VP-ITC MicroCalorimeter User’s Manual
of sites in the sample cell ) is in excess of 1000, you may obtain better values for the binding
constant at 580 RPM. The reason for this is that when binding is extremely tight, a significant
error is encountered near the equivalence point if the injected ligand solution does not mix
completely throughout the entire volume of the sample cell. Finally, when you are studying
particulate suspensions, which tend to settle from gravity, more stirring speed will be needed to
provide the energy to keep a uniform suspension.
6.5 Estimation of Required Concentrations
A frequently asked question is "I'm not sure of my system's binding constant, what concentrations should I
use?"
The following equation is a good approximation of the concentration of the material in the sample cell
( i.e. an enzyme ):
K
a
*
C
= 10 to 50
Where
K
a
is the binding (association) constant expressed as a positive power of 10 (i.e., 10) and C is the
molar concentration of the binding component in the sample cell. If
K
a
is unknown, a best guess will have
to suffice.
The molar concentration of the binding material in the syringe (i.e. an enzyme inhibitor) is estimated as
follows:
100
µl syringe:
20 * n * C
250
µl syringe:
7 * n * C
Where "
n
" is the Stoichiometry of the reaction and
C
is the molar concentration of the binding material in
the sample cell.
These are "rules of thumb" and not absolute. However, if
K
a
and n are unknown, best guess and perhaps a
range finding experiment should quickly allow the investigator to determine the correct experimental
conditions.
As an example, assume we have an enzyme in the cell with a binding constant of 10. The concentration of
enzyme should therefore be 10 * C = 10 to 50 or a concentration of between 10 to 5x10 molar. Assuming a
Stoichiometry of 1, the concentration of inhibitor in the 100 µl syringe should be 20-fold higher than the
concentration of the enzyme in the cell (20 x 1 x 10 or 2 x 10 molar, in the case of a 1 µ molar enzyme
solution). If it were desirable to use the 250 µl syringe, the concentration of inhibitor in this syringe should
be 7 times the concentration of enzyme in the cell.
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