+ produced in the body?

Answer 14

produced both in metabolism directly, and from the conversion of CO2 to HCO3-

Question 15

Do H+ and CO2 compete with O2 for binding to the heme group?

Answer 15

no they don’t, they are not transported in the same way

Question 16

How does pH affect the affinity of oxygen for haemoglobin? What does a shift to the right or left mean in the graph?

Answer 16

at a low pH the curve is shifted to the right (weaker binding) and at a low pH the curve is shifted to the left (stronger binding)

Question 17

At a lower pH is the affinity of Hb for O2 increased or decreased? Why?

Answer 17

decreased affinity because H+ stabilises Hb in the T state

Question 18

Why is pH lower in tissues?

Answer 18

due to metabolism that produces acids, e.g. lactic acid and CO2

Question 19

Why is it important that a lower pH lowers the affinity of Hb for O2?

Answer 19

this effect of H+ on lowering the affinity of Hb for O2 helps offload O2 from Hb to the tissues

Question 20

Where do hydrogen protons bind to Hb and stabilise the T state?

Answer 20

protonates His HC3, which then forms a salt bridge with Asp FG1 which leads to the release of O2 (in the tissues)

Question 21

What is the Bohr effect?

Answer 21

the increased efficiency of O2 transport as a result of the pH difference between the lungs and metabolic tissues

Question 22

Where are protons thought to bind?

Answer 22

the N-termini of the ɑ subunits, His HC3 of the β subunit and other amino acid residues

Question 23

How does the binding of O2 at 2.7kPa change at pH 7.4 to pH 7.2?

Answer 23

decreases from ~32% to ~20% and the extra O2 released supports continued metabolic activity in the tissue

Question 24

How is CO2 exported on hemoglobin?

Answer 24

exported in the form of a carbamate on the amino terminal residues of each of the polypeptide subunits

Question 25

How does CO2 binding increase stabilisation of the T state?

Answer 25

the formation of carbamate yields a proton that can contribute to the Bohr effect and the carbamate forms additional salt bridges

Question 26

Which state of hemoglobin does the release of CO2 into the lungs favour?

Answer 26

the R state as the carbamate groups are released which destabilises the T state

Question 27

What is BPG?

Answer 27

a molecule derived from an intermediate in glucose metabolism which interacts with Hb at a separate site to the O2-binding site and affects O2 binding (much like CO2 and H+)

Question 28

What is the concentration of BPG at sea level and at high altitudes?

Answer 28

5mM at sea level and 8mM at high altitudes

Question 29

Since BPG binds to a different site O2 binds to, what is it called?

Answer 29

a negative allosteric regulator of Hb function

Question 30

What is the structure of BPG and where does it bind? Does it stabilised or destabilise the T state?

Answer 30

small negatively charged molecule which binds to the positively charged central cavity of Hb (larger in the T state than in the R state)
stabilises the T state

Question 31

What effect does BPG have on oxygen binding?

Answer 31

decreases the affinity of HB for O2

Question 32

What effect does BPG have on the oxygen-hemoglobin binding curve? What does this mean?

Answer 32

shifts the curve to the right meaning that BPG make Hb bind oxygen more weakly

Question 33

Give an example of how concentration of BPG affects oxygen-binding.

Answer 33

at sea level, in an individual with 5 mM BPG, about 38% of O2 in saturated Hb (in the lungs) is delivered to the tissues
at high altitude, pO2 in lungs decreases to Hb is less saturated and O2 delivery decreases to 30%
if BGP increases to 8 mM affinity for O2 decreases and more O2 is delivered to the tissues (37% cf 38%)

Question 34

Where does carbon monoxide bind on hemoglobin and why is this an issue?

Answer 34

binds to the heme group with very high affinity and prevents oxygen from binding (competitive inhibitor)

Question 35

Why is it a problem that carbon monoxide stabilises hemoglobin’s high affinity R state?

Answer 35

because there is only a very small drop off in affinity of hemoglobin between the lungs and the tissues meaning that only a very small amount of O2 is released

Question 36

Should acidic conditions make hemoglobin bind oxygen more tightly or more weakly? Does that make sense in terms of what you know about exercise physiology?

Answer 36

acidic conditions should make hemoglobin bind oxygen more weakly
yes, it makes sense

Question 37

The atmosphere is ‘thinner’ at high altitude. So, people who live there need something to make their hemoglobin bind oxygen more tightly, right?

Answer 37

no, they require something that will release oxygen more readily in the tissues

Question 38

In pregnancy, maternal BPG levels can rise by ~30% and Foetal hemoglobin is different to adult hemoglobin. Why have we evolved like this - what is the selective advantage?

Answer 38

we have evolved like this so that more oxygen is available to the mother’s tissues to compensate for the lost oxygen which is being provided to the foetus
allows mothers to be active even when they are pregnant (this is the selective advantage)

Question 39

Do O2, CO and CO2 compete for the same binding site? Explain your answer.

Answer 39

no, CO2 binds to hemoglobin in the form of carbamate groups to the amino terminal residues of each of the polypeptide subunits
however, CO and O2 compete for the heme binding sites on the hemoglobin molecule