Sensitivity of Protein Sulfhydryl Repair Enzymes to Oxidative Stress

According to their demonstrated activities, the thiol-disulfide oxidoreductase (TDOR) enzyme systems [thioltransferase (glutaredoxin) and GSSG reductase; and thioredoxin and thioredoxin reductase] are expected to provide the primary cellular mechanism for protection and repair of sulfhydryl proteins under oxidative stress. Since all four enzymes have active site dithiol moieties, they may be vulnerable to oxidative damage themselves. Therefore, an hydroxyl radical generating system (chelated ferrous iron in combination with hydrogen peroxide) was used to document the relative sensitivity of each of the enzymes to oxidative stress in vitro. At particular concentrations of enzymes and oxidant system, all of the enzymes were deactivated nearly completely, but different patterns of susceptibility were observed. At the approximate physiological concentration of each enzyme thioredoxin and thioltransferase were largely deactivated with 1 mM Fe2+-ADP, 1 mM H2O2; whereas thioredoxin reductase and GSSG reductase were much less sensitive: 10 image thioredoxin (88% deactivated), 1 image thioltransferase (72%), 2 image thioredoxin reductase (5%), and 0.1 image GSSG reductase (17%). As the concentration of the oxidant system was decreased stepwise from 1 mM to 1 image to mimic conditions that may be associated with oxidative tissue injury in situ, deactivation of thioredoxin was decreased proportionately, whereas thioltransferase remained much more susceptible. As expected GSH and other radical scavengers protected thioltransferase from deactivation by Fe(ADP)-H2O2. To test the susceptibility of the TDOR enzymes to oxidative stress in a physiological-like setting, isolated perfused rabbit hearts were subjected to 30 min ischemia and 30 min reperfusion. The GSH/GSSG ratio and total dethiolase activity (thioltransferase and thioredoxin systems) remained unchanged relative to control hearts, indicating that overall redox status and sulfhydryl repair activity are maintained during moderate oxidative stress in situ.