Problem 3. Competition experiment.
0.5 nM radioligand L* is bound to 1 ml samples of the same membrane samples used in the above experiments in the presence or absence of varying amounts of either compound A or compound B. After establishing equilibrium, the following counts were bound to the membranes (after subtracting nonspecific binding):
|
Concentration A (nM)
|
CPM bound with A
|
Concentration B (nM)
|
CPM bound with B
|
|
0
|
73001
|
0
|
73001
|
|
0.05
|
72100
|
0.05
|
66365
|
|
0.1
|
71221
|
0.1
|
60835
|
|
0.3
|
67908
|
0.3
|
45626
|
|
0.5
|
64890
|
0.5
|
36501
|
|
1
|
58401
|
1
|
24334
|
|
3
|
41715
|
3
|
10429
|
|
5
|
32445
|
5
|
6636
|
|
10
|
20858
|
10
|
3476
|
|
30
|
8588
|
30
|
1197
|
|
100
|
2808
|
100
|
363
|
|
300
|
961
|
300
|
121
|
a) Which compound, A or B, is more a more potent displacer of the radioligand? Justify your answer why you think so.
b) Plot the fractional binding on a semilog plot and estimate the IC50s for both A and B.
c) Plot the results on a logit-log plot (pseudo-Hill plot) and determine the IC50s for both compounds A and B. Use these values for the next step. How close are the computed values to your estimate in part b?
d) Use the Cheng-Prusoff equation and the value of Kd for ligand L determined by the Scatchard plot in question 2 to calculate the Ki values for compounds A and B.