Biochemistry I Fall Term

Topic #2: Ligand Binding Calculations

Model reaction:     ML <=> M + L
Dissociation constant: Kd = [M][L]/[ML]
The Scatchard equation is:
n/L = n/Kd - n/Kd ,  where
n = [L]bound/[M]total;
L = [L]free;
n = # ligands/macromolecule, i.e.the stoichiometry;
Kd = the dissociation constant.
We have a 4 mg/ml solution of a protein (Mr = 40,000 Da). We want to determine Kd and the stoichiometry of its binding to a ligand, L. This is done in three steps.
1. Enter a value for [L]total in this box:
[L] added = mM
2. For the above value of [L]total, Calculate [L]free
[L]free = mM
    (Each calculated [L]free value has a small "experimental error" added to it.
Tabbing out of the volume entry slot or clicking anywhere on the page will also calculate [L]free.)

3. Record the values of [L]free you obtain. Then, calculate [L]bound, n, and n/L. Finally, graph the values on a Scatchard plot to determine Kd and n. You should get enough data so as to have 3 or 4 values of n/[L]free, both above and below the 1/Kd value.
(Hint: Graph the values as you obtain them in a saturation curve (n vs. [L]free); then as the shape of the curve becomes apparent, choose values for [L]total that fall into the appropriate range.)
Answers to this problem.

A sample Answer Sheet for a similar problem shows the format of the results and the graph required.

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