Inactivation of alcohol dehydrogenase (ADH) by ferryl derivatives of human hemoglobin

Abstract

In this paper, inactivation of alcohol dehydrogenase (ADH) by products of reactions of H 2O 2 with metHb has been studied. Inactivation of the enzyme was studied in two systems corresponding to two kinetic stages of the reaction. In the first system H 2O 2 was added to the mixture of metHb and ADH [the (metHb + ADH) + H 2O 2] system (ADH was present in the system since the moment of addition of H 2O 2 i. e. since the very beginning of the reaction of metHb with H 2O 2). In the second system ADH was added to the system 5 min after the initiation of the reaction of H 2O 2 with metHb [the (metHb + H 2O 2) 5 min + ADH] system. In the first case all the products of reaction of H 2O 2 with metHb (non-peroxyl and peroxyl radicals and non-radical products, viz. hydroperoxides and *HbFe(IV) = O) could react with the enzyme causing its inactivation. In the second system, enzyme reacted almost exclusively with non-radical products (though a small contribution of reactions with peroxyl radicals cannot be excluded). ADH inactivation was observed in both system. Hydrogen peroxide alone did not inactivate ADH at the concentrations employed evidencing that enzyme inactivation was due exclusively to products of reaction of H 2O 2 with metHb. The rate and extent of ADH inactivation were much higher in the first than in the second system. The dependence of ADH activity on the time of incubation with ferryl derivatives of Hb can be described by a sum of three exponentials in the first system and two exponentials in the second system. Reactions of appropriate forms of the ferryl derivatives of hemoglobin have been tentatively ascribed to these exponentials. The extent of the enzyme inactivation in the second system was dependent on the proton concentration, being at the highest at pH 7.4 and negligible at pH 6.0. The reaction of H 2O 2 with metHb resulted in the formation of cross-links of Hb subunits (dimers and trimers). The amount of the dimers formed was much lower in the first system i. e. when the radical forms dominated the reaction of inactivation.