Independent non-identical binding sites

Consider a macromolecule A that can bind several molecules of B. In the simplest case, where A possesses two binding sites for B, there are four possible species, A, AB, BA and BAB, whose concentrations depend on three binding constants  

Under conditions where cAttot cBitot, terms involving the total concentration of A do not occur in the analysis (as shown above), and it is therefore not possible to use Eqn. 9.24 to analyse the stoichiometry of the binding equilibrium. However, even under these experimental conditions, it is possible to obtain information about the number of binding sites, provided the binding constants of the two processes are sufficiently different in magnitude. 

Information about the minimum number of binding sites for B on the macromo-lecular species A can also be obtained if a signal can be measured which specifically monitors the concentration of A fully saturated with B (BAB in our scheme). For example, the enzyme DNA polymerase has two binding sites for metal ions, and both need to be occupied for the enzyme to be active. If it is assumed that the two 

In the special case of identical binding sites (KAssA = KAss2), the dependence of 012 on the total concentration of A (cAM) is weakly sigmoidal at low concentrations of B, and not hyperbolic this is a direct indication that A can bind more than one B. The total concentration of bound ligand (= 0 +02) follows a hyberbolic dependence, as expected since the sites are independent. 

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