Denaturation and Renaturation of Binding Sites of Bovine Serum Albumin for Methyl Orange

Abstract

The binding sites of bovine serum albumin for methyl orange are destroyed on denaturation with high concentrations of urea, and most of them, 14 out of 16, reform upon removal of the urea. The affinity of the binding sites formed on renaturation, however, is lower than that of the native protein for the anion in terms of the first binding constant, K₁, and also of the intrinsic binding constant, K. The bovine serum albumin molecule can also assume different conformations on the process of renaturation in different solvents as reflected by the binding parameters, even though the protein still has the residual constraint of the 17 disulfide bonds in the denatured form under our condition. From the thermodynamic and the spectroscopic aspects of the anion binding, the interaction of bovine serum albumin with methyl orange can be best explained in terms of binding of the anion in hydrophobic regions on the surface of the protein molecule.