Fluorescein Isothiocyanate Labeling of Recombinant Na+, K+-ATPase N-domain as a Tool for Testing Ligand Binding to the Nucleotide Binding Site

-ATPase (NKA) enzyme and reacts with a lysine residue. This results in the covalent labeling of NKA. The presence of the substrate ATP and eosin Y prevents NKA from being labeled by FITC. The interaction of ligands with enzyme active sites is of great interest in biomedicine and other areas of research. This study analyzes the binding of adenosine triphosphate (ATP), FITC, and eosin Y to a generated three-dimensional (3D) structural model of the NKA nucleotide-binding domain (N-domain) using molecular docking and molecular dynamics simulations. The recombinant N-domain was affinity-purified and tested for protection against FITC labeling mediated by ATP and eosin Y. FITC-labeled N-domain decreased, as observed by fluorescence emission under ultraviolet (UV) light in clear gels (sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE]), indicating competition between the ligands. We propose that the FITC competitive labeling assay of the recombinant N-domain is useful for testing molecules that potentially bind to the NKA nucleotide-binding site.