Ascorbate-dependent protection of human erythrocytes against oxidant stress generated by extracellular diazobenzene sulfonate.

Diazobenzene sulfonic acid (DABS) has been used to label thiols and amino groups on cell-surface proteins. However, we found that in addition to inhibiting an ascorbate-dependent trans-plasma membrane oxidoreductase in human erythrocytes, it also depleted alpha-tocopherol severely in the cell membrane. When erythrocytes were loaded with ascorbate, DABS-dependent loss of alpha-tocopherol was decreased, despite little change in intracellular ascorbate content. Sparing of alpha-tocopherol also was seen in erythrocyte ghosts resealed to contain ascorbate, although this was accompanied by loss of intravesicular ascorbate, probably due to the inability of ghosts to recycle ascorbate. A transmembrane transfer of electrons from ascorbate was confirmed by electron paramagnetic resonance spectroscopy, in which extracellular DABS was found to generate the ascorbate free radical within cells. When the membrane content of alpha-tocopherol was decreased to 20% of the initial value by DABS treatment, lipid peroxidation ensued, manifest by generation of F(2)-isoprostanes in the cell membranes. Intracellular ascorbate also strongly protected against F(2)-isoprostane formation. These results show that DABS causes an oxidant stress at the membrane surface that is transmitted within the cell, in part by an alpha-tocopherol-dependent mechanism, and that ascorbate recycling of alpha-tocopherol can protect against loss of alpha-tocopherol and the ensuing lipid peroxidation.