Lecture 1 Flashcards
What curve is given by simple binding?
Hyperbolic
What curve is given by co-operative binding?
Sigmoidal
What is Kd?
The equilibrium dissociation constant which is the concentration of ligand at half maximal binding
How would you work out the amount of drug needed to block 1/2 the binding sites from Kd in M?
Kd x 100 = g/l
g/l x litres in body = grams of drug required
What dictates the probability of a reaction to occur?
Gibbs free energy
What is Gibbs free energy given by?
G= -R T lnKeq
If Kd is large then affinity is…
low
If Kd is small then affinity is..
high
Give an example of a biological system where a large Kd is important for function
Selectin-carbohydrate interactions mediate the rolling of leukocytes in blood vessels, if the Kd was too high then the cells would stop rapidly causing the cells to burst
Give an example of a biological system where a small Kd is important for function
Antibody-antigen binding
What are the two main types of methods you can use to measure Kd?
Equilibrium methods
Kinetic methods
Outline how equilibrium methods measure Kd
They measure the free/total concentration of ligands and the concentration of the protein-ligand complex
Give three examples of equilibrium methods of measuring Kd
Equilibrium dialysis Isothermal titration calorimetry Analytical Ultracentrifugation NMR IR spectroscopy Fluorescence spectroscopy Absorbance spectroscopy
Outline the process of equilibrium dialysis
Separate a protein from ligand by a membrane which is only permeable to the ligand.
Leave to reach equilibrium
Measure the ligand concentration in compartment A using colour or radiobinding, then use this to work out the concentration of PL from the original amount of ligand added.
Use PL to work out Kd
What is the advantage of equilibrium dialysis
Direct
What are the disadvantages of equilibrium dialysis
Lots of protein required
Narrow range of Kd
Degradation could occur before equilibrium is reached
Protein/ligand could have stuck to the dialysis tube
Outline the basic concept of isothermal titration calorimetry in measuring Kd
It measures the enthalpy change by keeping a constant temperature between the two cells and measuring the difference in temperature between the reference cell and the jacket. This is then used to work out G which can be used to work out Kd
Outline the basic concept of analytical ultracentrifugation in measuring Kd
When a complex is formed the Mw will increase causing it to sediment more quickly.
