Molecular interactions between warfarin and human (HSA) or bovine (BSA) serum albumin evaluated by isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE)

[Display omitted] •Warfarin-albumin interactions have been evaluated by ITC, FS and FA/CE.•The strongest interaction step is about 5 × 104 for HSA and 1 × 105 for BSA.•Further high order interaction events for both albumins are detected only by FA/CE.•Binding parameters are affected by experimental conditions. Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding site of human (HSA) or bovine (BSA) serum albumin have been examined in strictly controlled experimental conditions (HEPES buffer 50 mM, pH 7.4 and 25 °C) by means of isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE). Each technique is based on measurements of a different property of the biochemical system, and then the results allow a critical discussion about the suitability of each approach to estimate the drug-protein binding parameters. The strongest interaction step is properly evaluated by the three assayed approaches being the derived binding constants strongly consistent: from 4 × 104 to 7 × 104 for HSA and from 0.8 × 105 to 1.2 × 105 for BSA. Binding enthalpy variations also show consistent results: −5.4 and −5.6 Kcal/mol for HSA and −4.3 and −3.7 Kcal/mol for BSA, as measured by ITC and FS, respectively. Further high order interaction events for both albumins are detected only by FA/CE.