19F NMR spectroscopic characterization of the interaction of niflumic acid with human serum albumin

The interaction of a non-steroidal anti-inflammatory drug, niflumic acid (NFA), with human serum albumin (HSA) has been investigated by ¹⁹F nuclear magnetic resonance (NMR) spectroscopy. A ¹⁹F NMR spectrum of NFA in a buffered (pH 7.4) solution of NaCl (0.1 mol L⁻¹) contained a single sharp signal of its CF₃ group 14.33 ppm from the internal reference 2,2,2-trifluoroethanol. Addition of 0.6 mmol L⁻¹ HSA to the NFA buffer solution caused splitting of the CF₃ signal into two broadened signals, shifted to the lower fields of 14.56 and 15.06 ppm, with an approximate intensity ratio of 1:3. Denaturation of HSA by addition of 3.0 mol L⁻¹ guanidine hydrochloride (GU) restored a single sharp signal of CF₃ at 14.38 ppm, indicating complete liberation of NFA from HSA as a result of its denaturation. These results suggest that the binding is reversible and occurs in at least two HSA regions. Competitive ¹⁹F NMR experiments using warfarin, dansyl-l-asparagine, and benzocaine (site I ligands), and l-tryptophan and ibuprofen (site II ligands) revealed that NFA binds to site I at two different regions, Ia and Ib, in the ratio 1:3. By use of ¹⁹F NMR with NFA as an ¹⁹F NMR probe the nonfluorinated site I-binding drugs sulfobromophthalein and iophenoxic acid were also found to bind sites Ia and Ib, respectively. These results illustrate the usefulness and convenience of ¹⁹F NMR for investigation of the HSA binding of both fluorinated and nonfluorinated drugs.