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 hexokinase inactivation induced by ascorbic acid/Fe(II) in rabbit erythrocytes. Our results suggest a model in which divalent iron is first oxidized to the trivalent state and then triggers the oxidation of ascorbic acid. The H202 formed during this process accelerates the formation of dehydroascorbic acid, which appears to be necessary and sufficient to induce hexokinase inactivation. This model was validated by showing that: (a) H202-decomposing enzymes, unlike scavengers of the hydroxyl radicals, reduced the extent of hexokinase inactivation; (b) when H202 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 H202 resulted in comparable levels of ascorbic 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), increased as a function of time. Taken together, these results demonstrate that the process of hexokinase inactivation induced by ascorbic acid/Fe(II) is mediated by dehydroascorbate and that iron and H202 have the sole function of accelerating its formation.
Fiorani
M
M
Istituto di Chimica Biologica Giorgio Fornaini, Università degli Studidi Urbino, Italy.
Saltarelli
R
R
De Sanctis
R
R
Palma
F
F
Ceccaroli
P
P
Stocchi
V
V
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002374:Catalase / Q000494:pharmacology; D003683:Dehydroascorbic Acid / Q000097:blood / Q000494:pharmacology*; D004912:Erythrocytes / Q000201:enzymology*; D005296:Ferrous Compounds / Q000494:pharmacology*; D005979:Glutathione Peroxidase / Q000494:pharmacology; D006593:Hexokinase / Q000037:antagonists & inhibitors / Q000097:blood*; D006861:Hydrogen Peroxide / Q000097:blood / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000097:blood; D007700:Kinetics; D011817:Rabbits
D005296:Ferrous Compounds; C051140:iron(II)-ascorbic acid complex; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D002374:Catalase; D005979:Glutathione Peroxidase; D006593:Hexokinase; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid