Spectrofluorimetric analysis of the binding of a target molecule to serum albumin: tricky aspects and tips

Protein binding heavily modulates drug activity. Therefore, the binding features need to be elucidated when chemistry researchers study new molecules (metal complexes) to be used as drugs. This paper concerns the experimental and data treatment aspects of the mechanistic analysis of the binding to a fluorescent protein (the golden standard serum albumin) by using direct fluorescence titrations. Fluorescence data are not rarely only qualitatively used, neglecting further treatments which could offer a precious detailed picture of the behavior of the drug. We aim to spread a mechanistic approach, discussing the critical aspects for correctly designing the experiments and treating the data. The researcher may confirm adduct formation and evaluate binding constants (Stern-Volmer KSV or other types of K). Also, we discuss here, with the help of literature examples, the correct use of temperature dependence of K to extract thermodynamic parameters, comment on enthalpy-entropy compensation, together with the use of synchronous spectra and exchange experiments to gain information on the binding type and site. We think that this tutorial/critical synopsis can be of help for the increasing community dealing with these experiments, which are valuable but often much more tricky than it might appear at first sight.