### Ligand Binding: Answer Sheet Example

Example Problem:
You are given a 4.0 mg/ml solution of a protein (Mr = 40,000 Da).
You do an equilibrium dialysis experiment at several concentrations of Ltotal.
a) Determine Kd and the stoichiometry of its binding to a ligand, L.
b) Submit your calculations and a graph of n/L. vs. n.
What we know from the problem:
1) Concentration of the protein = 4.0 mg/ml.
2) Protein Mr = 40,000 Da
What we found in the experiment:
Ltotal
(mM)
Lfree
(mM)
Lbound
(mM)
n

n/L
(mM-1)
``` 0.010
0.020
0.050
0.075
0.100
0.150
0.200
0.400
0.700
1.000
```
```0.005
0.011
0.029
0.045
0.061
0.103
0.142
0.333
0.625
0.901
```
```0.005
0.009
0.021
0.030
0.039
0.047
0.058
0.067
0.075
0.099
```
```0.05
0.09
0.21
0.30
0.39
0.47
0.58
0.67
0.75
0.99
```
```10.00
8.18
7.24
6.67
6.39
4.56
4.08
2.01
1.20
1.10
```
The protein concentration = (4 g/l)/(40,000 g/mol) = 0.10mM.
The last three columns were calculated using equations from the Ligand Binding page.

Graph of the calculated results:

The Kd = 0.10 mM.
The stoichiometry, n = 0.95-1.0.

9.27.04