Measurement of ligand binding with nuclear magnetic resonance spectroscopy

A method is described for measuring with nuclear magnetic resonance (NMR) spectroscopy the binding of ligands to particulate components of cells. This method is sensitive to intermediate-strength binding, which is difficult to measure with standard radiotracer binding assays, and is insensitive to 'tight' binding. Nonspecific binding and entrapment can be distinguished from specific binding even when a pharmacological agent is not available for competitive binding assays. Thus, the experimental approach presented provides a means of studying the possible physiological roles in neural tissue of metabolites that have not been implicated by neuropharmacological and neurophysiological experiments. The utility of this technique is discussed in terms of the information it has provided about the binding of enkephalin, epinephrine, acetylcholine and carnosine. This technique, although it uses technology new to most neuroscience laboratories, should be relatively easy to apply to existing research topics. The NMR spectrometer required for the binding assay is the type already found on most academic campuses, and the ligand need not have any special isotopic label. The particulate fraction can be prepared in the same way as for the radiotracer binding assays, which means that both tight and intermediate-strength ligand binding can be measured with the same preparation.