We are now in a position to deal with the main subject of this monograph, that is, the description of binding and linkage effects arising locally at the level of individual sites of a multi-site macromolecule. Consider a macromolecule containing N binding sites for a ligand X. We seek to resolve the thermodynamics of site-specific effects arising when binding of X can be monitored at each site separately. If the N sites are independent, then the description of site-specific effects becomes a trivial task, since each site behaves according to the rules outlined for a reference system in Section 2.1. When the sites are not only independent but also alike, then the macromolecule as a whole is a reference system. The case we are interested in is the one where all sites are different and linked. In this instance each separate site of the macromolecule is a subsystem open to interactions with other subsystems. Hence, the behavior of an individual site coupled to the rest of the macromolecule is expected to be rather complex, as it must reflect not only the binding properties of the site under consideration, but also the result of interactions with other sites.

The reference cycle and site-specific cooperativity

The simplest system of interest in the discussion of binding processes at the local level is that composed of a macromolecule M containing two binding sites for ligand X.