Equilibrium and Kinetic Studies of the Interaction between Orange I and Bovine Serum Albumin

The interaction between Orange I (sodium 4-[(4-hydroxy-1-naphthalenyl)azo]benzenesulfonate) and bovine serum albumin (BSA) at pH=7.0 and 25 °C has been studied by equilibrium dialysis, spectrophotometry, and the temperature-jump method. Equilibrium dialysis has revealed the existence of sixteen equivalent binding sites with the binding constant of K=9.7×103 M−1. All of them were found to be equivalent also in the sense of spectrophotometry. That is, Orange I interacts with BSA in a typically nonspecific manner. Five relaxations were found by the temperature-jump measurements. These relaxation times, distributed over the time range from 10 μs to 0.1 s, were determined over a wide range of reactant concentrations. The fastest relaxation becomes slow, but the four slower ones become fast, with the increase in the concentration of the free ligand. The data were interpreted by means of a combination of the bimolecular binding processes and the four isomerization modes of BSA. The results were discussed in the light of the role of the configurational adaptability of albumin molecules in the ligand-binding mechanism.