The effect of simultaneous binding of doxorubicin and cyclophosphamide on the human serum albumin structure

The interaction of human serum albumin (HSA) with doxorubicin and cyclophosphamide was analysed by using fluorescence and circular dichroism (CD) spectroscopy, electrophoretic light scattering (ELS) and molecular modelling. Based on the fluorescence quenching results we can assume the existence of HSA-Cyp-Dox complex in the solution. The Ksv and Ka values for the system obtained under physiological conditions were 1.603·104M−1 and 4.574·104M−1, respectively and proved only one binding site for the drugs. The reaction is spontaneous and hydrophobic or ionic interactions are predominant in the process. Results of circular dichroism spectroscopy exhibit that the presence of cyclophosphamide and doxorubicin in the binding center of the protein induce insubstantial alteration in their secondary structure. The Forster theory was employed to determine the donor – acceptor distance in the HSA system and it was found to be 3.67 nm. Zeta potential analysis confirmed existence of the hydrophobic interaction and some instability of the system under physiological conditions. According to molecular docking study in both cases doxorubicin binds to the same site of albumin with very similar free energy of biding. However, the origins of stabilization and the binding modes of Dox in the Sudlow site I of HSA are different.