Topic #2: Ligand Binding Calculations
Model reaction: ML <=> M +
L
Dissociation constant: Kd = [M][L]/[ML]
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The Scatchard equation is:
n/L = n/Kd -
n/Kd
, where
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n = [L]bound/[M]total;
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L = [L]free;
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n = # ligands/macromolecule, i.e.the stoichiometry;
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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. (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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