Some basic properties of ascorbate-dependent antioxidative-defence factors from rat liver cytosol

Abstract

Properties of the ascorbate-dependent antioxidative-defence factors from rat liver cytosol (Antonenkov and Sies, 1992, Biol. Chem. Hoppe-Seyler 373, 1111–1116) were investigated. The factors are assayed by their capacity to inhibit Fe/ADP-ascorbate (nonenzymatic) lipid peroxidation. Ammonium sulfate fractionation and gel-filtration of the dialysed cytosol on Sephadex G-200 or Sephacryl S-400 led to the separation of factors A and B, with approximate molecular masses of > 400 kDa and 60 kDa, respectively. After fractionation of cytosol, factors A and B required the presence of the thiol- reducing agent, dithioerythritol, for their activity; factor B more so than factor A. Inhibiting activity of factor B is displayed at higher ascorbate concentrations than that of factor A. Increasing the ionic strength strongly stimulated the inhibitory capacity of factor A, but only slightly that of factor B. Differences were observed in pH-dependence, though both factors were more active in neutral media (pH 6.2–6.8). Factors A and B had different time-courses of inhibition of TBARS formation and suppression of chemiluminescence intensity. Factor B was more stable in the course of thermal treatment. Repeated freezing and thawing did not affect the activity of both factors. However, factor A could be inactivated by ultrasonic treatment, whereas factor B was unaffected. A function of these factors could be to create more favourable conditions for the interaction of ascorbate as a very hydrophilic molecule with membrane-bound hydrophobic radicals.