Unfortunately, few analytical tools for evaluating protein-ligand interactions comprise all, or even most, of these properties. The most commonly used solution-phase methods for binding affinity determination are spectroscopic in nature, and typically measure nuclear magnetic resonance (NMR), ultraviolet light absorbance, circular dichroism, or fluorescence changes caused by protein-ligand interactions . These methods, especially ones based on NMR chemical shift changes, have the benefit in certain circumstances of indicating where the ligand is binding to its target . However, spectroscopic methods often require isotopic or fluorescence labeling of the ligand or receptor . Thermophysical techniques, such as isothermal or differential scanning calorimetry, require no chemical modification for their use, and in addition to measuring binding affinities these methods can also yield thermodynamic parameters of binding.
Though both spectroscopic techniques and calorimetric methods enable cofac-tors, buffers and metal ions to be included in the binding reaction, these methods are unfortunately very consumptive of purified protein. Partly to mitigate the difficulty of high protein consumption, binding affinity measurement techniques based on surface-immobilized receptors have been developed. These techniques include affinity chromatography and surface plasmon resonance (SPR) spectroscopy, with instruments using SPR and other biosensor techniques available commercially [6, 7]. While surface methods are operationally simple to execute and can yield useful kinetic parameters that describe binding interactions, such methods require chemical modification of the receptor for attachment, possibly occluding ligand binding sites or otherwise affecting binding interactions.
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