MicroCal VP-ITC User Manual page 47

Section 3: Running an ITC Experiment
It is well-known that ∆C p is a good indicator of changes in hydrophobic interactions with
binding, being negative if hydrophobic bonds are formed and positive if they are broken.
The critical parameter which determines the shape of the binding isotherm is the unitless constant
c, which is the product of the binding constant K times the total macromolecule concentration in
the cell at the start of the experiment, M tot , times the stoichiometry parameter, n.
c = KM tot n
Very large c values lead to very tight binding and the isotherm is rectangular in shape with the
height corresponding exactly to ∆H o and with the sharp drop occurring precisely at the
stoichiometric equivalence point n in the molar ratio X tot /M tot . The shape of this curve is
invariant with changes in K so long as the c value remains above ca. 5000. As c is reduced by
decreasing M tot (i.e., holding K, ∆H o and other parameters constant), the drop near the
equivalence point becomes broadened and the intercept at the Y axis becomes lower than the true
∆H o . In the limit of very low initial M tot concentration (cf., c=0.1), the isotherm becomes
featureless and traces a nearly horizontal line indicative of very weak binding. It is apparent from
looking at these isotherms that their shape is reasonably sensitive to binding constant only for c
values in the range 1 < c < 1000, corresponding to binding of intermediate strength. We will refer
to this range as the "experimental K window". When available, the middle of the window from c
= 5 to 500 is most ideal for measuring K.
The correct choice of macromolecule concentrations for an experiment depends both on the
objective of the experiment (i.e., whether you wish to determine a ∆H o of binding only, or
whether you wish to determine n and K in addition to ∆H o ) and upon the magnitude of K. While
considering your choice of starting concentration, it must also be remembered that the limiting
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