A series of Equilibrium Dialysis Experiments were set up where a fixed concentra
ID: 3163491 • Letter: A
Question
A series of Equilibrium Dialysis Experiments were set up where a fixed concentration and volume of the protein is contained within dialysis tubing and "dialyzed" until an equilibrium is reached against a solution containing a certain concentration of the ligand were set up with varying concentrations of ligand. This allows the "free" ligand to reach an equilibrium between the inside and outside regions of the experiment, while of course the "bound" ligand is contained entirely inside the dialysis tubing the following data was obtained:
Protein Concentration in Experiment: 0.918mg/mL
Polypeptide Molecular Weight of Protein: 55,800
Molecular Weight by Gel Filtration: 224,000
Data Obtained:
Tube #
Total Ligand Concentration Inside Tube
Total Ligand Concentration Outside Tube
1
12.4840mM
8.904mM
2
7.5000
4.208
3
5.3192
2.4832
4
3.9184
1.5064
5
3.2548
1.0948
6
2.3896
0.7016
7
1.6064
0.4264
8
1.0916
0.2596
9
0.5256
0.1136
Calculate the dissociation constant for the ligand binding to the protein.
How many binding sites per native molecule are there?
Tube #
Total Ligand Concentration Inside Tube
Total Ligand Concentration Outside Tube
1
12.4840mM
8.904mM
2
7.5000
4.208
3
5.3192
2.4832
4
3.9184
1.5064
5
3.2548
1.0948
6
2.3896
0.7016
7
1.6064
0.4264
8
1.0916
0.2596
9
0.5256
0.1136
Explanation / Answer
from the data we can get binding constant Ka=Kon/Koff
then dissociation constant= Kd=1/Ka
for tube 1: Ka= 12.4840/8.904=1.40
then Kd=1/1.40.714
for tube 2: Ka= 7.5/4.2= 1.78
Kd=1/1.78=.561
we get values similarly for other tubes.
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