VP-ITC MicroCalorimeter User’s Manual
VP-ITC sensitivity is ca. 0.1 µcal so for precise measurement each injection should have an
average of at least 3-5 µCal of heat absorbed or evolved into the 1.3 ml cell.
How these factors impinge on your choice of macromolecule concentration can be seen by
considering a particular example of the binding of 2'CMP to ribonuclease A, where the binding
constant is approximately 1 x 10
6
M
-1
and the ∆H
o
is approximately -15000 cal/mole for the
single binding site.
A. Measuring
∆
H
o
, K and n by deconvolution of total binding isotherm.
For a K of 10
6
, RNASE concentrations are in the experimental K window for the Molar range 10
-
6 <
M
tot
<
10
-3.
It requires at least 10 separate injections to define the total binding isotherms
and each injection must average ca. 5 µcal, so the total heat Q required in the 1.3 ml cell is 50
µcal, i.e.,
Q = 50 x 10
-6
cal = (15000 cal/mole) (M
tot
moles/l ) (1.3 x 10
-3
l )
Solving this equation for M
tot
gives a minimum concentration of ca. 3 x 10
-6
M. This
concentration is larger than the lowest concentration, 1 x 10
-6
, in the experimental K window so
the concentration range available in the K window becomes 3 x 10
-6
<
M <
10
-3
. Although any
value within this range is acceptable, it would lead to better estimates of parameters to choose
concentrations higher than the minimum of 3 x 10
-6
so that Q signals will be larger and c values
will be in the ideal range between 10 and 100. (i.e., 10
-5
<
M
tot
< 10
-4
).
B. Measuring only
∆
H
o
by single ligand injection into excess macromolecule.
To measure ∆H
o
by a single injection (i.e., without deconvolution of the total binding isotherm)
requires a c value large enough so the experimental intercept on the isotherm intercepts the Y
axis very close to the true ∆H
o
, i.e., c >
100. This means M
tot
> 10
-4
. Since there will be excess
macromolecule in the cell, the experimental heat Q will be determined by the amount of ligand
injected, i.e.,
Q = (15,000 cal/mole) (syringe conc.) (inj. vol.)
For example, a 10 ml injection of a 7 x 10
-5
M ligand solution would give the minimal 10 µcal of
heat. It is also possible to measure ∆H by injecting excess ligand into a very low concentration of
macromolecule.
Referring to case A above, once you have chosen M
tot
you must select the ligand concentration
X
tot
for the solution to be loaded into the syringe. This will depend on the volume of the syringe
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