Function of thioredoxin reductase as a peroxynitrite reductase using selenocystine or ebselen

The activity of mammalian thioredoxin reductase as a peroxynitrite reductas e was investigated. Peroxynitrite was infused to maintain a 0.2 mu M steady -state concentration in potassium phosphate buffer (pH 7.4). Benzoate hydro xylation and nitrite formation were used as indices of oxidation reactions of peroxynitrite and of peroxynitrite reduction, respectively. In the prese nce of NADPH (10 mu M), thioredoxin reductase at 50 nM alone did not signif icantly scavenge peroxynitrite, as shown by there being no significant effe ct on benzoate hydroxylation or nitrite formation. However, when selenocyst ine (1 mu M) or ebselen (2 mu M) was present in the reaction mixture, there was significant suppression of benzoate hydroxylation and an increase in n itrite formation until all the NADPH was oxidized. The addition of thioredo xin did not enhance these effects. In contrast, peroxynitrite reduction by ebselen complexed with BSA was enhanced by the presence of thioredoxin. eff iciently reduced ebselen selenoxide back to In parallel experiments, thiore doxin reductase ebselen.