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

Abstract

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported 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 and H2O2 to reinvestigate the mechanism of DMPO/.OH formation from the SOD and SOD mutants. The relative ratios of DMPO/.17OH and DMPO/.16OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H217O2 to H216O2. The reaction of the WT SOD with H217O2 in bicarbonate/CO2 buffer yielded 63% DMPO/.17OH and 37% DMPO/.16OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H217O2: DMPO/.17OH (64%); DMPO/.16OH (36%) from A4V and DMPO/.17OH (62%); and DMPO/.16OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/.OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/.OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/.OH or DEPMPO/.OH formed nor in the relative incorporation of oxygen from H2O2 or water.

Figure 1

Formation of DMPO/^⋅OH and DEPMPO/^⋅OH adducts during the reaction between H₂O₂ and WT-SOD or SOD mutants. (A) H₂O₂ (5 mM), DMPO (50 mM), DTPA (100 μ M), and WT-SOD (100 μg/ml) or A4V or G93A (100 μg/ml) were incubated in bicarbonate/CO₂ buffer (25 mM, pH 7.4). (B) H₂O₂ (5 mM), DEPMPO (50 mM), DTPA (100 μM), and WT-SOD (100 μg/ml) or A4V or G93A (100 μg/ml) were incubated in bicarbonate/CO₂ buffer (25 mM, pH 7.4). The spectra were recorded immediately after the reaction was initiated with the enzyme. Spectra are representative scans of three independent experiments.

Figure 2

The effect of ¹⁷O-labeled H₂O₂ on DMPO/^⋅OH formation. Incubation mixtures contained H₂O₂ (5 mM), DMPO (50 mM), and DTPA (100 μM) in a bicarbonate/CO₂ buffer. The reaction was initiated with either Fe(II)-EDTA (100 μM) or 100 μg of SOD or SOD mutants (G93A and A4V) as shown. Line positions from ¹⁷O and ¹⁶O couplings are shown by • and ○, respectively. The dotted lines show computer simulations of ESR spectra using the parameters a^N = 15.0 G and a^H = 15.0 G for DMPO/^⋅16OH and a^N = 15.0 G, a^H = 15.0 G, and a^17O = 4.6 G for DMPO/^⋅17OH.

Figure 3

The effect of ¹⁷O-labeled H₂O on DMPO/^⋅OH formation. (A, Upper) Incubation mixture contained Fe(II)-EDTA (100 μM), H₂O₂ (5 mM), and DMPO (50 mM) in a bicarbonate/CO₂ buffer made with H₂¹⁷O; (Lower) obtained at gain × 10. (B, Upper) Incubation mixtures contained H₂O₂ (5 mM), DMPO (50 mM), WT-SOD (100 μg/ml), and DTPA (100 μM) in a bicarbonate/H₂O₂ buffer made with H₂¹⁷O (pH 7.4); (Lower) same as Upper but obtained at gain × 10. The dotted lines show a computer simulation of ESR spectra using the parameters a^N = 15.0 G and a^H = 15.0 G for DMPO/^⋅16OH and a^N = 15.0 G, a^H = 15.0 G, and a^17O = 4.6 G for DMPO/^⋅17OH. (C) Conditions were the same as in B (Lower) using SOD mutants.

Figure 4

The effect of ¹⁷O-labeled H₂O₂ on DEPMPO/^⋅OH formation. Incubation mixtures contained H₂O₂ (5 mM), DEPMPO (20 mM), and DTPA (100 μM) in a bicarbonate/CO₂ buffer. The reaction was initiated with either Fe(II)-EDTA (100 μM) or 100 μg of SOD or SOD mutants (G93A and A4V) as shown. Line positions from ¹⁷O and ¹⁶O couplings are shown by • and ○, respectively. The dotted lines show a computer simulation of ESR spectra using the parameters a^N = 14.0 G, a^H = 13.2 G, a^P = 47.0 G for DEPMPO/^⋅16OH and a^N = 14.0 G, a^H = 13.1 G, a^P = 47.0 G, and a^17O = 4.1 G for DEPMPO/^⋅17OH).