User`s manual

56 Chapter 5: Software Description
Calorimetry Sciences Corp.
CSC 5300 N-ITC III 57
User’s Manual
In an experiment designed to measure K, the need to keep the product of K and C
prot
near
100 must be balanced against the need for a detectable heat. For this reason, very tight
binding constants cannot be determined directly unless ∆H is very large so that very low
concentrations of protein can be used. Competition experiments provide an alternative
means of determining tight binding energetics.
Appropriate experimental conditions for single site binding systems can be determined
with estimates of K and ∆H using the Experiment Design tab in the BindWorks program.
Enter the parameters as shown in Figure 5-29.
Note that for this experiment, in which a total of 200 μL of titrant will be added to the
initial 1300 μL of titrate, a ratio of 10:1 is selected for the titrant to titrate concentration.
This will give equal moles of protein and ligand after 12-13 injections so that sufcient
excess ligand can be added to give a post-saturation baseline.
A plot of the cumulative heat versus the injection number is shown to the right of input
values. Note that there is a sharp transition between the rising portion of the curve and the
plateau towards the end of the
titration. This sharp transition
indicates that K could not be
determined from these data.
While K cannot be determined
from this type of experiment,
the value of ∆H can be
determined even if the
concentration of protein in the
cell is uncertain because all of
the injected ligand binds. The
heat generated in each of the
initial injections is the product
of ∆H, the injection volume,
V
inj
, and the concentration of
ligand being injected, C
inj
.
Figure 5-29: The BindWorks design tab. The image shown
illustrates a poorly designed experiment.