Module 3

Question 1

Myoglobin & hemoglobin are . . .

Answer 1

homologues (paralogues) - perform different but related functions

Question 2

What is avidity?

Answer 2

the overall strength of binding b/w an antibody and an antigen

Question 3

What are the 2 functions of Mb?

Answer 3

storage of oxygen in muscles

release of oxygen for muscle contraction (when this happens very rapidly)

Question 4

How is the equilibrium dissociation constant (Kd) useful in expressing ligand binding?

Answer 4

it represents the concentration of free ligand at which the protein is 50% saturated (tells us what concentration of ligand we need to achieve saturation of our protein)

Question 5

Explain, using examples, the relationship between ligand binding affinity & the equilibrium dissociation constant, Kd.

Answer 5

Calmodulin is a protein found in rats

its ligand is Ca2+

has a Kd value of around 2*10^-5, LOW affinity (weak)

similar to weak enzyme-substrate interactions

On the other hand, avidin & biotin have a very small Kd value of 1*10^-15, meaning it has a very high affinity

Question 6

What is an implication of the high affinity b/w biotin & avidin?

Answer 6

o Biotin is needed for carboxylation

o Biotin binds the protein avidin found in raw egg white

o Biotin deficiency in humans is associated with the long-term consumption of diets rich in raw eggs

o Egg shell is POROUS – bacteria can enter through the egg shell and attack the developing embryo

o The avidin in the egg will extract the biotin from the bacteria & KILL them

Question 7

Could myoglobin transport O2?

Answer 7

o pO2 in lungs ~ 13 kPa; it BINDS oxygen well
o pO2 in tissues ~ 4 kPa; it will NOT release much at all

Question 8

T state

Answer 8

tense; more interactions, more stable, LOWER AFFINITY for O2

Question 9

R state

Answer 9

relaxed; less interactions, more flexible, HIGHER AFFINITY for O2

Question 10

How does the binding of oxygen to one subunit affect the rest of the protein?

Answer 10

The conformation can change rapidly from the T state to the R state as soon as oxygen is present, and it involves breaking salt bridges between the residues at the a1-B2 interface (alpha of protomer 1 and beta of protomer 2)

Question 11

Explain the structural basis of oxygen binding to Hb.

Answer 11

The T state is stabilized by a variety of salt-bridge interactions

Oxygen binding DESTABILIZES these interactions & allow a transition to the R state

There is an important salt-bridge interaction b/w His HC2 & Asp FG1

Question 12

What is nH according to complex analyses?

Answer 12

Complex analyses have shown that nH is related to the AVERAGE occupancy of the binding sites – not TOTAL occupancy

Question 13

How is H+ produced?

Answer 13

• when CO2 dissolves into water (conversion of CO2 to HCO3-), H+ is produced
• it is produced in metabolism directly

Question 14

How much of the tissue H+ and tissue CO2 does Hb transport?

Answer 14

About 40% of tissue H+ and 20% of tissue CO2 to the lungs + kidneys

Question 15

Explain the Bohr effect.

Answer 15

When Hb is at the tissues, H+ produced from metabolism & the dissociation of CO2 binds to Hb

this causes a conformational change BACK into T state

oxygen is released at low pHs at the tissues

Histidine, HC3, when it is protonated, is able to form a SALT bridge with Asp FG1

the pH difference b/w the lungs and metabolic tissues INCREASES the efficiency of the O2 transport

Question 16

Where are protons (H+) thought to bind?

Answer 16

• at histidine HC3 of beta subunits
• the N termini of the alpha-subunits
• other amino acid residues

Question 17

How do changes in pH influence the binding curve (for hemoglobin)?

Answer 17

o High pH causes it to shift to the left (higher affinity)

o pH > 7.4 - SHIFTS TO THE LEFT

o pH < 7.4 - SHIFTS TO THE RIGHT

Question 18

Describe how hemoglobin transport CO2?

Answer 18

• CO2 is produced by metabolism in tissues must be exported
• 15-20% of CO2 is exported in the form of CARBAMATE on the amino acid residues of each polypeptide unit
• formation of a carbamate yields ANOTHER proton that can contribute to Bohr effect
• the carbamate forms ADDITIONAL salt bridges, stabilizing T state
• when hemoglobin reaches lungs, carbamate is released, destabilizing the salt bridges that were formed & thus favouring the R state - allowing hemoglobin to pick up oxygen more efficiently

Question 19

How does Hb bind BPG?

Answer 19

It binds to the positively charged central of Hb

stabilizes the T state

decreases affinity of Hb for O2

Question 20

What is BPG?

Answer 20

EXTRA NEGATIVELY CHARGED molecule

reduces affinity of Hb for O2

produced from an intermediate in glucose metabolism

NEGATIVE allosteric regulator

present in RBCs

Question 21

Describe the functional effect of CO binding to Hb

Answer 21

Competitive inhibitor

binds more tightly than oxygen

CO increases affinity of the remaining Hb subunits for O2, so an Hb molecule that binds 2 CO molecules can bind O2 in the lungs, but it cannot readily release it in the tissues