A study of the interaction between fluorescein sodium salt and bovine serum albumin by steady-state fluorescence

The binding of fluorescein sodium salt with three kinds of commercially available bovine serum albumin (BSA) of different grades of purity was investigated at 288, 298 and 313 K by fluorescence and absorption measurements at pH 7.50. The association and dissociation constants Ka and Kd were determined by the quenching of BSA fluorescence in the presence of fluorescein sodium salt. The best results were obtained by fitting raw data by non-linear regression and Lineweaver–Burk equations. The modified Stern–Volmer and Scatchard plots gave less reliable data since the fitting was much more difficult.The agreement of the constants for the three sets of measurements coming from the different BSA was not as good as expected. BSA binding properties differ depending on the different BSA grades of purity. Actually, the binding constants found for the three BSAs used differed in the same set of interactions, even by keeping the experimental conditions constant. These results are a novelty in the field of BSA–ligand binding studies and should be taken into account for future binding studies using BSA. Actually, a large number of aspects should be considered including the grade of purity and the presence of BSA covalent and non-covalent dimers, trimers and oligomers in solution which can affect the goodness of the binding results.