User`s manual

54 Chapter 5: Software Description
Calorimetry Sciences Corp.
CSC 5300 N-ITC III 55
User’s Manual
Where X
b
is the amount of ligand bound per unit volume. Free ligand concentration can
be obtained by solving:
Where M is concentration of macromolecule. The cumulative heat effect is:
(For more details refer to J. Wyman, S. J. Gill Binding and Linkage. University Science
Books 1990.)
Designing an Experiment
A calorimetric binding experiment can be designed to measure only the enthalpy change,
∆H, and number of binding sites, n, or to measure the binding constant, K, in addition to
∆H and n. In the rst type of experiment, known as a stoichiometric binding experiment,
all of the injected ligand is bound to the protein until all of the binding sites are lled.
Consequently, all of the initial injections result in the same integrated heat until saturation
is reached. Subsequent injections then yield only the heat of dilution of the ligand.
In an experiment designed to measure K, there is curvature to the plot of the heat versus
the number of injections. It is the tting of this curvature that permits a determination
of K. In designing an experiment we want to identify conditions in which we expect
to observe this curvature. In order to achieve the curvature in the titration experiment
necessary to determine K, the product of K and the protein concentration, C
prot
, must be
between 10 and 1000 (see Wiseman, T., Williston, S., Brandts, J.F. and Lin, L.-N. (1989)
Anal. Biochem. 179, 131). If this product is too high, there is no curvature in the plot. If
this constant is too low, then the curvature is too gradual to allow a good determination
of K. Consequently, very low concentrations of protein are required to measure very tight
afnity constants.
Equation 5-7
X X M Y
t
− − ⋅ = 0
Equation 5-8
∑
∆=
i
i
i
i
HXß
Z
Vcell
Q