INACTIVATION OF CU,ZN-SUPEROXIDE DISMUTASE BY FREE-RADICALS DERIVED FROM ETHANOL-METABOLISM - A GAMMA-RADIOLYSIS STUDY

The reactions of free radicals derived from ethanol metabolism with Cu ,Zn SOD were studied. 1-Hydroxyethyl radicals were generated by gamma radiolysis of a N2O-saturated ethanolic solution (10(-2) M) in phospha te buffer (10(-3) M, pH 7.4). To generate acetyl radicals by gamma rad iolysis, 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 m ay be toxic toward various cellular constituents. We have previously r eported that HO. radicals reacting with either acetaldehyde or ethylen e glycol produce the same free radicals (Santiard et al., 1991, J. Chi m. Phys. 88, 967-976). The rate constant of reaction of 1-hydroxyethyl free radicals with Cu,Zn-SOD was measured separately by competition k inetics with the spin trapping agent alpha-(4-pyridyl 1-oxide) N-terbu tylnitrone (4-POBN), after having measured the rate constant of scaven ging of 1-hydroxyethyl free radicals by 4-POBN in the absence of SOD. We found k(1) (4-POBN + 1-hydroxyethyl radical) = 4.2 10(5) M(-1) s(-1 ) and k(R) (SOD + 1-hydroxyethyl radical) = 6.8 10(5) M(-1) s(-1). 1-H ydroxyethyl or acetyl radicals produced dose-dependent Cu,Zn-SOD inact ivation. The inactivation rate constant of Cu,Zn-SOD by 1-hydroxyethyl radicals is k(i) = 1.13 10(4) 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 wit h 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 radical s.