Hydroxyl radical production by H2O2 plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme.
To elaborate the catalytic activity of Cu2+ of Cu,Zn-superoxide dismutase (SOD) in the generation of hydroxyl radical (.OH) from H2O2, we investigated the mechanism of inactivation of alpha 1-protease inhibitor (alpha 1-PI), mediated by H2O2 and Cu,Zn-SOD. When alpha 1-PI was incubated with 500 units/ml Cu,Zn-SOD and 1.0 mM H2O2, 60% of anti-elastase activity of alpha 1-PI was lost within 90 min. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that free .OH was indeed generated in the reaction of Cu,Zn-SOD/H2O2; this was substantiated by the almost complete eradication of .OH by either ethanol or dimethyl sulfoxide accompanied by the generation of carbon-centered radicals. .OH production and alpha 1-PI inactivation in the H2O2/SOD system became apparent at 30 min or later. Dimethyl sulfoxide and 5,5-dimethyl-1-pyrroline N-oxide protected inactivation of alpha 1-PI significantly in this system, indicating that alpha 1-PI inactivation was mediated by .OH. SOD activity decreased rapidly during the reaction with H2O2 for the initial 30 min. Time-dependent changes in the ESR signal of SOD showed the destruction of ligands for Cu2+ in SOD by H2O2 within this initial period. Thus we conclude that inactivation of alpha 1-PI is mediated in the H2O2/Cu,Zn-SOD system via the generation of .OH by free Cu2+ released from oxidatively damaged SOD.
Sato
K
K
Department of Microbiology, Kumamoto University School of Medicine, Japan.
Akaike
T
T
Kohno
M
M
Ando
M
M
Maeda
H
H
D003497:Cyclic N-Oxides / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006878:Hydroxides / Q000378:metabolism*; D017665:Hydroxyl Radical; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D004369:Pentetic Acid / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013113:Spin Labels; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000378:metabolism*; D013997:Time Factors; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D003497:Cyclic N-Oxides; D005609:Free Radicals; D006878:Hydroxides; D007527:Isoenzymes; D011994:Recombinant Proteins; D013113:Spin Labels; D013463:Sulfuric Acid Esters; D000515:alpha 1-Antitrypsin; D017665:Hydroxyl Radical; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D004369:Pentetic Acid; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; C007482:dimethyl sulfate