On the basis of the fact that selenium (Se) from selenite binds to hemoglobin (Hb), we investigated the missing process in the selenium export from red blood cells (RBCs), i.e., the transfer of selenium bound to Hb to RBC membrane proteins. To elucidate the molecular events of the Hbassociated selenium export from RBC, an Hb-Se complex was synthesized from thiol-exchange of Cys-β93 in Hb with penicillamine-substituted glutathione selenotrisulfide, as a model of major metabolic intermediates, and then interactions between the Hb-Se complex and RBC inside-out vesicles (IOVs) were examined. Selenium bound to Hb was transferred to the IOV membrane on the basis of the intrinsic interactions between Hb and the cytoplasmic domains of Band 3 protein (CDB3). The observed selenium transfer was inhibited by the pretreatments of IOVs with iodoacetamide and the α-chymotrypsin digestion, indicating that the Hb mediates the selenium transfer to the thiol groups of CDB3. In addition, it was found that deoxygenated Hb with a high binding affinity for CDB3 more favorably transferred selenium to the IOV membranes than oxygenated Hb with a low affinity. When selenium export from RBC to the plasma was examined by continuously introducing nitrogen gas, the selenium export rate was promoted with an increase in the rate of deoxygenated Hb. Overall, these data suggested that Hb could possibly play a role in the selenium export from RBC treated with selenite in an oxygen-linked fashion.

In this study, we demonstrated a human serum albumin (HSA)-mediated selenium transfer; the selenium exported from red blood cells (RBCs) was bound to HSA through the selenotrisulfide and then transferred into the hepatocyte. After the treatment of the RBCs with selenite, the selenium efflux from the RBCs occurred in an HSA concentration-dependent manner. Pretreatment of HSA with iodoacetamide almost completely inhibited the selenium efflux from the RBC to the HSA solution. The selenium efflux experiment was carried out in an HSA solution (45 mg/mL), and subsequently the HSA solution was subjected to gel permeation chromatographic separation. The peak fraction of the selenium content was consistent with that of the HSA. The selenium bound to HSA in this solution was completely eliminated by a treatment with penicillamine (Pen), which resulted in the generation of penicillamine selenotrisulfide, PenSSeSPen. The selenium efflux from the RBCs was also occurred in a Pen solution, and PenSSeSPen was observed in the resulting Pen solution. The selenium exported from the RBC was thought to bind to the HSA via a selenotrisulfide linkage with its single free thiol. A model of the selenium-bound HSA was prepared by the reaction of the HSA with PenSSeSPen. The selenium from PenSSeSPen can bind to HSA by a thiol exchange between Pen and the free thiol of HSA, which produces the selenotrisulfide-containing HSA (HSA-SSeSPen). When HSA-SSeSPen was incubated with isolated rat hepatocytes, the selenium content in the hepatocytes increased along with its decrease in the incubation medium. To verify the results from the model experiments using HSA-SSeSPen, we conducted the HSA-mediated selenium transfer experiment from RBC treated with selenite to the hepatocytes. The selenotrisulfide-containing HSA was able to transport the selenium into the hepatocyte. Overall, the selenium transfer from the RBC to the hepatocytes involves a relay mechanism of thiol exchange that occurs between the selenotrisulfide and thiol compounds (selenotrisulfide relay mechanism: R-SSeS-R + HSA-SH --> HSA-SSeS-R + R'-SH --> R-SSeS-R').