Bovine serum albumin and alizarin yellow G — a model system for positive cooperative binding to macromolecules

The kinetics of the binding of alizarin yellow G to six nonequivalent binding sites on bovine serum albumin were investigated, using the stopped flow and T ‐jump techniques. The first step in the reaction sequence is very fast, and is followed by a slower step which is suggested to be an internal rearrangement of the collision complex formed in the first step. This temporal sequence shows that the strong cooperativity between the first and second binding sites is not due to the existence of different conformations in equilibrium in free albumin, but rather to a ligand‐induced change. Next, an additional, still slower transformation is observed. From the fact that this transformation is not rate‐determining for the formation of higher complexes we conclude that it corresponds to a branch reaction. The higher complexes are again formed with very high rate constants. The first complex has a high negative value of both Δ H and Δ S ° of formation. For the second and higher complexes, the heat of formation is much lower, and the standard entropy of formation is positive. The formation of these complexes is thus largely entropy‐driven. The difference between the thermodynamic parameters of the first and subsequent ligands is in accordance with the assumption that only the binding of the first ligand entails significant structural changes.