REEXAMINATION OF THE MECHANISM OF HYDROXYL RADICAL ADDUCTS FORMED FROM THE REACTION BETWEEN FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED CU,ZN SUPEROXIDE-DISMUTASE MUTANTS AND H2O2

Amyotrophic lateral sclerosis (ALS) involves the progressive degenerat ion of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reporte d to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5,5'-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water an d H2O2 to reinvestigate the mechanism of DMPO/(OH)-O-. formation from the SOD and SOD mutants. The relative ratios of DMPO/(OH)-O-. 17 and D MPO/(OH)-O-. 16 formed in the Fenton reaction were 90% and 10%, respec tively, reflecting the ratios of (H2O2)-O-17 to (H2O2)-O-16. The react ion of the WT SOD with (H2O2)-O-17 in bicarbonate/CO2 buffer yielded 6 3% DMPO/(OH)-O-. 17 and 37% DMPO/(OH)-O-. 16. Similar results were obt ained from the reaction between familial ALS SOD mutants and (H2O2)-O- 17:DMPO/(OH)-O-. 17 (64%); DMPO/(OH)-O-. 16 (36%) from A4V and DMPO/(O H)-O-. 17 (62%); and DMPO/(OH)-O-. 16 (38%) from G93A, These results w ere confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroli ne N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to ear lier reports, the present results indicate that a significant fraction of DMPO/(OH)-O-. formed during the reaction of SOD and familial ALS S OD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/(OH)-O-. presumably via DMPO ra dical cation, No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/(OH)-O-. or DEPMPO/(OH)-O-. form ed nor in the relative incorporation of oxygen from H2O2 or water.