Electrochemical site marker competitive method for probing the binding site and binding mode between bovine serum albumin and alizarin red S

A novel electrochemical site marker competitive method is proposed for the study of the binding site and the binding mode between bovine serum albumin (BSA) and alizarin red S (ARS). Two known site-selective markers for BSA, bilirubin (BR, for site I) and diazepam (DIA, for site II), were selected to bind to the two potential binding sites of BSA (site I and site II). The resulting BR–BSA or DIA–BSA complex was added into an ARS solution for binding site competition studies. From the current response of ARS after site competition, binding site I of BSA was identified as the binding site of BSA to ARS. Further investigation of the binding events by electrochemistry, fluorescence, and molecular docking techniques revealed that ARS was buried in the hydrophobic cavity of subdomain IIA, mainly through five hydrogen bonds between ARS and residues ARG(193), ARG(255), LYS(220), ARG(197), and ALA(289) of BSA. These efforts will aid in a better understanding of the toxicological action of active components in anthraquinone dyes.