Thiol Oxidase Activity of Bovine Lens Aldose Reductase Modified by Copper Ion

Abstract

Abstract: : Purpose. Copper is a trace element whose relevance in inducing cellular damage is well documented. In the cell the maintenance of low level of free copper is accomplished essentially through the presence of protein components able to efficiently chelate the metal ion. Abnormal high level of copper, as well as of other transition metal ions, can favour protein aggregation, which in the lens can be directly responsible for functional impairment. Despite being not a metal binding protein, bovine lens aldose reductase (ALR2) is extremely sensitive to Cu(II). The enzyme is readily inactivated by stoichiometric concentrations of the metal through an oxygen independent modification process. The modified enzyme (Cu-ALR2) retains two copper ions and carries a disulfide bond between Cys298 and Cys303. The ability of copper bound to ALR2 to intervene in redox processes has been evaluated. Methods. The modification of bovine lens ALR2 was accomplished by incubating at 25°C the purified enzyme in the presence of a copper:enzyme molar ratio of 2.5. The enzyme was then extensively dialyzed against EDTA. Copper content was evaluated by bathocuproine complexation. Thiol oxidation was evaluated by DTNB titration. Results.. When thiols are incubated in the presence of Cu-ALR2, a loss of reduced thiols is observed in a time dependent manner. The rate of thiol oxidation is proportional to the concentration of Cu-ALR2 and dependent on the nature of the thiol used and on the pH. While GSH and homocysteine appear to be quite insensitive to oxidation, cysteine, cysteamine and CysGly are more prone to oxidation as it occurs when free copper is use as catalyst. The presence of the disulfide bond on Cu-ALR2 is unnecessary for thiol oxidase activity. Indeed, when Cu(I) is used to modify ALR2, the obtained copper-modified enzyme, which doesn't contain any disulfide, is able to catalyze thiol oxidation. Conclusion. Cu-ALR2 acquires the ability to catalyze oxidation of different thiol compounds. This feature is associated with the retention of the redox reactivity of the bound copper.

Keywords: oxidation/oxidative or free radical damage