Cooperative Ligand Binding to Globular Protein(I). Binding of Monoanionic Azo Dyes to Hen Egg White Lysozyme

Binding of sodium 4-(2- or 4-hydroxy-1-naphthylazo)benzenesulfonate and sodium 4-[4-(amino or dialkylamino)phenylazo]benzenesulfonate (alkyl: methyl, ethyl, or butyl) to hen egg white lysozyme has been studied by an equilibrium dialysis method at pH = 7.0 (0.1 mol dm−3 phosphate buffer) and 25 °C. In the case of the (2- or 4-hydroxy-1-naphthylazo)benzenesulfonate system, a single cooperative binding stage has been observed with a maximum binding number of 10. In the case of the 4-[4-(dialkylamino)phenylazo]benzenesulfonate systems, two cooperative binding stages have been observed for the dyes having alkyl groups longer than a methyl group. The maximum binding numbers of the first stage are 14 and 13 for 4-[4-(diethylamino)phenylazo]benzenesulfonate and 4-[4-(dibutylamino)phenylazo]benzenesulfonate, respectively, and those of the second stage were the same as those for the first one. The binding isotherms have been well interpreted by the stepwise binding model, which takes account of the free energy decrease due to interactions between bound ligands and of its reduction as the binding step proceeds, and binding constants have been evaluated. On the basis of the binding constants and the free energy changes of binding, the detailed binding mechanism is discussed.