Chemistry Reference
In-Depth Information
The NOE pumping experiment permits fast and direct detection of ligand binding
to target macromolecules without regard for the size of the macromolecule [24].
This method relies on NOE transference magnetization from the protein to the
ligand by applying a diffusion filter, which eliminates the signals of the ligand.
Consequently, ligand molecules in fast exchange which experience magnetization
transfer from the protein can be detected. For example, NOE pumping has been
used to detect salicylic acid binding to human serum albumin (HSA) in the
presence of non-binding compounds [29].
In the reverse NOE pumping experiment (RNP) a transverse relaxation filter is
first applied to attenuate the receptor signal while preserving and inverting the
ligand signals. During a mixing time, ligand magnetization is partially transferred
to the protein through intermolecular cross-relaxation. A reference experiment in
which no magnetization transfer takes place is subtracted. In the difference
spectrum, only signals of compounds which interact with the protein are observed
[24, 30].
The method has been demonstrated with HSA solutions containing the non-
binding small molecule glucose and a series of unbranched fatty acid molecules
that are known ligands. Only the fatty acids has beene observed in the difference
spectrum. The amount of signal pumped increased with fatty acid chain length,
indicative of increasing affinity, suggesting that a series of ligands can be ranked
by affinity, using this technique [15, 30].
For identification of weak affinity ligands, the NOE pumping experiment is more
robust than the standard diffusion experiments. NOE pumping and reverse NOE
pumping are very sensitive experiments for primary NMR screening and the latter
is considered to be more sensitive.
Saturation Transfer Difference (STD-NMR)
Saturation transfer difference (STD) NMR spectroscopy, represents one of the
most sensitive and versatile ligand-observed NMR screening methods, which is
used to detect ligands with binding affinity typically in the micromolar to
milimolar range [31]. The basis of this methodology is that the magnetization
