Topic #2: Ligand Binding Calculations
ML <=> M + L
Dissociation constant: Kd = [M][L]/[ML]
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.
(Each calculated [L]free value has a small "experimental error" added to it.
- The Scatchard equation is:
n/L = n/Kd - n/Kd
- n = [L]bound/[M]total;
- L = [L]free;
- n = # ligands/macromolecule, i.e.the stoichiometry;
- Kd = the dissociation constant.
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.