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 g roups on cell-surface proteins. However, we found that in addition to inhib iting an ascorbate-dependent trans-plasma membrane oxidoreductase in human erythrocytes, it also depleted alpha-tocopherol severely in the cell membra ne. When erythrocytes were loaded with ascorbate, DABS-dependent loss of al pha-tocopherol was decreased, despite little change in intracellular ascorb ate content. Sparing of alpha-tocopherol also was seen in erythrocyte ghost s resealed to contain ascorbate, although this was accompanied by loss of i ntravesicular ascorbate, probably due to the inability of ghosts to recycle ascorbate. A transmembriine transfer of electrons from ascorbate was confi rmed by electron paramagnetic resonance spectroscopy, in which extracellula r DABS was found to generate the ascorbate free radical within cells. When the membrane content of alpha-tocopherol was decreased to 20% of the initia l value by DABS treatment, lipid peroxidation ensued, manifest by generatio n of F-2-isoprostanes in the cell membranes. Intracellular ascorbate also s trongly protected against F-2-isoprostane formation. These results show tha t DABS causes an oxidant stress at the membrane surface that is transmitted within the cell, in part by an alpha-tocopherol-dependent mechanism, and t hat ascorbate recycling of alpha-tocopherol can protect against loss of alp ha-tocopherol and the ensuing lipid peroxidation. BIOCHEM PHARMACOL 60;1:41 -53, 2000. (C) 2000 Elsevier Science Inc.