• binding thermodynamics;
  • bovine serum albumin;
  • rolitetracycline;
  • spectroscopy;
  • tetracycline;
  • warfarin

An understanding of the detailed energetics and mechanism of the binding of drugs with target proteins is essential for devising guidelines to synthesize new drugs. Binding of the antibiotic drugs tetracycline and rolitetracycline with serum albumin has been studied by a combination of isothermal titration calorimetry, differential scanning calorimetry, steady-state and time-resolved fluorescence, and circular dichroism spectroscopies. Both tetracycline and rolitetracycline bind to bovine serum albumin in a sequential manner with first binding being the major binding event with an association constant of the order of 104 for tetracycline and 103 for rolitetracycline, respectively. Ionic strength dependence and binding in the presence of tetrabutylammonium bromide and sucrose indicate involvement of a mix of hydrophobic, ionic, and hydrogen bonding interactions. The isothermal titration calorimetry results for the binding of these drugs to bovine serum albumin in the presence of warfarin and in the presence of each other indicate that both these drugs share binding site 2 on bovine serum albumin. The differential scanning calorimetry results provide quantitative information on the effect of drugs on the stability of bovine serum albumin. A comparison of isothermal titration calorimetry and fluorescence results demonstrates that the former technique has been able to explain the sequential binding events that can be missed by the fluorescence measurements.