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Flashcards in Lecture 1 Deck (18):
1

What curve is given by simple binding?

Hyperbolic

2

What curve is given by co-operative binding?

Sigmoidal

3

What is Kd?

The equilibrium dissociation constant which is the concentration of ligand at half maximal binding

4

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

5

What dictates the probability of a reaction to occur?

Gibbs free energy

6

What is Gibbs free energy given by?

G= -R T lnKeq

7

If Kd is large then affinity is...

low

8

If Kd is small then affinity is..

high

9

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

10

Give an example of a biological system where a small Kd is important for function

Antibody-antigen binding

11

What are the two main types of methods you can use to measure Kd?

Equilibrium methods
Kinetic methods

12

Outline how equilibrium methods measure Kd

They measure the free/total concentration of ligands and the concentration of the protein-ligand complex

13

Give three examples of equilibrium methods of measuring Kd

Equilibrium dialysis
Isothermal titration calorimetry
Analytical Ultracentrifugation
NMR
IR spectroscopy
Fluorescence spectroscopy
Absorbance spectroscopy

14

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

15

What is the advantage of equilibrium dialysis

Direct

16

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

17

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

18

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.