Serum albumin as a primary non-covalent binding protein for nitro-oleic acid

This work explores the interaction of 9/10-nitro-oleic acid (NO₂-OA) with human serum albumin (HSA). The molecular mechanism of the biological action of NO₂-OA is to our knowledge based on a reversible covalent reaction-Michael addition of nucleophilic amino acid residues of proteins. Since HSA is an important fatty acid transporter, a key question is whether NO₂-OA can bind covalently or non-covalently to HSA, similarly to oleic acid (OA), which can interact with the FA1-FA7 binding sites of the HSA molecule. ¹H NMR studies and competition analysis with OA and the drugs ibuprofen and warfarin were used to investigate a potential non-covalent binding mode. NO₂-OA/HSA binding was confirmed to compete with warfarin for FA-7 with significantly higher affinity. NO₂-OA competes with ibuprofen for FA-3 and FA-6, however, in contrast to the situation with warfarin, the binding affinities are not significantly different. The described interactions are based exclusively on non-covalent binding. No covalent binding of NO₂-OA to HSA was detected by MS/MS. More detailed studies based on MALDI-TOF-MS and Ellman's assay indicated that HSA can be covalently modified in the presence of NO₂-OA to a very limited extent. It was also shown that NO₂-OA has a higher affinity to HSA than that of OA.