The carbonylation and covalent dimerization of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity is inhibited by the radical scavenger tempol

Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of various diseases via mechanisms that are not completely understood. Recently, we reported that high doses of tempol moderately increased survival in a rat model of ALS (amyotrophic lateral sclerosis) while decreasing the levels of oxidized hSOD1 (human Cu,Zn-superoxide dismutase) in spinal cord tissues. To better understand such a protective effect in vivo , we studied the effects of tempol on hSOD1 oxidation in vitro . The chosen oxidizing system was the bicarbonate-dependent peroxidase activity of hSOD1 that consumes H2O2 to produce carbonate radical, which oxidizes the enzyme. Most of the experiments were performed with 30 μM hSOD1, 25 mM bicarbonate, 1 mM H2O2, 0.1 mM DTPA (diethylenetriaminepenta-acetic acid) and 50 mM phosphate buffer at a final pH of 7.4. The results showed that tempol (5–75 μM) does not inhibit hSOD1 turnover, but decreases its resulting oxidation to carbonylated and covalently dimerized forms. Tempol acted by scavenging the carbonate radical produced and by recombining with hSOD1-derived radicals. As a result, tempol was consumed nearly stoichiometrically with hSOD1 monomers. MS analyses of turned-over hSOD1 and of a related peptide oxidized by the carbonate radical indicated the formation of a relatively unstable adduct between tempol and hSOD1-Trp32•. Tempol consumption by the bicarbonate-dependent peroxidase activity of hSOD1 may be one of the reasons why high doses of tempol were required to afford protection in an ALS rat model. Overall, the results of the present study confirm that tempol can protect against protein oxidation and the ensuing consequences. Abbreviations: ALS, amyotrophic lateral sclerosis; bSOD1, bovine cytosolic Cu,Zn-superoxide dismutase 1; DHR, dihydrorhodamine 123; DMPO, 5,5-dimethylpyrroline-N-oxide; DTPA, diethylenetriaminepenta-acetic acid; ESI, electrospray ionization; FTC, fluorescein 5-thiosemicarbazide; hSOD1, cytosolic Cu,Zn-superoxide dismutase; MALDI, matrix-assisted laser-desorption ionization; MS/MS, tandem MS; SOD, Cu,Zn-superoxide dismutase; tempol, 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl; TOF/TOF, tandem time-of-flight