The effects of anions on the solution structure of Na,K-ATPase

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 physiolo gical pH. using anion concentrations of 0.1 muM to 1 mM with final protein concentration of 0.5 to 1 mg/ml. Absorption spectra and Fourier transform i nfrared (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 moi ety. Evidence for this came from major spectral changes (intensity variatio ns) 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 x 103 M-1 for aspirin and K=1.04 x 104 m(-1) for ascorbate complexes. The anion interact ion resulted in major protein secondary structural changes from that of the alpha -helix 19.8%; beta -pleated sheet 25.6%; turn 9.1%; beta -antiparall el 7.5% and random 38% in the free Na,KA-TPasc to that of the alpha -helix 24-26%; beta -pleated 17-18%; turn 8%; beta -antipleated 5-3% and random 45 .0% in the anion-ATPase complexes.