Conformational effects in the interaction of phenylbutazone with albumin studied by circular dichroism.

The binding of phenylbutazone (PB) to human serum albumin (HSA) at different pH and in the presence of different NaSCN and urea concentrations that alter the conformation of the protein was examined qualitatively on the basis of extrinsic elliptical strength at 288 nm by means of circular dichroism (CD). The values of the binding index expressed as a ratio of [theta]max/[theta]pH7.4(288) at each extrinsic rotational strength in the presence of various concentrations of NaSCN, urea and hydrogen ion were directly proportional to the alpha-helix content based on the peptide backbone alteration of HSA by NaSCN, urea and hydrogen ion except for the pH range of 5.0 to 10.0. The values in the pH range of 7.4 to 10.0 depended on the concentration of hydrogen ion and not on the alpha-helix content, showing a significant effect of the hydrogen ion on the tertiary conformation with respect to the binding sites of the amino acid chain rather than the peptide backbone of HSA. The increases in the binding index observed in the pH range of 7.4 to 10.0 were not observed at all in the case of NaSCN and urea at the concentrations studied. It was demonstrated that the binding of PB to HSA increased with the change in the tertiary conformation caused by hydrogen ions but decreased with that in the secondary conformation caused by a concentration change of NaSCN and urea. Thus, the binding was closely associated with skeletal conformational alterations as well as changes in the binding sites of the amino acid chains of the protein.