Anions interact with protein to induce structural changes at ligand binding sites. The effects of anion complexation include structural stabilization and promote cation-protein interaction. This study was designed to examine the interaction of aspirin and ascorbate anions with the Na⁺, K⁺-dependent adenosine triphosphatase (Na,K-ATPase) in H²O and D²O solutions at physiological pH, using anion concentrations of 0.1 µM to 1 µM with final protein concentration of 0.5 to 1 mg/ml. Absorption spectra and Fourier transform infrared (FTIR) difference spectroscopy with its self-deconvolution, second derivative resolution enhancement and curve-fitting procedures were applied to characterize the anion binding mode, binding constant, and the protein secondary structure in the anion-ATPase complexes.

Spectroscopic evidence showed that the anion interaction is mainly through the polypeptide C=O and C-N groups with minor perturbation of the lipid moiety. Evidence for this came from major spectral changes (intensity variations)of the protein amide I and amide II vibrations at 1651 and 1550 cm^-1, respectively. The anion-ATPase binding constants were K=6.45 × 10³ M^-1 for aspirin and K=1.04 × 10⁴ M^-1 for ascorbate complexes. The anion interaction resulted in major protein secondary structural changes from that of the α-helix 19.8%; β-pleated sheet 25.6%; turn 9.1%; β-antiparallel 7.5% and random 38% in the free Na,K-ATPase to that of the α-helix 24-26%; β-pleated 17-18%; turn 8%; β-antiparallel 5-3% and random 45.0% in the anion-ATPase complexes.