VP-ITC MicroCalorimeter User’s Manual
VP-ITC sensitivity is ca. 0.1
µ
cal so for precise measurement each injection should have an
average of at least 3-5
µ
Cal 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 106 M-1 and the
∆
Ho is approximately -15000 cal/mole for the
single binding site.
A. Measuring
∆
Ho, K and n by deconvolution of total binding isotherm
.
For a K of 106, RNASE concentrations are in the experimental K window for the Molar range 10-
6 < Mtot < 10-3. It requires at least 10 separate injections to define the total binding isotherms
and each injection must average ca. 5
µ
cal, so the total heat Q required in the 1.3 ml cell is 50
µ
cal, i.e.,
Q = 50 x 10-6 cal = (15000 cal/mole) (Mtot moles/l ) (1.3 x 10-3 l )
Solving this equation for Mtot 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 < Mtot < 10-4 ).
B. Measuring only
∆
Ho by single ligand injection into excess macromolecule
.
To measure
∆
Ho 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
∆
Ho, i.e., c > 100. This means Mtot > 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
µ
cal 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 Mtot you must select the ligand concentration
Xtot for the solution to be loaded into the syringe. This will depend on the volume of the syringe
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