Recognition of anthracycline binding domains in bovine serum albumin and design of a free fatty acid sensor protein.

ABSTRACT

The mode of binding of N-acylated doxorubicin derivatives to bovine serum albumin (BSA) has been determined by spectrophotometric analysis. A water-insoluble derivative containing a N-hydroxysuccinimide ester moiety on the sugar amino group side-chain is found to react very rapidly with a specific domain (BSAA600) located in the NH2-terminal half of the BSA molecule. A stable covalent protein-anthracycline complex with 11 molar stoichiometry and containing the albumin monomer is formed. This specific association between albumin and doxorubicin derivative is accompanied by large changes in the spectral characteristics of the anthracycline chromophore. A new strong absorption band at 600 nm is associated to ionization of the chromophore phenolic groups. Titration experiments indicate that the pKa of the protein bound anthracycline is about 3 pH units lower than the pKa of free doxorubicin in aqueous buffer indicating chromophore localization in a basic microenvironment on the albumin molecule. For N-acylated doxorubicin derivatives which associate non-covalently to the BSAA600 domain, the strength of binding is found to be controlled by ionic as well as hydrophobic protein-anthracycline interactions. Water-soluble derivatives containing a side-chain carboxylic group bind with Kd approximately 10 microM, which is at least 100-fold more strongly than doxorubicin. The anthracycline chromophore is displaced from the BSAA600 domain in a non-competitive manner by fatty acids ranging in chain length from C6 to C18 and at a fatty acid/BSA molar ratio < 2. We therefore propose a model for the anthracycline binding domain in which the chromophore resides near the opening of the hydrophobic channel into which the fatty acid hydrocarbon chain is inserted. The clusters of basic amino acid residues located at this site may form the basic anthracycline microenvironment. Our results demonstrate that doxorubicin derivatives with a sugar amino group side-chain are well suited as probes for investigations on protein-anthracycline interactions. The practical application of the covalent BSA-DOX complex as a free fatty acid sensor protein for detection of enzymatic release of fatty acids in liposomal and cell membranes is suggested.