Chemistry Reference
In-Depth Information
D
obs
=
x
free.
D
free
+
x
bound
.D
bound
,
(7)
where
x
i is the mole fraction.
Thus, when the difference in size between receptor and ligand is large enough, it
can be assumed that the diffusion coefficient of the receptor is not largely
modified in the free or bound state. However, the diffusion coefficient of the
ligand decreases substantially in the bound state, allowing the separation of
resonance signals of the complexed molecules from the mixture of the other faster
diffusing free ligands [27, 35].
The diffusion ordered spectroscopy (DOSY) NMR technique was introduced by C.S.
Johnson Jr. and co-workers [46-48] and has been directly applied to determine the
affinity of ligands without the need of titration with ligands. In addition, DOSY can be
further combined with other NMR techniques that allows the structural identification
of the interacting molecules [45, 49]. Moreover, DOSY-NMR is a powerful tool for
analysis of mixtures and identification of receptor binding ligands from a pool of low
molecular weight compounds [24]. This technique allows the NMR signals of discrete
compounds to be resolved based on variance of translational molecular diffusion rates
as well as chemical shifts. Resonances arising from individual chemical species align
exactly along the diffusion dimension making it possible to solve mixture components
whose diffusion coefficients differ only by a few percent. This makes it of great
interest as a tool for analyzing mixtures.
However, NMR diffusion has been used as a tool for spectroscopically isolating
and identifying ligands with receptor binding affinity from a mixture of potential
ligands. The concept of separating compounds by receptor affinity used in the
PFG experiment is reminiscent of affinity chromatography methods and has been
termed ''Affinity NMR'' [24]. The broad applicability of PFG-NMR diffusion
measurements is useful since its simplicity makes it flexible to modification.
Accuracy in diffusion measurements is improved with modified pulse sequences
that permit selective editing or subtraction of undesirable signals, such as the
protein background [45].
