ROLE OF DEHYDROASCORBATE IN RABBIT ERYTHROCYTE HEXOKINASE INACTIVATION INDUCED BY ASCORBIC ACID FE(II)/

In this study we investigated the species involved in the process of h exokinase inactivation induced by ascorbic acid/Fe(II) in rabbit eryth rocytes. Our results suggest a model in which divalent iron is first o xidized to the trivalent state and then triggers the oxidation of asco rbic acid. The H2O2 formed during this process accelerates the formati on of dehydroascorbic acid, which appears to be necessary and sufficie nt to induce hexokinase inactivation. This model was validated by show ing that: (a) H2O2-decomposing enzymes, unlike scavengers of the hydro xyl radicals, reduced the extent of hexokinase inactivation; (b) when H2O2 was used instead of ascorbate/Fe(II), it was unable, even at very high concentrations, to inhibit hexokinase activity; (c) replacing Fe (II) with either Fe(III) or H2O2 resulted in comparable levels of asco rbic. acid-induced hexokinase inactivation; (d) expression of maximal hexokinase inhibiting activity was also triggered via enzyme-catalyzed oxidation of ascorbic acid or direct addition of dehydroascorbic acid ; (e) the level of dehydroascorbic acid, which was actively generated in the external medium upon addition of ascorbic acid/Fe(II), increase d as a function of time. Taken together, these results demonstrate tha t the process of hexokinase inactivation induced by ascorbic acid/Fe(I I) is mediated by dehydroascorbate and that iron and H2O2 have the sol e function of accelerating its formation. (C) 1996 Academic Press, Inc .