Antibiotic doxorubicin and its derivative bind milk β-lactoglobulin

beta-Lactoglobulin (beta-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as doxorubicin and its derivatives. We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with beta-lactoglobulin in aqueous solution at physiological conditions, using FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling. Structural analysis showed that DOX and FDOX bind beta-LG via both hydrophilic and hydrophobic contacts with overall binding constants of KDOX-beta-LG = 1.0 (+/- 0.4) x 10(4) M-1 and KFDOX-beta-LG = 2.5 (+/- 0.5) x 10(4) M-1 and the number of drug molecules bound per protein (n) 1.2 for DOX and 0.6 for FDOX. Molecular modeling showed the participation of several amino acids in the drug-protein complexes with the free binding energy of -8.12 kcal/mol for DOX-beta-LG and -7.74 kcal/mol for FDOX-beta-LG complexes. DOX and FDOX do not share similar binding sites with beta-LG. Protein conformation showed minor alterations with reduction of beta-sheet from 58% (free protein) to 57-51% in the drug-beta-LG complexes. beta-LG can transport doxorubicin and its derivative in vitro. 2012 Elsevier B.V. All rights reserved.