Inactivation of Cu,Zn-superoxide dismutase by free radicals derived from ethanol metabolism: A γ radiolysis study

The reactions of free radicals derived from ethanol metabolism with Cu,Zn SOD were studied. 1-Hydroxyethyl radicals were generated by γ radiolysis of a N2O-saturated ethanolic solution (10-2 M) in phosphate buffer (10-3 M, pH 7.4). To generate acetyl radicals by γ radiolysis, we used ethylene glycol (10-2 M) in phosphate buffer (10-3 M, pH 7.4). This allows us to avoid the use of acetaldehyde, which may be toxic toward various cellular constituents. We have previously reported that HO radicals reacting with either acetaldehyde or ethylene glycol produce the same free radicals (Santiard et al., 1991, J. Chim. Phys. 88, 967–976). The rate constant of reaction of 1-hydroxyethyl free radicals with Cu,Zn-SOD was measured separately by competition kinetics with the spin trapping agent α-(4-pyridyl 1-oxide) N-terbutylnitrone (4-POBN), after having measured the rate constant of scavenging of 1-hydroxyethyl free radicals by 4-POBN in the absence of SOD. We found k1 (4-POBN + 1-hydroxyethyl radical) = 4.2 105 M−1 s−1 and kR (SOD + 1-hydroxyethyl radical) = 6.8 105 M−1 −1). 1Hydroxyethyl or acetyl radicals produced dose-dependent Cu,Zn-SOD inactivation. The inactivation rate constant of Cu,Zn-SOD by 1-hydroxyethyl radicals is k1 = 1.13 104 M−1 s−1 Free radicals derived from ethanol metabolism can thus react SOD leading to enzyme inactivation, besides the fact that the reactivities of 1-hydroxyethyl radicals with 4-POBN and with proteins such as Cu,Zn SOD are of the same order of magnitude could explain the difficulties to trap in vivo these radicals.