Effects of phenylbutazone and mefenamic acid on two classes of binding sites of 2-(4'-hydroxyphenylazo)benzoic acid on bovine serum albumin characterized by absorption spectra and circular dichroism spectra.

The competitive binding of phenylbutazone (PB) and mefenamic acid (MF) with 2-(4'-hydroxyphenylazo)benzoic acid (HABA) on bovine serum albumin (BSA) was studied by the investigation of the effects of these drugs on two bound forms of HABA, the azo and hydrazone forms. PB displaced preferentially the hydrazone form. MF displaced both the azo and hydrazone forms, though the azo form was preferentially displaced at the small molar ratio of MF to BSA. The binding constants of PB and MF obtained from the convenient spectrophotometry were dependent on the concentration of added drugs. These facts reflect the selective displacement of the azo and hydrazone forms. In connection with the absorption spectra of bound HABA, induced circular dichroism (CD) spectra of HABA-BSA complex were investigated. The induced CD spectra varied with the change of the molar ratio of HABA to BSA, indicating the presence of at least three different perturbed HABA molecules by BSA. The addition of PB and MF caused dramatic changes of the induced CD spectra of HABA-BSA complex. These spectral changes were discussed with the displacement of the azo and hydrazone forms.