Decrease in 2,2,6,6-Tetramethyl-Piperidine-1-Oxyl (TEMPO) EPR Signal in Peroxynitrite-Treated Erythrocyte Membranes.

The treatment of erythrocyte membranes with peroxynitrite (ONOO-), a cytotoxic species formed in vivo by the almost completely diffusion controlled reaction of nitric oxide (NO*) and the superoxide anion (O2*-), led to the loss of the EPR signal of the nitroxide radical 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO). The decrease in the TEMPO EPR signal was peroxynitrite concentration dependent in the studied peroxynitrite concentration range (100-1000 microM). The absence of such a phenomenon in the control membranes (not treated with peroxynitrite) and in a buffer treated with peroxynitrite indicates that the effect must be caused by nitroxide radicals reacting with the products of peroxynitrite reactions with membrane components. To find out which membrane components are responsible for the decrease in EPR signal, this effect was studied in simple model systems (protein and lipid suspensions). The same phenomenon was observed in both lipid and protein systems treated with peroxynitrite, but in protein solutions the effect was greater and occurred for lower peroxynitrite concentrations. A clear effect of the loss of the EPR signal was observed for both erythrocyte membranes and bovine serum albumin (BSA) solution for a peroxynitrite concentration of 100 microM, while in the case of linolenic acid suspension, a significant difference between control and peroxynitrite-treated samples was achieved for a peroxynitrite concentration of 1000 microM. A comparison of the results obtained for the lipid and protein systems suggests that the reaction of nitroxide radicals with protein derived species plays the main role in the observed decrease in the TEMPO EPR signal in peroxynitrite treated erythrocyte membranes.