Equilibrium and kinetic studies of the binding of tri- and tetra-anionic ligands to bovine serum albumin.
The binding of tri- and tetra-anionic azo dyes (Amaranth, Ponceau 4R, and Ponceau 6R) to bovine serum albumin (BSA) at pH = 7.0 and 25 degrees C has been studied by equilibrium dialysis, spectrophotometry, and by stopped-flow and temperature-jump methods. Equilibrium dialysis revealed that BSA has one primary binding site and about two secondary sites for each dye. The values of the binding constant for the primary site show that the stability of the complex at the primary site progressively increases with an increase in the number and the density of anionic charges on ligand. Kinetic data have been found to be consistent with a scheme in which a rapid bimolecular binding is followed by two isomerizations of the complex (in the case of Amaranth) or by one isomerization (in the cases of Ponceau 4R and Ponceau 6R). Equilibrium and rate constants for each step of the scheme were determined. From the results it was found that the increment of the number and the density of anionic charges on ligand accelerates the forward process of the final isomerization step but retards the backward one of it, resulting in the enhancement of the stability of the complex at the primary site. On the basis of these results and the structure of the ligands, the detailed binding mechanism has been discussed in the light of the electrostatic interaction between the ligands and the binding site on BSA.
Murakami
K
K
Department of Chemistry, Faculty of Education, Yamaguchi University, Yoshida, Japan.
D000818:Animals; D000838:Anions; D001665:Binding Sites; D002417:Cattle; D004560:Electricity; D007700:Kinetics; D008024:Ligands; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013057:Spectrum Analysis; D013696:Temperature
D000838:Anions; D008024:Ligands; D012710:Serum Albumin, Bovine