Module 3 Flashcards
Myoglobin & hemoglobin are . . .
homologues (paralogues) - perform different but related functions
What is avidity?
the overall strength of binding b/w an antibody and an antigen
What are the 2 functions of Mb?
storage of oxygen in muscles
release of oxygen for muscle contraction (when this happens very rapidly)
How is the equilibrium dissociation constant (Kd) useful in expressing ligand binding?
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)
Explain, using examples, the relationship between ligand binding affinity & the equilibrium dissociation constant, Kd.
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
What is an implication of the high affinity b/w biotin & avidin?
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
Could myoglobin transport O2?
o pO2 in lungs ~ 13 kPa; it BINDS oxygen well
o pO2 in tissues ~ 4 kPa; it will NOT release much at all
T state
tense; more interactions, more stable, LOWER AFFINITY for O2
R state
relaxed; less interactions, more flexible, HIGHER AFFINITY for O2
How does the binding of oxygen to one subunit affect the rest of the protein?
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)
Explain the structural basis of oxygen binding to Hb.
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
What is nH according to complex analyses?
Complex analyses have shown that nH is related to the AVERAGE occupancy of the binding sites – not TOTAL occupancy
How is H+ produced?
- when CO2 dissolves into water (conversion of CO2 to HCO3-), H+ is produced
- it is produced in metabolism directly
How much of the tissue H+ and tissue CO2 does Hb transport?
About 40% of tissue H+ and 20% of tissue CO2 to the lungs + kidneys
Explain the Bohr effect.
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
Where are protons (H+) thought to bind?
- at histidine HC3 of beta subunits
- the N termini of the alpha-subunits
- other amino acid residues
How do changes in pH influence the binding curve (for hemoglobin)?
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
Describe how hemoglobin transport CO2?
- 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
How does Hb bind BPG?
It binds to the positively charged central of Hb
stabilizes the T state
decreases affinity of Hb for O2
What is BPG?
EXTRA NEGATIVELY CHARGED molecule
reduces affinity of Hb for O2
produced from an intermediate in glucose metabolism
NEGATIVE allosteric regulator
present in RBCs
Describe the functional effect of CO binding to Hb
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
