answer 7-4: Conclusions A ligand binding replacement, whenever possible, is a very informative experiment, that may provide important information on the selectivity of the protein for its two ligands; moreover this type of experiment may be resorted to: 1) To measure the affinity of a ligand whose binding yields a poor signal. Suppose that P and PX have exactly the same absorbance spectrum: binding cannot be recorded by absorbance. If however PY has a different absorbance spectrum from P and PX, the replacement of Y by X can be followed by absorbance spectroscopy. 2) To measure the dissociation equilibrium constant of high and very high affinity ligands. Indeed we demonstrated that X50 = Kx (Ky + [Y]) / Ky > Kx and the higher the concentration of the competing ligand Y, the higher the X50. Thus in the case of the very high affinity ligand X, whose dissociation constant is too high to be reliably messured at the protein concentrations required by the sensitivity of the instrument used, one may proceed as follows: (i) measure the dissociation constant of ligand Y; (ii) measure the dissociation of ligand X in the presence of ligand Y at different concentrations; and (iii) obtain Kx from global fitting of the experiments. Caution: reaching the replacement equilibrium with very high affinity ligands may require long incubation times! A replacement experiment, however, may present some limitations. The most important limitation occurs when we record ligand replacement of multiple site, cooperative proteins. In this case the reaction scheme is as follows: PYn + nX <==> PYn-1 + Y + nX <==> PYn-1X + Y + (n-1)X <==> ... <==> PXn + nY In this reaction scheme the protein releases one ligand at a time and has either all its binding sites liganded or all liganded except one. Thus the properties of the unliganded protein are never tested. As a consequences it is often observed that ligand replacement is non-cooperative, even though both ligands by themselves would bind cooperatively: cooperativity is usually not observable in ligand replacements. This tutorial is complete. |