MicroCal VP-ITC User Manual page 48

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.
H o , K and n by deconvolution of total binding isotherm.
∆
A. Measuring
For a K of 10 6 , RNASE concentrations are in the experimental K window for the Molar range 10 -
6 < M -3. It requires at least 10 separate injections to define the total binding isotherms
tot < 10
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
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.,
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
H o by single ligand injection into excess macromolecule.
∆
Q = (15,000 cal/mole) (syringe conc.) (inj. vol.)
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