ASCORBATE IS THE MAJOR ELECTRON-DONOR FOR A TRANSMEMBRANE OXIDOREDUCTASE OF HUMAN ERYTHROCYTES

Ascorbic acid is an important antioxidant in human blood. Erythrocytes contribute to the antioxidant capacity of blood by regenerating ascor bate and possibly by exporting ascorbate-derived reducing equivalents through a transmembrane oxidoreductase. The role of ascorbate as an el ectron donor to the latter enzyme was tested in human erythrocytes and ghosts using nitroblue tetrazolium as an electron acceptor. Although nitroblue tetrazolium was not directly reduced by ascorbate, erythrocy te ghosts facilitated reduction of nitroblue tetrazolium in the presen ce of ascorbate and ascorbate derivatives containing a reducing double bond. The resulting blue monoformazan product was deposited directly in ghost membranes. Ascorbate-induced monoformazan deposition showed s everal features of an enzyme-mediated process, including hyperbolic de pendence on substrate and acceptor concentrations, as well as sensitiv ity to enzyme proteolysis, detergent solubilization, and sulfhydryl re agents. Incubation of intact erythrocytes with nitroblue tetrazolium c aused deposition of the monoformazan in ghost membranes prepared from the cells. This deposition reflected the intracellular ascorbate conte nt and was inhibited by extracellular ferricyanide, a known electron a cceptor for the transmembrane oxidoreductase. Although nitroblue tetra zolium did not cross the cell membrane, like the cell-impermeant ferri cyanide, it oxidized intracellular [C-14]ascorbate to [C-14]dehydroasc orbate, which then exited the cells. In resealed ghosts, both monoform azan deposition and ferricyanide reduction were proportional to the in travesicular ascorbate concentration. NADH was only about half as effe ctive as a donor for the enzyme as ascorbate in both open and resealed ghosts. These results suggest that not only can ascorbate donate elec trons to a transmembrane oxidoreductase, but that it may be the major donor in intact erythrocytes.