Abstract

The synthesis of the 1,4-sulfonaphthylhydrazone of hellebrigenin (HSNH) from hellebrigenin and 1,4-sulfonaphthylhydrazine is described. This compound is a specific inhibitor of the (Na⁺ + K⁺)-ATPase and serves as a hydrophobic probe of the enzyme. The effects of various ligands on the fluorescence intensity (I_f) due to the enzyme-HSNH system were examined, and many of the effects on I_f were considered to emanate from the cardiotonic steroid site on the enzyme. Among the monovalent cations only Na⁺ enhanced I_f, while other monovalent cations (K⁺, Rb⁺, Li⁺, Cs⁺, Tl⁺, and NH₄⁺) suppressed it; the order of effectiveness of the suppressing ions paralleled the order of affinities for the K⁺ site. ATP or Mg⁺⁺ plus phosphate markedly diminished I_f. The effects of ligands were antagonized by hellibrigenin and were abolished or decreased if the enzyme was minimally denatured. The effects of the above ligands were specific for HSNH, since fluorescence due to 8-anilino-naphthalene-1-sulfonic acid or a biologically inactive analogue of HSNH, the 1,4-sulfonaphthylhydrazone of dianhydrostrophanthidin, was not affected by the cations and the effect of ATP was less.