Furthermore, the discussion here considers only equilibrium properties of the interactions, not anything about their kinetics, although many biological systems operate far from equilibrium. We assume, reasonably, but mostly without evidence, that at least in a relative way the values determined in vitro reflect values that would pertain in vivo. Thus, there is in general no practical way to relate in vitro values to in vivo values. Although values of affinity and specificity measured in vitro are valid for the conditions in which they are determined, those conditions typically differ dramatically from conditions in vivo, which in fact can be known at best only approximately. The reader will have noticed by now the unsubtle segue from biology-in vivo-to biochemistry-implicitly and explicitly in vitro. (To be philosophical about it, we would be strictly correct in saying that a fundamental property of specificity is that no target has a fixed value of specificity.) However, it is an operational definition in the sense that it depends on the identities of ligands A and A’, and its value therefore cannot be considered to be an inherent property of target B. This is a thermodynamically rigorous definition of specificity, just like the ΔG values on which it is based. However, there is one way to quantify specificity that is straightforward, thermodynamically sound, and entirely general, and should be in much wider use: the difference in free energy change, ΔΔG, between ΔG for binding of ligand A with target B and ΔG for binding of ligand A’ with target B. Some definitions are inconsistent with each other and/or not thermodynamically sound. Where affinity appears familiar and straightforward, specificity appears to be more elusive, subject to interpretation in different ways in different contexts or by different authors, as indeed it is in the literature.
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