Biochemistry I Fall Term

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.

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9.27.04